Angeliki Platania

Tom Sexton
email: sexton@igbmc.fr

Mikhail Spivakov

Chromatin architecture and topology in pluripotent stem cells

Keywords: 4C, epigenomics, browser, chromatin loops, quantile normalization, biological replicates

There are several people who have contributed to the conception and execution of my thesis and I would like to thank them.

First I wish to thank my supervisor, Tom Sexton, for accepting me in his laboratory and giving me the chance to work on such a challenging project. His motivation, crazy ideas and enthusiasm were an inspiration for me. Thank you for the guidance throughout this journey, for believing in me and helping me improve as a scientist. Except from a great scientist, Tom is a very cool and nice person. He has always been there when things went bad, helping me overcome several challenges and reminding me that the priority is to take care of my health and my well-being. I would do it all over again and I am extremely grateful for that! Besides Tom, I would like to thank the rest of my committee members for agreeing to evaluate my work and attend my thesis presentation in the middle of summer. Pr. Wendy Bickmore I still cannot believe that you accepted to be a part of my jury. You have been an inspiration to me and it will be a great honor to discuss my work with you. Dr. Luca Giorgetti I had the chance to meet you in several conferences and I was always impressed after our short talks during poster sessions. I am looking forward to discuss my results with you. Dr. Isabela Sumara thank you for being so kind and postponing your holidays in order to attend my presentation. I am looking forward for your comments. Pr. Kerstin Bystricky thank you for discovering the ANCHOR system, for all the valuable advice that you gave me since the very beginning of my thesis and for being part of my TAC committee. Last, I would also like to thank Dr. Evi Soutoglou, who was also part of my TAC, and for all the great discussions that we had over the years.

I am grateful to all the Sexton lab members, former and current. Cathie, ma collègue d'amour, thank you for doing your magics with your golden hands during the last and most critical years of my thesis. You are adorable and it was such a pleasure working with you. Nezih, we went through this thesis together and my days wouldn't have been the same without you. Thank you for all the great discussions, the 4C brainstorming and the delicious Turkish dishes that you shared with me! A special mention to Gui for his patience and contribution to the project. Thank you for all the effort that you put on developing the GP-Tool and analyzing my data. Natalia, my module and Sox2 mate, thank you for sharing your ideas with me and for bringing breakfast and chocolates to cheer us up. Dominique and Manon you did an excellent job while establishing the ANCHOR system in the lab and I am very grateful for that. Anne, Audrey, Yousra and Sanjay thank you for welcoming me in the team the first months of the Ph.D. Of course, the fresh blood in the lab Kubra and Thomas, thank you for reading and correcting my French summaries. I hope that we will have the opportunity to work together soon! Moreover, I wish to thank all the people from the IGBMC facilities, and especially Marion and Amelie that took care of my cell lines, as well as Erwan, that spent hours with me at the microscope trying to see the ANCHOR spots and helping me setup my experiments. Last, a special thanks goes to Tineke Lenstra and Marit de Kort for sharing their expertise on the MS2 system and to Jennifer Mitchell for all the great advice and reagents that shared with me throughout the years.

To the amazing group of talented girls that started their Ph.D. with me, thank you so much for everything! I consider you as truly good friends. A special mention goes to Sirine, who became one of my favorite people. I cannot imagine how my life would have been without you. Thank you for your support (inside and outside the lab), kindness, love, energy and all your DIY recipes. You are an amazing and inspiring woman. Karima, my Mediterranean friend, thank you for all the discussions about spiritual life, for being there when I needed you, for the delicious dishes and for sharing with me your tisane expertise. Another special thanks goes to Diana and Kamar. You are two talented goddesses and I am really happy to met you. Keep being so amazing! I feel extremely fortunate that during these years I had the chance to meet some amazing people outside the lab. Lina, ζουζούνα, I lived some of my favorite moments ever with you. You are one of the best bar-, concert-, party-, running-, coffee-mates. Thank you for being you, simply. Katerina, τσιτσίνι, thank you for being so sweet and supporting me during all these years. Charlie, my first French friend, thank you for all the franco-grec discussions and the great time that we had together. I could not neglect thanking my dear friends Julien and Florine for all the moments that we lived together, for hosting me at their beautiful house and for preparing my birthday cakes the last two years. Special thanks goes to Gab, who never said no to the overloaded plates that I served him, as well as Marie, Stephane, Nico, Yiannis and Liza for all the soirées and the wonderful time that we spent together. Now, I wish to thank several people that are by my side for many years and without whom this thesis would not have happened. I could write a full page expressing my gratitude for each one individually but I have to keep it short. Faye, I am so proud to be your best friend. Thank you for being next to me at every single step of my life. You have the best smile and best sense of humor. Vaso, I feel so lucky for having you in my life since day zero -literally. Your love and your support gave me strength at the most difficult moments. Adam, what can I say for you? My life during the university and Ph.D. years wouldn't have been the same without you. I wouldn't exchange you for 1 million camels! Thank you for all the holidays that we spent together, your critical comments on my project and for being so amazing. Giorgo (aka Kavvadia), you are the brightest star. Thank you for always being there, the right moment with the right thing to say. I cannot find the right words to express my gratitude to Konstantina, Katerina, Elina, Dimitra and Ioanna. Since 2010, we have lived EVERYTHING together.

Thank you for being so wonderful, funny, openminded and supportive. Thank you for your true love and for accepting me as I am, with my good and bad moods.

One of the most important people in my life the last years is Julien. You showed me a whole new side of life that I had never imagined before and I am grateful for that. Thank you for being next to me, loving and supporting me every single day (yes, during the writing period as well!).

Thank you for all the things and places that you made me discover. The best is yet to come! Of course, I want to thank Simone, Marc and Loic, for welcoming me into their family, for all the great diners that have shared with me and for their support.

I would like to express my gratitude to my parents, Eugenia and Antonis as well as my brother Stratos and my sister Maura. Thank you for your unconditional love, for all the great moments that you offered me from my childhood until today and for all the sacrifices that you have done for me. Without you I wouldn't have been where and who I am today. Mother, you are the strongest and sweetest woman in the world. This thesis project and all that are about to come are dedicated to you! Antonis, I am so grateful for having you in my life. Thank you for your love and for being next to me during the most happy, important, difficult times of my life.

Stratos and Maura, my beloved siblings, I cannot imagine how my life would have been without you. You are the biggest loves of my life. We might be living apart during the last years, but every time that the three of us are together is as if we haven't spent one single day separately. I would also like to acknowledge my extended family, my aunts (Sofia and Gioula), uncles (Vangelis and Thanasis) and of course my beloved cousins (Dionisis, Vasilis, Athina, Angeliki, Dimitra, Mirto) for their love, mentality, energy and support. I am extremely happy and proud for being part of this big and noisy Greek family. Last but not least, I wish to thank my grandparents who do not speak English, so the next lines are written in Greek just for them:

Γιαγιάκα μου και παππούλη μου σας λατρεύω και μου λείπετε πολύ. Χωρίς εσάς δίπλα μου δεν θα κατάφερνα τίποτα. Ανυπομονώ να γυρίσω πίσω και να σας δω από κοντά.

List of tables

Summary

Genomes must be highly compacted within cell nuclei, but in a manner ensuring accessibility of appropriate genes to the transcriptional machinery, and accommodating marked gene program changes during development and cell reprogramming. While we have a deep view of genome organization in space from microscopy and biochemical approaches on large populations of fixed cells, fundamental questions about chromatin dynamics in living cells are completely unaddressed, despite growing appreciation that dynamics likely play key roles in the regulation of gene expression. In this study I monitored enhancer-promoter communication in real time, coupled to MS2-labeled nascent mRNA, and characterized chromatin dynamics at these loci. To address these questions, I incorporated the parS-ParB (also termed ANCHOR) system, originally developed in yeast, into mouse embryonic stem cells (ESCs) to label the endogenous Sox2 promoter and its distal (>100 kb downstream) enhancer (SCR, for Sox2 control region) for single-cell live tracking.

The real time-behavior of these elements was dissected in wild-type ESCs, during early differentiation stages when Sox2 expression is lost, in mutants of critical regulatory elements within the SCR, and in ESCs in response to pharmacological inhibition of transcriptional initiation or elongation. I found that Sox2 and SCR are frequently in close proximity, and significantly closer than control regions, but also observed an apparent uncoupling of separation distances with transcriptional firing. Rather, transcriptional activity associated with changes in local chromatin dynamics. Both the gene and the enhancer are more constrained than the control regions. Further, perturbation of Sox2 expression upon differentiation, SCR mutation and inhibition of transcriptional initiation by RNA Pol II resulted in decreased apparent diffusive speed at Sox2 promoter. Although alterations in local chromatin dynamics correlated with gene activity, loci participating in active transcriptional bursts were not found to have different chromatin dynamics to non-transcribing alleles within the same experiment.

I propose that "poised" loci have similar dynamics to transcribing loci, and that alterations in mobility accompany a transition from a refractory state to a poised, transcriptionally competent state, to be assessed in future experiments. Collectively, these findings bolster the importance of assessing chromatin dynamics at transcription sites as a means of understanding the nuclear microenvironment and regulatory principles.

Summary in French

Les génomes doivent être très compactés à l'intérieur des noyaux cellulaires, mais d'une manière qui puisse garantir l'accessibilité des gènes cibles à la machinerie transcriptionnelle et qui permette des changements importants du programme génétique au cours du développement et de la reprogrammation cellulaire. La topologie de la chromatine présente un intérêt particulier pour le contrôle de l'expression des gènes, car de nombreux gènes sont régulés par des éléments activateurs, appelés amplificateurs ou enhancers. Les enhancers peuvent être localisés à des distances de l'ordre de la mégabase de leurs gènes cibles ou dans les introns de gènes dont ils ne régulent pas l'expression (Shen et al., 2012;[START_REF] Thurman | The accessible chromatin landscape of the human genome[END_REF]. Des variantes de la technique de capture de la conformation des chromosomes (3C) [START_REF] Dekker | The three "C" s of chromosome conformation capture: controls, controls, controls[END_REF]Denker & De Laat, 2016) ont montré que les enhancers se trouvent à proximité physique directe de leurs gènes cibles, vraisemblablement grâce à la formation de boucles chromatiniennes.

Bien que l'on sache peu de choses sur la manière dont les enhancers régulent l'expression des gènes, il est suggéré que ces boucles augmentent la concentration locale de facteurs de transcription liés aux séquences promotrices et aux enhancers, fournissant ainsi un environnement permettant la transcription [START_REF] Tolhuis | Looping and interaction between hypersensitive sites in the active β-globin locus[END_REF]. Ainsi, des études récentes suggèrent que jusqu'à la moitié des gènes ne dépendent pas de leur enhancer le plus proche (Lettice et al., 2003). De manière plus frappante, un grand nombre de contacts entre enhancers et promoteurs semblent être établis avant l'activation du gène, suggérant qu'un signal inconnu serait nécessaire dans certains contextes pour permettre la formation d'une boucle chromatinienne favorisant la transcription (Palstra et al., 2003).

Actuellement, nous disposons d'une vue approfondie de l'organisation du génome dans l'espace grâce à la microscopie et aux approches biochimiques sur de grandes populations de cellules fixées. Néanmoins, alors qu'il est de plus en plus suggéré que la dynamique de la chromatine joue des rôles clés dans la régulation de l'expression des gènes, les questions fondamentales sur la dynamique de la chromatine dans les cellules vivantes sont encore très peu étudiées.

Le gène Sox2 est impliqué dans le maintien de la pluripotence et dans l'autorenouvèlement des cellules souches embryonnaires (CSE), ainsi que dans la détermination du destin cellulaire, la reprogrammation cellulaire, la régénération des tissus adultes et le cancer dans de multiples tissus tels que le poumon et le sein [START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF]. Une étude récente sur le locus Sox2 a montré qu'une région distale (région de contrôle de Sox2, SCR) située >100 kb en aval de Sox2, forme une grande boucle chromatinienne spécifiquement requise pour la transcription de Sox2 dans les CSE (Zhou et al., 2014). Néanmoins, dans le cadre de cette boucle chromatinienne, aucune information n'est disponible quant à la dynamique de la chromatine, la variabilité de cellule à cellule, la façon dont la formation de cette boucle peut influencer la régulation de Sox2.

Dans cette étude, j'ai suivi en temps réel la communication enhancer-promoteur et caractérisé la dynamique de la chromatine à ces loci. Pour réaliser cela, j'ai incorporé le système parS-ParB (également appelé ANCHOR) [START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF], initialement développé chez la levure, dans des CSE de souris afin de marquer le promoteur endogène de Sox2 et son enhancer distal (SCR) pour suivre en direct leur localisation. En parallèle, j'ai construit une lignée témoin dans laquelle les séquences ANCHOR sont décalées de 60 kb afin qu'elles puissent conserver la même distance génomique sans toutefois former de boucle chromatinienne.

Après avoir généré la lignée CSE ANCHOR à double insertion, j'ai montré que le système ANCHOR ne perturbe ni l'expression de Sox2, ni la formation de la boucle chromatinienne.

Plus précisément, j'ai confirmé entre autres par RT-qPCR en ciblant des allèles spécifiques, que la transcription de Sox2, la pluripotence et le potentiel de différenciation des CSE ne sont affectés ni par l'insertion des séquences parS, ni par le recrutement des protéines fluorescentes ParB. De plus, j'ai confirmé par 4C (Simonis et al., 2006;Van De Werken et al., 2012) que la boucle enhancer-promoteur n'est pas affectée par ces outils dans les CSE et que, comme pour les cellules de type sauvage, la boucle est perdue lors de la différenciation neuronale précoce.

Les délétions alléliques spécifiques de différentes sous parties de la SCR ont montré leur contribution différentielle à l'expression de Sox2 (laboratoire Mitchell, communication personnelle). J'ai donc créé deux lignées dérivées de ma lignée cellulaire ANCHOR de type sauvage en supprimant différents composants de l'enhancer. De la même façon que pour la lignée cellulaire ANCHOR de type sauvage, j'ai caractérisé des clones dans lesquels différentes sous parties de la SCR sont absentes et j'ai effectué des expériences 4C spécifiques à l'allèle (en ciblant SCR ) pour évaluer la capacité de ces cellules à former une boucle chromatinienne indispensable à l'expression de Sox2. Ces expériences ont montré que des délétions situées dans l'enhancer entraînent une faible perte d'interaction entre Sox2 et SCR. Afin de tester directement la relation entre le statut de l'ARN Pol II (ARN polymérase II) et le mouvement de la chromatine dans des cellules vivantes, nous avons utilisé des petites molécules inhibitrices pour bloquer le cycle de transcription à deux étapes différentes : à l'initiation, avec le triptolide, ainsi qu'à l'élongation de l'ARN Pol II, avec le flavopiridol [START_REF] Bensaude | Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity?[END_REF].

Afin de comprendre la relation entre les interactions promoteur-enhancer et la mobilité de la chromatine sur les paramètres d'activation de la transcription, j'ai utilisé le système MS2/MCP [START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF], qui permet la localisation d'ARNm uniques. J'ai inséré dans le génome de la lignée ANCHOR 24 fois la séquence MS2 et incorporé du MCP marqué par fluorescence pour l'imagerie simultanée en direct du statut transcriptionnel de Sox2. Après avoir établi et testé ces lignées cellulaires, j'ai obtenu et analysé un grand nombre de films courts de cellules individuelles pour examiner la dynamique de la chromatine dans des conditions de type sauvage dans les CSE, au cours des premiers stades de différenciation lorsque l'expression de Sox2 est perdue, dans les sous-délétions de l'enhancer qui créent des perturbations de l'expression de Sox2 et ont un effet léger sur le bouclage et, enfin, dans les CSE en réponse à l'inhibition pharmacologique de l'initiation ou de l'élongation transcriptionnelle. Cela a permis d'évaluer la variabilité d'une cellule à l'autre et de mesurer les paramètres de diffusion. En collaboration avec l'équipe de Nacho Molina (IGBMC, Strasbourg), nous avons développé une méthode pour mesurer précisément les paramètres de diffusion de la chromatine à partir de la microscopie en direct. Plus précisément, en effectuant une analyse de suivi de point unique, nous avons extrait deux paramètres : le coefficient de diffusion (D), qui reflète la vitesse de la diffusion; et le paramètre de diffusion anormale α, qui pour les petites valeurs (α<1) indique que la diffusion est limitée à des volumes plus confinés.

J'ai démontré que Sox2 et SCR sont fréquemment à proximité, et significativement plus proches que les régions de contrôle. Néanmoins, je n'ai pas mis en évidence de corrélation directe entre les distances de séparation entre Sox2 et SCR et l'activité transcriptionnelle. En effet, l'activité transcriptionnelle est plutôt associée à des changements dans la dynamique locale de la chromatine. Le gène et l'enhancer subissent quant à eux des contraintes plus importantes que les régions de contrôles. La perturbation de l'expression de Sox2 lors de la différenciation, la mutation du SCR et l'inhibition de l'initiation transcriptionnelle par l'ARN Pol II ont entraîné une diminution de la vitesse apparente de diffusion au niveau du promoteur de Sox2. Bien que les altérations de la dynamique locale de la chromatine soient en corrélation avec l'activité transcriptionelle des gènes, les loci transcriptionnellement actifs ne présentaient pas une dynamique de la chromatine différente de celle des loci transcriptionnellement inactifs au cours de la même expérience. Je propose que les loci « en pause » aient une dynamique similaire à celle des loci transcriptionnellement actifs, et que les altérations de la mobilité accompagnent la transition d'un état réfractaire à un état transcriptionnellement compétent, à évaluer dans des expériences futures. Collectivement, ces résultats renforcent l'importance de l'évaluation de la dynamique de la chromatine aux sites de transcription comme moyen de comprendre le microenvironnement nucléaire et les règles qui régissent son organisation.

Revisite des modèles de boucle d'enhancer

Un grand nombre de recherches suggèrent que le bouclage enhancer-promoteur est nécessaire à l'expression des gènes [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF][START_REF] Chen | Dynamic interplay between enhancer-promoter topology and gene activity[END_REF][START_REF] Deng | Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor[END_REF][START_REF] Deng | Reactivation of developmentally silenced globin genes by forced chromatin looping[END_REF]Palstra et al., 2003). Cependant, des expériences récentes d'imagerie ont démontré, au moins pour certains loci spécifiques, des enhancers à de grandes distances des gènes cibles activés (Alexander et al., 2019 ;Benabdallah et al., 2019 ;[START_REF] Lim | Visualization of Transvection in Living Drosophila Embryos[END_REF], découplant ainsi la proximité enhancer-promoteur de l'expression génique. Dans cette étude, je montre que les distances Sox2-SCR sont significativement plus proches (médiane=146 nm) que les régions témoins de séparation génomique équivalente (médiane=195 nm), en accord avec les données Hi-C (Bonev et al., 2017). Néanmoins, les paires Sox2-SCR et de contrôle ont été trouvées à proximité immédiate, comme on pourrait s'y attendre pour des séparations génomiques de ~115 kb. L'imagerie à haut débit récente sur des cellules fixées a révélé un degré remarquable d'hétérogénéité et de variabilité entre les cellules dans l'organisation spatiale du génome [START_REF] Bintu | Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells[END_REF][START_REF] Cattoni | Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions[END_REF][START_REF] Finn | Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization[END_REF][START_REF] Giorgetti | Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription[END_REF] [START_REF] Williams | Pausing of RNA Polymerase II Regulates Mammalian Developmental Potential through Control of Signaling Networks[END_REF].

Les perturbations qui convertissent les loci Sox2 en un état plus réfractaire peuvent le faire en modifiant le microenvironnement nucléaire, avec des effets ultérieurs sur la dynamique de la chromatine. Par exemple, la différenciation entraîne des changements de masse dans la charge de Pol II au niveau du promoteur, ainsi qu'une perte de liaison TF (facteur de transcription) à la fois au niveau du promoteur et du SCR; ces changements peuvent réduire directement les contraintes de mobilité de la chromatine locale, ou indirectement via la dissociation des hubs transcriptionnelles. Des expériences de triple marquage réalisées sur des périodes beaucoup plus longues pourraient également permettre de découvrir si des loci de type sauvage font la transition entre les états "réfractaire" et "en pause" proposés, déterminés par des altérations à plus long terme des paramètres de diffusion locaux. Il est intéressant de noter que la dynamique de la chromatine n'a pratiquement pas été modifiée par le traitement avec un inhibiteur de l'élongation de la transcription, le flavopiridol, mais que la dynamique du promoteur a été sensible au traitement avec un inhibiteur de l'initiation de la transcription, le triptolide, ce qui suggère que le recrutement de Pol II et/ou la fusion du promoteur, mais pas l'élongation processive, sont les étapes déterminantes de la mobilité locale de la chromatine.

Comparaison avec les résultats précédents

Pendant ma thèse, un autre laboratoire a publié ses travaux, dans lesquels ils ont interrogé l'organisation spatiale et l'activité de Sox2 et SCR dans des CSE F1 (Alexander et al., 2019).

En utilisant des opérateurs cuO et tetO pour marquer le promoteur et l'enhancer de l'allèle 129, les auteurs ont démontré que Sox2 et SCR sont séparés par des distances beaucoup plus grandes (moyenne ~340 nm), par rapport à celles rapportées dans mon étude, dans le noyau des CSE.

J'ai directement évalué cette divergence en introduisant ANCHOR dans les mêmes emplacements génomiques exacts de cette étude, et j'ai constaté que ces régions étaient aussi fréquemment proximales. Je propose donc que les grandes distances de séparation rapportées par les auteurs (Alexander et al., 2019) soient dues à des différences techniques, peut-être causées par les grands opérateurs répétitifs tet/cu. En effet, les auteurs ont inclus une lignée témoin, où un fragment de 111 kb a été supprimé entre les paires cuO et tetO, laissant une attache de 14 kb entre elles (Alexander et al., 2019). Curieusement, la distance de séparation moyenne observée était de ~250 nm et jamais inférieure à 100 nm, comme cela a été précédemment rapporté pour des paires de sondes DNA FISH séparées par quelques dizaines (~30-60) de kilobases [START_REF] Giorgetti | Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription[END_REF]. Je suppose que les grandes distances de séparation rapportées par Alexander et al. (Alexander et al., 2019) sont dues à l'intégration de copies multiples de séquences répétitives d'opérateurs et à la liaison stable des répresseurs marqués par fluorescence.

En outre, dans cette étude, en utilisant un système Sox2-MS2, nous avons mesuré des fréquences transcriptionnelles beaucoup plus élevées par rapport à ce qui a été rapporté précédemment (Alexander et al., 2019). Ces différences pourraient être causées par le marqueur de sélection de résistance qui était maintenu dans le locus dans l'autre étude (excisé dans mes cellules Anchor_Sox2-MS2), et pourrait perturber davantage la transcription de Sox2 et la stabilité de l'ARNm. En effet, j'ai observé que la lignée conservant le gène marqueur Les paramètres de diffusion locale comme "mesures" de l'activité régulatrice ?

À ma connaissance, ce travail est la première étude d'imagerie en direct qui décrit les distances de séparation et les propriétés de diffusion d'une paire enhancer-promoteur, simultanément à la transcription du gène, et en réponse à des inhibiteurs de la transcription. Des études précédentes ont également abordé le lien entre la dynamique de la chromatine et l'expression des gènes [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF], atteignant des résultats apparemment contradictoires. Mes résultats confirment et contredisent à la fois ces résultats précédents dans une certaine mesure. Tout d'abord, comme décrit ci-dessus pour mes propres résultats, le paramètre de diffusion anormale (α) semble lié à "l'activité", avec une plus grande contrainte dans les régions actives, en accord avec un rapport récent, où il a été démontré que la transcription coïncide avec le confinement d'un gène producteur d'ARNm dans des volumes nucléaires plus petits [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF].

Plus précisément, j'ai également constaté que le promoteur de Sox2, mais pas le SCR, diffuse plus rapidement (D app plus élevé) lors de l'activation. Ce résultat concorde partiellement avec un rapport récent: un gène et un enhancer activés lors de la différenciation se déplacent plus rapidement [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF], interprété dans cette étude comme une augmentation de l'énergie thermique via le processus de la transcription active. Mes résultats, ainsi que d'autres [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF], soulignent l'importance d'évaluer la dynamique de la chromatine aux sites de transcription comme moyen de comprendre le microenvironnement nucléaire et les principes de régulation. Néanmoins, D app et α sont des paramètres diffusifs assez généraux. Par exemple, un α réduit peut s'expliquer par de multiples mécanismes, tels que le confinement à un volume réduit ou "l'attraction" vers des régions spécifiques, qu'il est impossible de distinguer. J'attends avec impatience les progrès de la théorie physique permettant de définir des paramètres diffusifs inférés plus précis, ce qui pourrait donner un meilleur aperçu et permettre une analyse encore plus précise de la dynamique locale de la chromatine. Si l'on ajoute à cela des outils d'imagerie plus sophistiqués et de meilleures méthodes de manipulation du noyau (par exemple, l'ablation de protéines régulatrices clés à l'aide du système de dégradation de l'auxine (N. Q. [START_REF] Liu | WAPL maintains a cohesin loading cycle to preserve cell-typespecific distal gene regulation[END_REF][START_REF] Nora | Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Article Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization[END_REF][START_REF] Rao | Cohesin Loss Eliminates All Loop Domains[END_REF], ou la manipulation des propriétés de séparation des phases liquide-liquide (Y. [START_REF] Shin | Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome[END_REF], la compréhension globale des aspects quadridimensionnels de la régulation transcriptionnelle devient de plus en plus réalisable.

Introduction Transcription

Eukaryotic genomes are much more than linear sequences of DNA. The DNA helix must be condensed in the form of chromatin to fit within the cell nucleus, which has a diameter only about one-tenth that of a human hair. The fundamental unit of chromatin is the nucleosome core particle, which consists of 147 base pairs (bp) of DNA wrapped 1.7 turns around a histone octamer. Each histone octamer is composed of two copies of H2A and H2B (forming two H2A/H2B dimers) and two copies of H3 and H4 (forming one H3/H4 tetramer) [START_REF] Kornberg | Chromatin Structure : A Repeating Unit of Histones and DNA[END_REF][START_REF] Kornberg | Chromatin Structure: Oligomers of the Histones[END_REF][START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 Å resolution[END_REF][START_REF] Olins | Spheroid chromatin units (v bodies)[END_REF] (Figure 1). Further packaging of DNA is brought about by formation of the chromatosome core particle , composed of a linker histone H1 bound to the nucleosome [START_REF] Simpson | Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histones[END_REF]. The spatial organization of chromatin not only serves as a way to compact DNA but also affects nuclear processes involving DNA, including transcription, DNA replication and DNA repair. from [START_REF] Fyodorov | Emerging roles of linker histones in regulating chromatin structure and function[END_REF].

Transcription cycle

Transcription is the process by which the information from a DNA template is copied into a new RNA molecule. In eukaryotes, RNA Polymerase II (Pol II) transcribes messenger RNAs (mRNAs) from protein-coding genes. Access to key cis-regulatory binding sites is restricted by nucleosomes, thus Pol II collaborates with transcriptional regulators -also known as activators or repressors -, general transcription factors (GTFs) including TATA-binding protein (TBP), TFII-A, -B, -D, -E, -F, and -H and co-activators, like the Mediator complex [START_REF] Esnault | Mediator-Dependent Recruitment of TFIIH Modules in Preinitiation Complex[END_REF][START_REF] Kim | A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II[END_REF][START_REF] Plaschka | Architecture of the RNA polymerase II-Mediator core initiation complex[END_REF][START_REF] Workman | Nucleosome core displacement in vitro via a metastable transcription factor-nucleosome complex[END_REF], chromatin modifiers and remodelers, in order to get access to the promoter and form the preinitiation complex (PIC) [START_REF] Lorch | Histone Octamer Transfer by a Chromatin-Remodeling Complex[END_REF][START_REF] Mizuguchi | ATP-Driven Exchange of Histone H2AZ Variant Catalyzed by SWR1 Chromatin Remodeling Complex[END_REF][START_REF] Narlikar | Cooperation between Complexes that Regulate Chromatin Structure and Transcription Chromatin structure creates barriers for each step[END_REF][START_REF] Zhang | Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss[END_REF]. Eukaryotic transcription is a complex and highly dynamic event, exhibiting regulation at multiple steps.

Regulation of transcriptional output requires the concerted assembly of trans-acting chromatin factors and transcription factors (TFs) on proximal and distal regulatory DNA elements, termed "promoters" and "enhancers", respectively (discussed in detail later on in the Introduction).

Both promoters and enhancers contain binding sites for gene-specific TFs that determine when a gene will be activated throughout development [START_REF] Lambert | The Human Transcription Factors[END_REF][START_REF] Shlyueva | Transcriptional enhancers: from properties to genome-wide predictions[END_REF].

The transcription cycle can be divided in eight major steps: chromatin opening, pre-initiation complex (PIC) formation, initiation, promoter-proximal pausing, pause release, productive elongation, termination and recycling (Figure 2). The tight packaging of DNA into chromatin can present an obstacle to transcription by rendering recognition sites inaccessible. Hence, nucleosome displacement or ejection seems to be a critical event in the process of transcription [START_REF] Mueller | Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction[END_REF][START_REF] Petesch | Rapid, Transcription-Independent Loss of Nucleosomes over a Large Chromatin Domain at Hsp70 Loci[END_REF]. Such changes in chromatin accessibility are believed to be mediated by a combination of regulatory proteins, including a special class of TFs called pioneer factors [START_REF] Mayran | Pioneer transcription factors shape the epigenetic landscape[END_REF][START_REF] Zaret | Pioneer transcription factors: Establishing competence for gene expression[END_REF], histone acetylation, chromatin-remodeling complexes, and perhaps the histone variant H2A.Z [START_REF] Esnault | Mediator-Dependent Recruitment of TFIIH Modules in Preinitiation Complex[END_REF][START_REF] Kim | A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II[END_REF][START_REF] Lorch | Histone Octamer Transfer by a Chromatin-Remodeling Complex[END_REF][START_REF] Mizuguchi | ATP-Driven Exchange of Histone H2AZ Variant Catalyzed by SWR1 Chromatin Remodeling Complex[END_REF][START_REF] Narlikar | Cooperation between Complexes that Regulate Chromatin Structure and Transcription Chromatin structure creates barriers for each step[END_REF][START_REF] Plaschka | Architecture of the RNA polymerase II-Mediator core initiation complex[END_REF][START_REF] Workman | Nucleosome core displacement in vitro via a metastable transcription factor-nucleosome complex[END_REF][START_REF] Zhang | Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss[END_REF] (Figure 3).

Binding of these regulatory factors serves as a mechanism of selection between the cell-type specific genes that can be transcribed out of a large pool of regulatory DNA elements that are available in the genome. After this series of modifications, the recognition sites that were buried in chromatin are exposed to TFs and RNA Polymerase II. Gene-specific TFs associate with sequence-specific sites on the promoter region [START_REF] Lambert | The Human Transcription Factors[END_REF]. This binding triggers the recruitment of GTFs and RNA Pol II, resulting in the proper docking of a functional pre-initiation complex [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF]. After the assembly of the PIC, engaged Pol II unwinds locally the DNA to form the transcription bubble and initiates RNA synthesis. As the RNA extends further, contacts with the promoter and the promoter-bound GTFs are broken, resulting in promoter escape and transition to elongation (Y. J. [START_REF] Kim | A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II[END_REF][START_REF] Liu | Two Cyclin-Dependent Kinases Promote RNA Polymerase II Transcription and Formation of the Scaffold Complex[END_REF]. In many eukaryotic genes, RNA Pol II undergoes promoter-proximal pausing within the first 20-60 bp before it starts productive elongation [START_REF] Krumm | The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region[END_REF] Leighton J. [START_REF] Core | Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters. Reform and Revolt in the City of Dreaming Spires[END_REF][START_REF] Muse | RNA polymerase is poised for activation across the genome[END_REF][START_REF] Rougvie | The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged[END_REF][START_REF] Zeitlinger | RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo[END_REF].

The phenomenon of Pol II pausing is widespread across metazoans and is mediated by two pausing-inducing factors: the negative elongation factor (NELF) and the DRB sensitivityinducing factor (DSIF) [START_REF] Muse | RNA polymerase is poised for activation across the genome[END_REF][START_REF] Vos | Structure of paused transcription complex Pol II-DSIF-NELF[END_REF][START_REF] Wada | DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs[END_REF][START_REF] Wu | NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila[END_REF][START_REF] Yamaguchi | NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation[END_REF].

Paused Pol II is fully competent to resume elongation, remaining stably engaged with the nascent RNA. However, productive transcription requires escape of paused Pol II from the promoter and transition to rapid elongation of the transcript. Pause-release of RNA Poll II is triggered by the recruitment of the positive transcription elongation factor b (P-TEFb) -which consists of CDK9 and cyclin T1-(N. F. [START_REF] Marshall | Purification of P-TEFb, a transcription factor required for the transition into productive elongation[END_REF][START_REF] Ni | P-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo[END_REF][START_REF] Rahl | C-Myc regulates transcriptional pause release[END_REF]; treatment of cells with the P-TEFb inhibitor flavopiridol dramatically decreases global transcription [START_REF] Chao | Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo[END_REF][START_REF] Jonkers | Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons[END_REF]. Cyclin-dependent kinase 9 (CDK9) subunit of P-TEFb phosphorylates the C-terminal domain (CTD) of RNA Pol II and the DSIF-NELF complex, causing NELF to dissociate from Pol II and transforming DSIF to a positive association factor that associates with Pol II that traverses through the gene body [START_REF] Harlen | The code and beyond: Transcription regulation by the RNA polymerase II carboxy-terminal domain[END_REF][START_REF] Lu | Phaseseparation mechanism for C-terminal hyperphosphorylation of RNA polymerase II[END_REF][START_REF] Wada | Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro[END_REF]. H2A.Z nucleosomes are less stable than H2A-nucleosomes and therefore might be more easily ejected from promoters in certain temporal contexts. Adapted from [START_REF] Cairns | The logic of chromatin architecture and remodelling at promoters[END_REF].

As previously described, folding promoter DNA around histones to form nucleosomes renders important recognition sites inaccessible. Thus, promoters undergo nucleosome removal to allow the recruitment of the transcription machinery [START_REF] Lorch | Histone Octamer Transfer by a Chromatin-Remodeling Complex[END_REF][START_REF] Mizuguchi | ATP-Driven Exchange of Histone H2AZ Variant Catalyzed by SWR1 Chromatin Remodeling Complex[END_REF][START_REF] Narlikar | Cooperation between Complexes that Regulate Chromatin Structure and Transcription Chromatin structure creates barriers for each step[END_REF][START_REF] Zhang | Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss[END_REF] (Figure 3). Genes with paused Pol II have been shown

to remove nucleosomes and open promoters before gene activation [START_REF] Costlow | High-resolution mapping of DNase I-hypersensitive sites of Drosophila heat shock genes in Drosophila melanogaster and Saccharomyces cerevisiae[END_REF][START_REF] Wu | The 5' ends of drosophila heat shock genes in chromatin are hypersensitive to DNase I[END_REF]. In addition, it has been demonstrated that paused genes possess low levels of nucleosome occupancy that depends on the presence of promoter-paused polymerase [START_REF] Gilchrist | Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation[END_REF][START_REF] Gilchrist | NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly[END_REF]. The relationship between paused polymerase and promoter-proximal nucleosome organization has been resolved in D. melanogaster [START_REF] Gilchrist | Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation[END_REF]:

depletion of NELF largely reduced promoter-proximal Pol II occupancy at highly regulated genes and led to an increase in nucleosome occupancy downstream of the TSS at these genes.

These findings suggested that pausing could serve as a mechanism to maintain open and regulatory factor-accessible promoters, hence facilitating the binding of the transcription machinery and future gene expression. Besides the chromatin opening function of paused Pol II, another potential role of pausing is to ensure rapid and/or synchronous gene activation. The presence of a preloaded Pol II already bound by transcriptional activators and co-activators would allow a rapid switch into productive elongation. In support of this idea, pausing has been identified at highly inducible genes and a number of genes that are implicated in early embryonic development at Drosophila melanogaster [START_REF] Boettiger | Synchronous and stochastic patterns of gene activation in the drosophila embryo[END_REF][START_REF] Hendrix | Promoter elements associated with RNA Pol II stalling in the Drosophila embryo[END_REF][START_REF] Muse | RNA polymerase is poised for activation across the genome[END_REF][START_REF] Zeitlinger | RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo[END_REF].

However, it is important to mention that pausing before activation is not a feature of all rapidly inducible genes [START_REF] Kininis | Genomic Analyses of Transcription Factor Binding, Histone Acetylation, and Gene Expression Reveal Mechanistically Distinct Classes of Estrogen-Regulated Promoters[END_REF].

Finally, after pause release, at the 3' end of the gene, the nascent RNA is released, cleaved and polyadenylated [START_REF] Proudfoot | Transcriptional termination in mammals: Stopping the RNA polymerase II juggernaut[END_REF]. The process of polyadenylation is important for later nuclear export, translation and stability of the mRNA transcript. After cleavage, Pol II terminates transcription, dissociates from the template, and eventually can be recycled to start a new round of the transcriptional cycle (Leighton J. [START_REF] Core | Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters. Reform and Revolt in the City of Dreaming Spires[END_REF][START_REF] Nojima | Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing[END_REF][START_REF] Yudkovsky | A transcription reinitiation intermediate that is stabilized by activator[END_REF].

Transcriptional bursting

As noted earlier, gene expression is a highly regulated process. At the same time, single-cell analysis has revealed that gene expression is a stochastic process and that stable phenotypes in a population are derived from variable single-cell gene expression patterns [START_REF] Blake | Noise in eukaryotic gene expression[END_REF][START_REF] Gregor | Probing the Limits to Positional Information[END_REF]. Traditional biochemical technologies, such as chromatin immunoprecipitation (ChIP), have been used to elucidate the steps of transcription in millions of cells simultaneously [START_REF] Rhee | Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution[END_REF][START_REF] Shang | Coactivators such as CBP, p300, pCAF, and possibly SRC1 and AIB1 possess intrinsic histone acetyltransferase (HAT) activi[END_REF]. However, the behavior measured on the population level does not reflect accurately the behavior in individual cells [START_REF] Lickwar | Genomewide protein-DNA binding dynamics suggest a molecular clutch for transcription factor function[END_REF][START_REF] Stenoien | FRAP reveals that mobility of oestrogen receptor-α is ligandand proteasome-dependent[END_REF].

Visualization of transcription in individual cells provided evidence that genes are often transcribed in discontinuous bursts, with periods of gene activity followed by periods of inactivity [START_REF] Boettiger | Synchronous and stochastic patterns of gene activation in the drosophila embryo[END_REF][START_REF] Chubb | Transcriptional Pulsing of a Developmental Gene[END_REF][START_REF] Golding | Real-time kinetics of gene activity in individual bacteria[END_REF][START_REF] Karpova | Concurrent fast and slow cycling of a transcriptional activator at an endogenous promoter[END_REF][START_REF] Larson | Real-time observation of transcription initiation and elongation on an endogenous yeast gene[END_REF][START_REF] Raj | Stochastic mRNA synthesis in mammalian cells[END_REF][START_REF] Suter | Mammalian genes are transcribed with widely different bursting kinetics[END_REF][START_REF] Yunger | Single-allele analysis of transcription kinetics in living mammalian cells[END_REF]. These bursts were first described in the late 1980s where [START_REF] Mcknight | Post-replicative nonribosomal transcription units in D. melanogaster embryos[END_REF] observed some "fiber-free gaps" located within transcription units in Drosophila embryos. Since then, bursting has been demonstrated to be ubiquitous from bacteria to human cells [START_REF] Boettiger | Synchronous and stochastic patterns of gene activation in the drosophila embryo[END_REF][START_REF] Chubb | Transcriptional Pulsing of a Developmental Gene[END_REF][START_REF] Golding | Real-time kinetics of gene activity in individual bacteria[END_REF][START_REF] Suter | Mammalian genes are transcribed with widely different bursting kinetics[END_REF][START_REF] Yunger | Single-allele analysis of transcription kinetics in living mammalian cells[END_REF]. Live-cell recordings [START_REF] Chubb | Transcriptional Pulsing of a Developmental Gene[END_REF][START_REF] Fukaya | Enhancer Control of Transcriptional Bursting[END_REF][START_REF] Suter | Mammalian genes are transcribed with widely different bursting kinetics[END_REF] and single-molecule fluorescence in situ hybridization (smFISH) [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF][START_REF] Fukaya | Enhancer Control of Transcriptional Bursting[END_REF][START_REF] Raj | Stochastic mRNA synthesis in mammalian cells[END_REF] have been used to characterize transcriptional bursting. All these observations suggested that any aspect of gene bursting can be regulated, including burst duration, amplitude and frequency (number of bursts per unit time) [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF][START_REF] Dar | Transcriptional burst frequency and burst size are equally modulated across the human genome[END_REF][START_REF] Fukaya | Enhancer Control of Transcriptional Bursting[END_REF][START_REF] Suter | Mammalian genes are transcribed with widely different bursting kinetics[END_REF] (Figure 4).

Figure 4: Description of bursting parameters. Adapted from [START_REF] Lim | Imaging transcriptional dynamics[END_REF].

Bursting is often described using a two-state model, where a promoter switches between two states, on and off, and transcription can occur only at the on state. Despite being widely applied in modeling, several studies on mammalian cells reported the presence of a refractory period between bursts: after responding to a stimulus, the template is no longer permissive for transcription [START_REF] Harper | Dynamic analysis of stochastic transcription cycles[END_REF][START_REF] Li | Frequency Modulation of Transcriptional Bursting Enables Sensitive and Rapid Gene Regulation[END_REF][START_REF] Molina | Stimulusinduced modulation of transcriptional bursting in a single mammalian gene[END_REF][START_REF] Suter | Mammalian genes are transcribed with widely different bursting kinetics[END_REF]. The presence of this reset time is of great importance for several genes since they acquire a "memory" in the reactivation process [START_REF] Harper | Dynamic analysis of stochastic transcription cycles[END_REF][START_REF] Suter | Mammalian genes are transcribed with widely different bursting kinetics[END_REF]. This observation suggests the existence of two major subpopulations across a cell population, in which a fraction of cells are poised for induction while another fraction are in a refractory state. This heterogeneity in the cell state allows the population to have both a rapid and a longer maintained response. Furthermore, having a refractory period indicates that several biochemical reactions are involved in order to reactivate a previously fired gene [START_REF] Harper | Dynamic analysis of stochastic transcription cycles[END_REF][START_REF] Suter | Mammalian genes are transcribed with widely different bursting kinetics[END_REF].

Several seminal studies have given valuable insight that points toward a role for chromatin, transcription factors and promoter-enhancer communications in regulating bursting. A proposed mechanism underlying gene bursting is the state of chromatin [START_REF] Becskei | Contributions of low molecule number and chromosomal positioning to stochastic gene expression[END_REF][START_REF] Raj | Stochastic mRNA synthesis in mammalian cells[END_REF][START_REF] Raser | Control of stochasticity in eukaryotic gene expression[END_REF]. In this model, the promoter's active and inactive states may reflect different chromatin conformations, of which only some are permissive for transcription, thus controlling the timing of gene firing and inactivation [START_REF] Hager | Transcription Dynamics[END_REF]. Another model suggests that bursting properties are influenced by the kinetics of transcription-factor binding (J. Chen et al., 2014;[START_REF] Karpova | Dynamic interactions of a transcription factor with DNA are accelerated by a chromatin remodeller[END_REF][START_REF] Lin | Combinatorial gene regulation by modulation of relative pulse timing[END_REF][START_REF] Loffreda | Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity[END_REF][START_REF] Mir | Dense bicoid hubs accentuate binding along the morphogen gradient[END_REF][START_REF] Senecal | Transcription factors modulate c-Fos transcriptional bursts[END_REF][START_REF] Swinstead | Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions[END_REF][START_REF] Johnson | Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor-regulated Promoter[END_REF].

Recent advances in in vivo fluorescence microscopy, such as fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) [START_REF] Scalettar | FRAP and FCS Studies of Self-Diffusion and Mutual Diffusion in Entangled DNA Solutions[END_REF], have made it possible to resolve the binding and the diffusion of molecules in the nucleus of living cells: transcription factors were found to bind to DNA in a dynamic manner and have short residence times [START_REF] Karpova | Dynamic interactions of a transcription factor with DNA are accelerated by a chromatin remodeller[END_REF][START_REF] Phair | Global Nature of Dynamic Protein-Chromatin Interactions In Vivo: Three-Dimensional Genome Scanning and Dynamic Interaction Networks of Chromatin Proteins[END_REF][START_REF] Swinstead | Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions[END_REF][START_REF] Johnson | Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor-regulated Promoter[END_REF], while the core histones (H2A, H2B, H3 and H4) display a more stable binding [START_REF] Kimura | Kinetics of core histones in living human cells: Little exchange of H3 and H4 and some rapid exchange of H2B[END_REF][START_REF] Phair | Global Nature of Dynamic Protein-Chromatin Interactions In Vivo: Three-Dimensional Genome Scanning and Dynamic Interaction Networks of Chromatin Proteins[END_REF]. Since TFs show high turnover, it has been assumed that the activity of target genes depends on the TF on-rate and thus local TF concentration. Consistently, by utilizing a combination of single molecule observations of RNA in fixed and living cells with computational modeling, it has been demonstrated that TF concentration regulates the burst frequency [START_REF] Mir | Dense bicoid hubs accentuate binding along the morphogen gradient[END_REF][START_REF] Senecal | Transcription factors modulate c-Fos transcriptional bursts[END_REF]. A supplementary way of regulating gene bursting is through modulation of the TF dwell time (J. Chen et al., 2014;[START_REF] Lickwar | Genomewide protein-DNA binding dynamics suggest a molecular clutch for transcription factor function[END_REF][START_REF] Lin | Combinatorial gene regulation by modulation of relative pulse timing[END_REF][START_REF] Loffreda | Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity[END_REF][START_REF] Swinstead | Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions[END_REF]. Transcription factor acetylation [START_REF] Loffreda | Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity[END_REF], affinity of the TF binding site [START_REF] Suter | Mammalian genes are transcribed with widely different bursting kinetics[END_REF] or cooperative binding of other regulators (J. Chen et al., 2014;[START_REF] Lin | Combinatorial gene regulation by modulation of relative pulse timing[END_REF][START_REF] Swinstead | Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions[END_REF] determine the residence time of TFs. Long dwell time of a transcription factor might increase the number of polymerases that are recruited to the target gene, thereby increasing the burst duration of the gene. These models of transcriptional bursting have focused on biochemical activities at gene promoters, and are further confounded by their complex interplay with distal regulatory elements. The models proposed above may operate simultaneously on the same gene, providing cells the ability to regulate different bursting parameters independently, hence tuning the levels and the heterogeneity of gene expression between cells.

Distal regulatory elements

Enhancers

Early transgenic studies have revealed that gene expression during development is weak in the absence of additional, often distal, cis-acting regulatory elements [START_REF] Banerji | A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes[END_REF][START_REF] Banerji | Expression of a β-globin gene is enhanced by remote SV40 DNA sequences[END_REF][START_REF] Benoist | In vivo sequence requirements of the SV40 early promoter region[END_REF][START_REF] Gillies | A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene[END_REF][START_REF] Moreau | The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants P.Moreau[END_REF]. These elements, named enhancers, were first described in the Xenopus oocyte where a remote upstream sequence, originally termed modulator, positively affected the expression of the sea urchin H2A histone gene [START_REF] Grosschedl | Spacer DNA sequences upstream of the T-A-T-A-A-A-T-A sequence are essential for promotion of H2A histone gene transcription in vivo[END_REF]. Deletion of this element resulted in a 15-to 20-fold decrease of H2A gene expression. Shortly after this initial observation, plasmid-based assays revealed that the tandem 72 bp simian virus 40 (SV40) DNA repeat located 150 bp upstream from the cap site of the SV40 early gene was essential for SV40 expression [START_REF] Benoist | In vivo sequence requirements of the SV40 early promoter region[END_REF]. Deletion of this upstream sequence reduced early gene expression of T antigen to undetectable levels and concomitantly abolished virus viability. Likewise, the same 72 bp repeated DNA element was shown to increase the expression of a β-globin gene in HeLa cells by more than two orders of magnitude even when it was located 3300 bp downstream from the promoter [START_REF] Banerji | Expression of a β-globin gene is enhanced by remote SV40 DNA sequences[END_REF]. The first endogenous enhancer found in mammalian genomes was the immunoglobulin (Ig) heavy-chain enhancer [START_REF] Banerji | A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes[END_REF][START_REF] Gillies | A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene[END_REF][START_REF] Mercola | Transcriptional enhancer elements in the mouse immunoglobulin heavy chain locus[END_REF][START_REF] Neuberger | Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells[END_REF]. Notably, the Ig enhancer is the first example of an enhancer that functions in a tissue-or cell-type specific manner. Of the various cell-lines tested, Ig enhancer activity was observed only in B-lymphocyte-derived myeloma cells [START_REF] Banerji | A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes[END_REF][START_REF] Gillies | A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene[END_REF].

Properties and classification

Enhancers appear to increase or activate the rate of transcription in a position-and orientationindependent manner with respect to their target genes [START_REF] Banerji | A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes[END_REF][START_REF] Benoist | In vivo sequence requirements of the SV40 early promoter region[END_REF][START_REF] Fromm | Simian virus 40 early-and late-region promoter functions are enhanced by the 72-base-pair repeat inserted at distant locations and inverted orientations[END_REF][START_REF] Moreau | The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants P.Moreau[END_REF]. This flexibility is the defining hallmark of enhancers. In many cases, enhancers are found in the non-coding part of the genome, at great genomic distances from their target genes (sometimes more than hundreds of kilobases) and can bypass more proximal genes to regulate their targets (Amano et al., 2009;[START_REF] Mifsud | Mapping long-range promoter contacts in human cells with highresolution capture Hi-C[END_REF]Sanyal, Lajoie, Jain, & Dekker, 2012;Schoenfelder et al., 2015) (Figure 5). One of the most characteristic examples reported so far, is a mouse limb bud enhancer of the Sonic Hedgehog (Shh) gene, called ZRS (ZPA regulatory sequence), which is found in the intron of another gene, approximately 1 megabase (Mb) upstream of the Shh promoter (Amano et al., 2009;Lettice et al., 2003;[START_REF] Sagai | Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb[END_REF]. and upregulating its expression. Adapted from [START_REF] Shlyueva | Transcriptional enhancers: from properties to genome-wide predictions[END_REF].

Similar to promoters, enhancers contain DNA recognition motifs that attract sequence-specific TFs, RNA Pol II and GTFs [START_REF] Koch | Transcription initiation platforms and GTF recruitment at tissuespecific enhancers and promoters[END_REF][START_REF] Lambert | The Human Transcription Factors[END_REF]. These proteins recruit coactivators and co-repressors, such that the combination of all bound factors will ultimately determine the activity of the enhancer in response to developmental cues. Moreover, active enhancers exhibit DNase I hypersensitivity (HS), which reflects an "open" chromatin statedepleted of nucleosomes -as a result of the binding of several transcription factors [START_REF] Boyle | High-Resolution Mapping and Characterization of Open Chromatin across the Genome[END_REF]Roadmap Epigenomics Consortium et al., 2015;[START_REF] Thurman | The accessible chromatin landscape of the human genome[END_REF]. Nucleosomes flanking the enhancers show characteristic post-translational histone modifications (Creyghton et al., 2010;Rada-Iglesias et al., 2011;[START_REF] Zentner | Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions[END_REF]. Depending on these modifications, enhancers can be divided into several states: active, inactive/poised and intermediate/primed. Active enhancers display high levels of histone H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac), a major substrate for the histone acetyltransferase CBP/p300 (Creyghton et al., 2010;[START_REF] Jin | Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation[END_REF]Rada-Iglesias et al., 2011;[START_REF] Tie | CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing[END_REF] (Figure 6A), as well as the "alternative" marks histone H3 lysine 64 acetylation (H3K64ac) and histone H3 lysine 122 acetylation (H3K122ac) recently identified in mouse embryonic stem cells (mESCs) (Pradeepa et al., 2016). Inactive or poised enhancers are marked by H3K4me1 and H3K27me3, a mark enriched in Polycomb (PcG)-associated and transcriptionally repressed regions (Creyghton et al., 2010;Rada-Iglesias et al., 2011) (Figure 6B). In addition, enhancers that have not yet been activated but are primed for activation either at a later developmental stage or in response to external stimuli can be premarked by H3K4me1 [START_REF] Zentner | Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions[END_REF] (Figure 6C). enhancers can be marked by H3K4me1. Adapted from [START_REF] Shlyueva | Transcriptional enhancers: from properties to genome-wide predictions[END_REF].

Identification and validation

The advent of high-throughput and deep sequencing methods has enabled the systematic study of potential regulatory elements: more than one million putative enhancers have been documented in several mouse and human tissues and cell types [START_REF] Altshuler | An integrated map of genetic variation from 1,092 human genomes[END_REF][START_REF] Dunham | An integrated encyclopedia of DNA elements in the human genome[END_REF]Roadmap Epigenomics Consortium et al., 2015;Shen et al., 2012;[START_REF] Thurman | The accessible chromatin landscape of the human genome[END_REF]. Several approaches have been used in order to predict and functionally assess transcriptional enhancers, including the analysis of sequence motifs, chromatin structure and histone modifications, the binding of TFs and cofactors, as well as parallel and/or genomewide screens of enhancer activity.

As mentioned above, enhancers are bound by a large range of transcription factors and cofactors. Given this association, computational analysis of TF binding motifs combined with the assessment of evolutionary DNA sequence conservation was used as an approach for predicting putative enhancer sequences [START_REF] Berman | Exploiting transcription factor binding site clustering to identify cisregulatory modules involved in pattern formation in the Drosophila genome[END_REF][START_REF] Del Bene | In vivo validation of a computationally predicted conserved Ath5 target gene set[END_REF][START_REF] Kheradpour | Reliable prediction of regulator targets using 12 Drosophila genomes[END_REF][START_REF] Pennacchio | In vivo enhancer analysis of human conserved non-coding sequences[END_REF]. However, even though these types of approaches provide a first overview of the regulatory elements across the genome, they seem to identify only a proportion of enhancers. For instance, it has been demonstrated that only a small fraction of the conserved motif matches are bound by TFs in vivo in a given tissue or stage and that transcription factor binding can be context-dependent and rely on the formation of complexes with other proteins [START_REF] Birney | Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project[END_REF]Z. Liu et al., 2014;[START_REF] Slattery | Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins[END_REF][START_REF] Yáñez-Cuna | Uncovering cis-regulatory sequence requirements for context-specific transcription factor binding[END_REF]. As the role of motifs is to recruit TFs, chromatin immunoprecipitation followed by either microarray analysis (ChIP-on-chip) or deep sequencing (ChIP-seq) enables the determination of in vivo TF binding sites in an unbiased, genome-wide and systematic manner [START_REF] Johnson | Genome-Wide Mapping of in Vivo Protein-DNA Interactions[END_REF][START_REF] Robertson | Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing[END_REF] (Figure 7A). Alternatively, ChIP-seq has been commonly used as a method for identifying the in vivo binding sites of transcriptional cofactors, such as the histone acetyltransferase p300 [START_REF] May | Large-scale discovery of enhancers from human heart tissue[END_REF][START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF][START_REF] Visel | A high-resolution enhancer atlas of the developing telencephalon[END_REF] (Figure 7C). Binding of p300 has enabled the genome-wide prediction of enhancers in mice and humans [START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]Rada-Iglesias et al., 2011;[START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF], while a great amount of the p300-binding sites tested using episomal mouse reporter assays demonstrated reproducible enhancer activity [START_REF] Blow | ChIP-seq Identification of Weakly Conserved Heart Enhancers[END_REF][START_REF] May | Large-scale discovery of enhancers from human heart tissue[END_REF][START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF][START_REF] Visel | A high-resolution enhancer atlas of the developing telencephalon[END_REF].

A complementary method in identifying enhancers takes advantage of their chromatin accessibility. Active enhancers have been shown to persist in a nucleosome-deprived chromatin state and display hypersensitivity to DNase I digestion [START_REF] Boyle | High-Resolution Mapping and Characterization of Open Chromatin across the Genome[END_REF][START_REF] Thurman | The accessible chromatin landscape of the human genome[END_REF]. Next-generation sequence-based techniques, such as DNase-seq [START_REF] Boyle | High-Resolution Mapping and Characterization of Open Chromatin across the Genome[END_REF], have been utilized to determine enhancers independently of any given TF binding motif or TF binding (Figure 7B). As described above, nucleosomes flanking active enhancers show a characteristic pattern of post-translational histone modifications (Creyghton et al., 2010;Rada-Iglesias et al., 2011;[START_REF] Zentner | Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions[END_REF]. Genome-wide prediction of enhancers using epigenetic marks has been used extensively in a plethora of biological contexts to predict regulatory elements [START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF]Heintzman et al., 2009[START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]Rada-Iglesias et al., 2011;[START_REF] Roh | Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping[END_REF] and it agrees well with enhancer activity assays (Arnold et al., 2013;[START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF][START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]. However, none of the known histone marks correlates perfectly with enhancer activity (Arnold et al., 2013;[START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF]. Tissuespecific analysis of chromatin state showed that several active mesodermal enhancers in D.

melanogaster embryos displayed heterogeneous histone modifications and were not predicted by the presence of H3K27ac [START_REF] Bonn | Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development[END_REF]. Furthermore, it is becoming increasingly apparent that regulatory elements can be devoid of classical enhancer chromatin marks but still display enhancer functions (Pradeepa et al., 2016;[START_REF] Rajagopal | High-throughput mapping of regulatory DNA[END_REF]. In particular, a new class of active functional enhancers was found in mouse ESCs that is marked by H3K122ac, but lacking H3K27ac (Pradeepa et al., 2016). Adapted from [START_REF] Shlyueva | Transcriptional enhancers: from properties to genome-wide predictions[END_REF].

Despite the fact that a genomic region has been suggested by DNase-seq or ChIP-seq to act as an enhancer, its physiological relevance needs to be established in the appropriate developmental context. Predicted enhancers should be further validated in functional assays.

Enhancer sequences are frequently tested for their ability to activate transcription by simply cloning the candidate enhancer sequence upstream of a minimal promoter driving the expression of a reporter gene (such as lacZ, GFP or luciferase) and measuring reporter gene expression in cell lines or transgenic animal models. This activity is the hallmark property of enhancers [START_REF] Banerji | Expression of a β-globin gene is enhanced by remote SV40 DNA sequences[END_REF][START_REF] Benoist | In vivo sequence requirements of the SV40 early promoter region[END_REF] and is broadly used to evaluate predicted enhancers [START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]Rada-Iglesias et al., 2011;[START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF][START_REF] Zinzen | Combinatorial binding predicts spatio-temporal cis-regulatory activity[END_REF] in transgenic D. melanogaster and mouse embryos [START_REF] Visel | VISTA Enhancer Browser -A database of tissue-specific human enhancers[END_REF]. However, such classical plasmid-based approaches are mainly low throughput and are mainly used to test previously predicted enhancers [START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]Rada-Iglesias et al., 2011;[START_REF] Visel | ChIP-seq accurately predicts tissue-specific activity of enhancers[END_REF]. Self-transcribing active regulatory region sequencing (STARR-seq) is a plasmid-based approach, whereby enhancer sequences mediate their own transcription (Arnold et al., 2013).

This method exploits the fact that enhancers can exert control in a position-and orientationindependent manner [START_REF] Banerji | A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes[END_REF][START_REF] Benoist | In vivo sequence requirements of the SV40 early promoter region[END_REF][START_REF] Fromm | Simian virus 40 early-and late-region promoter functions are enhanced by the 72-base-pair repeat inserted at distant locations and inverted orientations[END_REF][START_REF] Moreau | The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants P.Moreau[END_REF] and inserts the candidate elements downstream of a reporter gene driven by a minimal promoter (Arnold et al., 2013). STARR-seq is of special importance since it allows genome-wide screens of enhancer activity. More recently, CRISPR-Cas9-based approaches have been used to examine enhancer function [START_REF] Fulco | Systematic mapping of functional enhancer-promoter connections with CRISPR interference[END_REF][START_REF] Klann | CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome[END_REF]. For example, targeting KRAB-dCas9 (KRAB effector domain fused to catalytically dead Cas9) [START_REF] Thakore | Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements[END_REF] to the MYC locus in K562 cells, identified seven putative MYC enhancers.

Despite this enormous progress, several seminal questions remain unanswered: notably, what percentage of enhancers predicted by sequence-and chromatin-based methods can truly function as enhancers in vivo. By using ChIP-STARR-seq in human ESCs, an assay that combines antibodies against TFs or histone marks with STARR-seq plasmid, it has been shown that only 12% of H3K27ac ChIP-seq peaks displayed enhancer activity [START_REF] Barakat | Functional Dissection of the Enhancer Repertoire in Human Embryonic Stem Cells[END_REF], which indicates that genome-wide assays might have overvalued the number of functional enhancers. In addition, a high-throughput CRISPR-Cas9-based approach was used to identify the regulatory elements controlling the expression of the ESC-specific genes, Nanog, Rpp25, Tdgf1 and Zfp42 [START_REF] Rajagopal | High-throughput mapping of regulatory DNA[END_REF]. Strikingly, the authors identified a novel class of cis-regulatory elements that did not coincide with any known markers of regulatory activity, including H3K27ac and H3K4me1. These findings suggest that, to identify enhancers, a more comprehensive analysis of histone marks is required than has previously been considered.

Notably, the same study [START_REF] Rajagopal | High-throughput mapping of regulatory DNA[END_REF], and a CRISPR-Cas9-mediated deletion screening for cis-regulatory elements in the human POU5F1 locus [START_REF] Diao | A tiling-deletionbased genetic screen for cis-regulatory element identification in mammalian cells[END_REF], showed that promoters can function as enhancers for neighboring genes. Actually, enhancers and promoters are highly related and show important similarities in structure and function [START_REF] Boyle | High-Resolution Mapping and Characterization of Open Chromatin across the Genome[END_REF][START_REF] Koch | Transcription initiation platforms and GTF recruitment at tissuespecific enhancers and promoters[END_REF][START_REF] Thurman | The accessible chromatin landscape of the human genome[END_REF]. Both these regulatory elements exhibit DNase I hypersensitivity, which results from the depletion of nucleosomes [START_REF] Boyle | High-Resolution Mapping and Characterization of Open Chromatin across the Genome[END_REF][START_REF] Thurman | The accessible chromatin landscape of the human genome[END_REF]. Similar to promoters, enhancers are bound by general transcription factors and; cofactors, recruit RNAPII and initiate transcription (see below) (T. K. Kim et al., 2010;[START_REF] Koch | Transcription initiation platforms and GTF recruitment at tissuespecific enhancers and promoters[END_REF]. Nevertheless, the fact the promoters can behave as enhancer-like elements for neighboring genes [START_REF] Dao | Genome-wide characterization of mammalian promoters with distal enhancer functions[END_REF][START_REF] Diao | A tiling-deletionbased genetic screen for cis-regulatory element identification in mammalian cells[END_REF][START_REF] Rajagopal | High-throughput mapping of regulatory DNA[END_REF] adds additional complexity to the distinction of these two regulatory elements and argues that the classical definitions of the promoter and the enhancer might need to be revised.

Apart from that, the functional roles for most of the chromatin modifications associated with active enhancers are not fully understood. There is no clear evidence that histone marks such as H3K4me1 or H3K27ac are necessary or even mechanistically involved in transcription [START_REF] Pengelly | A Histone Mutant Reproduces the Phenotype Caused by Loss of Histone-Modifying Factor Polycomb[END_REF]. Interestingly, loss of H3K4me1 from enhancers in a series of MLL3 and MLL4 (MLL3/MLL4) histone methyltransferase catalytic mutant mouse ESC lines leads to a partial reduction of H3K27ac from enhancers but has only a minor effect on transcription from either enhancers or promoters, implying that H3K4me1 is dispensable for maintenance of gene expression programs in mouse ESCs [START_REF] Dorighi | Mll3 and Mll4 Facilitate Enhancer RNA Synthesis and Transcription from Promoters Independently of H3K4 Monomethylation[END_REF].

Enhancer RNAs

A great number of studies has demonstrated that enhancers become activated as transcribed units, adding a new layer of complexity to genome regulation. Evidence of enhancer-derived transcription was first documented in human erythroid cells, in the locus control region (LCR) that regulates the transcription of the downstream beta-globin gene clusters, located 10-50 kb away [START_REF] Collis | Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression[END_REF][START_REF] Ling | HS2 enhancer function is blocked by a transcriptional terminator inserted between the enhancer and the promoter[END_REF][START_REF] Tuan | Transcription of the hypersensitive site HS2 enhancer in erythroid cells[END_REF].

The LCR was shown to comprise five erythroid-specific DNase I hypersensitivity sites (HS) and most importantly, transcriptional initiation sites were found inside these regions [START_REF] Collis | Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression[END_REF][START_REF] Tuan | Transcription of the hypersensitive site HS2 enhancer in erythroid cells[END_REF]. A few years later, separate studies reported direct RNA Pol II recruitment and production of enhancer RNAs (eRNAs) from functionally active enhancers in several mammalian cell types, including cortical neurons and T cells (de Santa et al., 2010; T.

K. Kim et al., 2010;[START_REF] Koch | Transcription initiation platforms and GTF recruitment at tissuespecific enhancers and promoters[END_REF]. RNA Pol II is recruited to cell-specific enhancers [START_REF] Koch | Transcription initiation platforms and GTF recruitment at tissuespecific enhancers and promoters[END_REF] and responds dynamically upon stimulus-induced activation (for example by membrane depolarization or endotoxin activation) (de Santa et al., 2010; T. K. Kim et al., 2010).

As large numbers of eRNA transcripts have been detected, more comprehensive insights into their features and regulation have been gained. eRNA-producing enhancers display high levels of H3K4me1, H3K27ac, H3K79me2, low levels of H3K4me3, and are depleted for H3K27me3 [START_REF] Djebali | Landscape of transcription in human cells[END_REF]Heintzman et al., 2009). eRNAs are generally not spliced and the majority, although not all, lack polyadenylated tails [START_REF] De Santa | A large fraction of extragenic RNA Pol II transcription sites overlap enhancers[END_REF][START_REF] Djebali | Landscape of transcription in human cells[END_REF]T. K. Kim et al., 2010;[START_REF] Koch | Transcription initiation platforms and GTF recruitment at tissuespecific enhancers and promoters[END_REF]. By using genome-wide binding profiles, it has been demonstrated that eRNA-producing enhancers are extensively occupied by TFs, such as p53 [START_REF] Melo | ERNAs Are Required for p53-Dependent Enhancer Activity and Gene Transcription[END_REF] or (E2)-bound estrogen receptor α (ERα) in MCF-7 human breast cancer cells (W. [START_REF] Li | Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation[END_REF], and that the expression levels of the produced eRNAs dynamically change in response to signal transduction events [START_REF] De Santa | A large fraction of extragenic RNA Pol II transcription sites overlap enhancers[END_REF][START_REF] Hah | A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells[END_REF]; T. K. Kim et al., 2010;[START_REF] Lam | Rev-Erbs repress macrophage gene expression by inhibiting enhancerdirected transcription[END_REF]W. Li et al., 2013;[START_REF] Melo | ERNAs Are Required for p53-Dependent Enhancer Activity and Gene Transcription[END_REF][START_REF] Mousavi | ERNAs Promote Transcription by Establishing Chromatin Accessibility at Defined Genomic Loci[END_REF][START_REF] Wang | Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA[END_REF]. Most importantly, signal-dependent changes in eRNA expression levels at enhancers are strongly correlated with changes at the level of mRNA synthesis at nearby genes (T. K. Kim et al., 2010;[START_REF] Lam | Rev-Erbs repress macrophage gene expression by inhibiting enhancerdirected transcription[END_REF]W. Li et al., 2013), suggesting that, at least for some enhancers, a stimulus-induced eRNA is linked to proper gene activation of neighboring coding genes. Collectively, enhancer transcription is a genome-wide phenomenon across multiple cell types which does not appear to be a random process resulting from the irregular/accidental binding of RNA Pol II to the open chromatin of enhancers [START_REF] Hah | A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells[END_REF]T. K. Kim et al., 2010;Rada-Iglesias et al., 2011). When considering the functional significance of enhancer transcription, two possible scenarios are emerging: first, the act of eRNA transcription, not the features of the eRNA transcript itself, have a specific activating function; second, the eRNA per se plays a functional role in regulating gene expression. In support of the latter model, artificial recruitment of eRNA to promoters [START_REF] Melo | ERNAs Are Required for p53-Dependent Enhancer Activity and Gene Transcription[END_REF] or enhancers [START_REF] Li | Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation[END_REF] stimulated reporter transcription, and reporter activity is reduced when the eRNA-producing sequence is inverted, altering the sequence of the eRNA without affecting binding sites for TFs [START_REF] Lam | Rev-Erbs repress macrophage gene expression by inhibiting enhancerdirected transcription[END_REF]. However, these experimental strategies have only been applied to reporter systems, and the relative contribution of endogenous enhancer transcription and/or eRNA binding at promoters to transcriptional regulation is poorly characterized and may be contextdependent.

Super-Enhancers

As previously noted, mammalian genomes contain a great amount of active transcriptional enhancers that can be detected based on enhancer-specific chromatin features like chromatin accessibility, the binding of cell-type specific transcription factors and the accumulation of active histone marks, notably H3K27ac [START_REF] Long | Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution[END_REF]. Such studies resulted in the identification of a new class of regulatory elements, the so-called "superenhancers" [START_REF] Hah | Inflammation-sensitive super enhancers form domains of coordinately regulated enhancer RNAs[END_REF][START_REF] Hay | Genetic dissection of the α-globin super-enhancer in vivo[END_REF][START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF][START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF][START_REF] Shin | Hierarchy within the mammary STAT5-driven Wap super-enhancer[END_REF][START_REF] Whyte | Master transcription factors and mediator establish superenhancers at key cell identity genes[END_REF]Zhou et al., 2014). Super-enhancers (SEs) share many features with typical enhancers but in a considerably larger scale: they are typically an order of magnitude larger than typical enhancers and can be distinguished by the very dense accumulation of master regulators, the Mediator co-activator complex, p300 and H3K27ac [START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF][START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF][START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF][START_REF] Whyte | Master transcription factors and mediator establish superenhancers at key cell identity genes[END_REF] (Figure 8).

Super-enhancers exhibit high levels of RNA Pol II binding and produce quite high amounts of eRNAs compared to typical enhancers [START_REF] Hah | Inflammation-sensitive super enhancers form domains of coordinately regulated enhancer RNAs[END_REF][START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF][START_REF] Hnisz | Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers[END_REF] which are dynamically regulated upon cellular signaling [START_REF] Hah | Inflammation-sensitive super enhancers form domains of coordinately regulated enhancer RNAs[END_REF]. Recent studies suggest that eRNAs may be involved or required for gene activation [START_REF] Hah | Enhancer transcripts mark active estrogen receptor binding sites[END_REF]W. Li et al., 2013;[START_REF] Mousavi | ERNAs Promote Transcription by Establishing Chromatin Accessibility at Defined Genomic Loci[END_REF], therefore high-levels of SE transcripts, in concert with master TFs, could contribute to the transcriptional activation of their associated genes and the establishment of a cell-type specific regulatory network. Various studies in a wide variety of mammalian cell types, have reported the association of super-enhancers with highly cell-type specific genes known to play major roles in their biological identities [START_REF] Hay | Genetic dissection of the α-globin super-enhancer in vivo[END_REF][START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF][START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF][START_REF] Shin | Hierarchy within the mammary STAT5-driven Wap super-enhancer[END_REF][START_REF] Vahedi | Super-enhancers delineate disease-associated regulatory nodes in T cells[END_REF][START_REF] Whyte | Master transcription factors and mediator establish superenhancers at key cell identity genes[END_REF]. Given the importance of super-enhancers in the control of cell identity, it is not surprising that key oncogenes and other important genes for tumorigenesis reside in enhancer-dense genomic regions [START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF][START_REF] Hnisz | Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers[END_REF][START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF][START_REF] Mansour | An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element[END_REF] and disease-associated sequence variation has been observed

within super-enhancers of disease-relevant cell types [START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF]. It is also interesting to mention that although the definition of SEs is recent, locus control regions (LCRs) characterized in early studies, such as the LCR of β-globin [START_REF] Grosveld | Positionindependent, high-level expression of the human β-globin gene in transgenic mice[END_REF] and the enhancer element of the immunoglobin heavy chain [START_REF] Banerji | A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes[END_REF][START_REF] Gillies | A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene[END_REF][START_REF] Mercola | Transcriptional enhancer elements in the mouse immunoglobulin heavy chain locus[END_REF], also correspond to this class of regulatory element. Collectively, these results suggest that super-enhancers have evolved to robustly control genes that play critical roles in cellular physiology and disease [START_REF] Hay | Genetic dissection of the α-globin super-enhancer in vivo[END_REF][START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF][START_REF] Hnisz | Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers[END_REF][START_REF] Lovén | Selective inhibition of tumor oncogenes by disruption of super-enhancers[END_REF][START_REF] Mansour | An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element[END_REF][START_REF] Whyte | Master transcription factors and mediator establish superenhancers at key cell identity genes[END_REF]. Despite the fact that super-enhancers have been identified in a plethora of different cell types, there are conflicting results as to whether SEs represent a new category of regulatory elements, different from conventional enhancers, and on the importance of individual elements within them [START_REF] Hay | Genetic dissection of the α-globin super-enhancer in vivo[END_REF]Moorthy et al., 2017;[START_REF] Shin | Hierarchy within the mammary STAT5-driven Wap super-enhancer[END_REF][START_REF] Thomas | Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements[END_REF]. At some loci, each constituent element contributes independently and in an additive rather than synergistic fashion to gene expression [START_REF] Hay | Genetic dissection of the α-globin super-enhancer in vivo[END_REF]. At other loci, some constituent enhancers were shown to be more essential than others and to control the activation of other elements within the same SE in order to secure full expression of the target genes [START_REF] Hnisz | Convergence of Developmental and Oncogenic Signaling Pathways at Transcriptional Super-Enhancers[END_REF][START_REF] Shin | Hierarchy within the mammary STAT5-driven Wap super-enhancer[END_REF][START_REF] Thomas | Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements[END_REF].

Multiple enhancers promote developmental robustness

With our understanding of the complexity of genome organization, it is becoming evident that many enhancers do not function in isolation. Numerous studies have demonstrated that developmental genes are controlled by multiple enhancers with both overlapping and separate spatial and temporal activities [START_REF] Frankel | Phenotypic robustness conferred by apparently redundant transcriptional enhancers[END_REF][START_REF] Hay | Genetic dissection of the α-globin super-enhancer in vivo[END_REF]Hong, Hendrix, & Levine, 2008;[START_REF] Marinić | An Integrated Holo-Enhancer Unit Defines Tissue and Gene Specificity of the Fgf8 Regulatory Landscape[END_REF][START_REF] Osterwalder | Enhancer redundancy provides phenotypic robustness in mammalian development[END_REF]. This finding raises two important questions: What are the relationships between simultaneously active enhancers?

What is the purpose of multiple-enhancer regulation of single genes?

Multiple enhancers can cooperate in the same tissue, where they produce additive, synergistic or redundant interactions to drive high levels of expression of their shared target gene [START_REF] Bender | Targeted deletion of 5′HS1 and 5′HS4 of the β-globin locus control region reveals additive activity of the DNaseI hypersensitive sites[END_REF][START_REF] Frankel | Phenotypic robustness conferred by apparently redundant transcriptional enhancers[END_REF][START_REF] Hay | Genetic dissection of the α-globin super-enhancer in vivo[END_REF][START_REF] Hong | Shadow enhancers as a source of evolutionary novelty[END_REF][START_REF] Long | Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder[END_REF][START_REF] Marinić | An Integrated Holo-Enhancer Unit Defines Tissue and Gene Specificity of the Fgf8 Regulatory Landscape[END_REF][START_REF] Perry | Shadow enhancers foster robustness of drosophila gastrulation[END_REF][START_REF] Shin | Hierarchy within the mammary STAT5-driven Wap super-enhancer[END_REF] (Figure 9A).

Some characteristic examples in this category are the α-and β-globin genes [START_REF] Bender | Targeted deletion of 5′HS1 and 5′HS4 of the β-globin locus control region reveals additive activity of the DNaseI hypersensitive sites[END_REF][START_REF] Hay | Genetic dissection of the α-globin super-enhancer in vivo[END_REF]. Interestingly, genome-wide profiling of promoter contacts in human and mouse cells with high-resolution promoter-capture HiC, showed that the number of promoter-interacting enhancers is positively related with the level of gene expression [START_REF] Mifsud | Mapping long-range promoter contacts in human cells with highresolution capture Hi-C[END_REF]Schoenfelder et al., 2015), which supports the idea that additive enhancers drive higher levels of expression compared to individually acting enhancers. Another mode of function in gene activation is enhancer redundancy, as has been described for some "shadow enhancers" in Drosophila melanogaster [START_REF] Frankel | Phenotypic robustness conferred by apparently redundant transcriptional enhancers[END_REF][START_REF] Hong | Shadow enhancers as a source of evolutionary novelty[END_REF][START_REF] Perry | Shadow enhancers foster robustness of drosophila gastrulation[END_REF][START_REF] Perry | Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo[END_REF]. By performing whole-genome ChIP-chip assays in the early Drosophila embryo [START_REF] Hong | Shadow enhancers as a source of evolutionary novelty[END_REF], Mike Levine and colleagues discovered some remote "secondary" enhancers that appear to produce similar patterns of expression to these generated by "primary" enhancers. The overlapping function of primary and shadow enhancers buffers the expression of critical patterning genes and confers robustness against environmental and genetic variation, which is a common feature of natural populations [START_REF] Frankel | Phenotypic robustness conferred by apparently redundant transcriptional enhancers[END_REF][START_REF] Perry | Shadow enhancers foster robustness of drosophila gastrulation[END_REF]. Importantly, redundancy between enhancers could explain why deletions of some cis-regulatory elements lead to dramatic phenotypes [START_REF] Bahr | A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies[END_REF][START_REF] Gonen | Sex reversal following deletion of a single distal enhancer of Sox9[END_REF]Moorthy et al., 2017;[START_REF] Sagai | Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb[END_REF]Zhou et al., 2014), while others show weak or no effects (Moorthy et al., 2017;[START_REF] Osterwalder | Enhancer redundancy provides phenotypic robustness in mammalian development[END_REF]. developmental gene in a tissue-specific manner. Adapted from [START_REF] Krijger | Regulation of disease-associated gene expression in the 3D genome[END_REF].

Alternatively, genes with a complex tissue-specific developmental expression pattern, such as Shh [START_REF] Jeong | A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies longrange ventral forebrain enhancers[END_REF]Lettice et al., 2003;[START_REF] Sagai | A cluster of three long-range enhancers directs regional Shh expression in the epithelial linings[END_REF], Myc [START_REF] Uslu | Long-range enhancers regulating Myc expression are required for normal facial morphogenesis[END_REF] and the Hox gene clusters [START_REF] Andrey | A Switch Between Topological Domains Underlies HoxD Genes Collinearity in Mouse Limbs[END_REF][START_REF] Montavon | A regulatory archipelago controls hox genes transcription in digits[END_REF], are controlled by multiple enhancers with non-redundant spatiotemporal activities spread over tens to hundreds of kilobases upstream or downstream of the target gene, facilitating vast combinatorial complexity of gene-expression programs (Figure 9B). Together, these studies highlight a key role of multiple enhancers in guaranteeing precise fine-tuning of gene expression during development.

Role of enhancers in disease

The release of the human genome sequence in combination with genome-wide association studies (GWAS) and chromatin signatures of enhancers have revealed that many disease-and trait-genetic variants systematically lie within enhancers [START_REF] Dunham | An integrated encyclopedia of DNA elements in the human genome[END_REF][START_REF] Maurano | Systematic localization of common diseaseassociated variation in regulatory DNA[END_REF]Roadmap Epigenomics Consortium et al., 2015). Indeed, it has been demonstrated that non-coding mutations and genome rearrangements that perturb enhancer-promoter communication can underlie disease susceptibility and developmental abnormalities [START_REF] Bahr | A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies[END_REF][START_REF] Benko | Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence[END_REF][START_REF] Bhatia | Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia[END_REF][START_REF] Franke | Formation of new chromatin domains determines pathogenicity of genomic duplications[END_REF][START_REF] Gonen | Sex reversal following deletion of a single distal enhancer of Sox9[END_REF][START_REF] Kioussis | β-Globin gene inactivation by DNA translocation in γβ-thalassaemi[END_REF]Lettice et al., 2003[START_REF] Lettice | Opposing Functions of the ETS Factor Family Define Shh Spatial Expression in Limb Buds and Underlie Polydactyly[END_REF][START_REF] Long | Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder[END_REF][START_REF] Lupiáñez | Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Article Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions[END_REF][START_REF] Mansour | An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element[END_REF][START_REF] Sagai | Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb[END_REF][START_REF] Uslu | Long-range enhancers regulating Myc expression are required for normal facial morphogenesis[END_REF]. Mutations in enhancers and non-coding DNA can provoke severe Mendelian and common genetic disease, a class of conditions referred as "enhanceropathies" [START_REF] Smith | Enhancer biology and enhanceropathies[END_REF]. The deleterious effects of disruptions in enhancers can result from deletions, duplications, rearrangements, inversions or point mutations. In the present section, I discuss several examples in which distant-acting gene enhancers were directly shown to play a causative role in disease (Table 1).

SOX9 is a pivotal regulator of morphogenesis in the developing embryo, particularly in craniofacial development, chondrogenesis and sex determination [START_REF] Lee | Sox9 function in craniofacial development and disease[END_REF].

Heterozygous loss-of-function mutations in the SOX9 gene cause campomelic dysplasia (CD), a severe, often lethal, congenital dysplasia characterized by bowing of the long bones of the legs, sex reversal and craniofacial defects [START_REF] Wagner | Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the <em>SRY</em>-related gene <em>SOX9<[END_REF]. Pierre Robin sequence (PRS) is a congenital craniofacial disorder associated with micrognathia, glossoptosis and upper airway obstruction [START_REF] Robin | A fall of the base of the tongue considered as a new cause of nasopharyngeal respiratory impairment: Pierre Robin sequence, a translation[END_REF]. Because of the phenotypic overlap between the craniofacial malformations of campomelic dysplasia and Pierre Robin sequence, and the existence of several non-coding mutations, including translocations and microdeletions, within the 2-Mb gene desert upstream of SOX9, it has been hypothesized that PRS may be caused by dysregulation of SOX9 [START_REF] Amarillo | Familial Microdeletion of 17q24.3 Upstream of SOX9 Is Associated With Isolated Pierre Robin Sequence Due to Position Effect[END_REF][START_REF] Bagheri-Fam | Long-range upstream and downstream enhancers control distinct subsets of the complex spatiotemporal Sox9 expression pattern[END_REF]. Indeed, it has been subsequently shown that Pierre Robin sequence is caused by developmental misexpression of SOX9 due to deletion of extreme long-range tissue-specific enhancers, located 1.25 and 1.45 Mb centromeric of SOX9 [START_REF] Benko | Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence[END_REF][START_REF] Long | Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder[END_REF].

As mentioned earlier, key developmental genes might have functions in more than one tissue and be surrounded and regulated by multiple tissue-specific cis-regulatory elements, spread over hundreds of kilobases from the TSS. In mammals, Sox9 is a direct target of SRY (Sexdetermining Region of Y chromosome), a pivotal factor for testis and subsequent male development [START_REF] Capel | Vertebrate sex determination: Evolutionary plasticity of a fundamental switch[END_REF]). An in vivo screen for gonad enhancers in the 2-Mb gene desert upstream of Sox9, uncovered a regulatory element, the enhancer 13 (Enh13), located 565 Kb upstream of the TSS which appeared to be essential for initiating mouse testis development [START_REF] Gonen | Sex reversal following deletion of a single distal enhancer of Sox9[END_REF]. Surprisingly, while XY embryos carrying a heterozygous deletion of Enh13 (Enh13 +/-) undergo normal testis development, XY Enh13 -/-embryos produce fully sexreversed ovaries identical to those of XX wild-type embryos, demonstrating that deletion of a single distal-enhancer results in sex reversal [START_REF] Gonen | Sex reversal following deletion of a single distal enhancer of Sox9[END_REF].

Deletions of key tissue-specific enhancers have been shown to be causative for several other disorders, including: cleft lip with or without cleft palate (CL/P) craniofacial malformation resulting from deletion of a distal enhancer controlling Myc expression in the future upper lip [START_REF] Uslu | Long-range enhancers regulating Myc expression are required for normal facial morphogenesis[END_REF]; truncated limbs in mice resulting from deletion of a limb-specific Shh enhancer [START_REF] Sagai | Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb[END_REF] (Figure 10) and β-thalassemia due to the inability of red blood cells to produce mature globin after deletion of the hemoglobin subunit beta (HBB) LCR [START_REF] Kioussis | β-Globin gene inactivation by DNA translocation in γβ-thalassaemi[END_REF][START_REF] Tuan | An erythroid-specific, developmental-stage-independent enhancer far upstream of the human "β-like globin" genes[END_REF].

Besides the pathological consequences of the removal of enhancer elements, repositioning of distal enhancers can lead to human disorders. A representative example is α-thalassaemia, a haemoglobinopathy -like β-thalassemia-caused by imbalances in the ratio of α-globin in erythroid cells. While many patients diagnosed with α-thalassaemia were carrying globincoding mutations [START_REF] Kan | Deletion of α-globin genes in haemoglobin-H disease demonstrates multiple α-globin structural loci[END_REF], a subset of individuals did not exhibit any abnormalities in the protein-coding sequence. It was eventually identified that a single-nucleotide polymorphism (SNP) in the non-coding region between the α-globin genes and their cognate enhancer, creates a novel binding site for the erythroid TF GATA1, forming a cryptic promoter [START_REF] De Gobbi | A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter[END_REF]. This new element interferes with normal activation of α-globin, by reallocating the enhancer away from α-globin genes.

Point mutations in enhancers can also produce disease, considering that enhancers are the elements where tissue-specific TFs bind [START_REF] Lambert | The Human Transcription Factors[END_REF]. Among others, aniridia [START_REF] Bhatia | Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia[END_REF][START_REF] Lauderdale | 3' deletions cause aniridia by preventing PAX6 gene expression[END_REF] and preaxial polydactyly (Lettice et al., 2003[START_REF] Lettice | Opposing Functions of the ETS Factor Family Define Shh Spatial Expression in Limb Buds and Underlie Polydactyly[END_REF] are of great importance. Aniridia is a congenital eye disorder caused by haploinsufficiency of the developmental regulator PAX6, predominantly through loss-offunction mutations or gene deletions and in some cases due to disruption of the genomic region downstream of PAX6 [START_REF] Lauderdale | 3' deletions cause aniridia by preventing PAX6 gene expression[END_REF]. A de novo single point mutation (chr11:

31,685,945 G->T) in an ultra-conserved enhancer (SIMO) located 150 kb telomeric of PAX6 was identified in an affected individual [START_REF] Bhatia | Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia[END_REF]. SIMO was shown to be critical for PAX6 expression in developing ocular tissues as the single point mutation in this element was sufficient to disrupt an autoregulatory PAX6 binding site, cause loss of enhancer activity and deficient maintenance of PAX6 expression [START_REF] Bhatia | Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia[END_REF]. In the same line, point mutations residing within the limb-specific enhancer ZRS, located approximately 850 kb away from the Shh, lead to a congenital limb malformation called preaxial polydactyly (PPD) (Lettice et al., 2003[START_REF] Lettice | Opposing Functions of the ETS Factor Family Define Shh Spatial Expression in Limb Buds and Underlie Polydactyly[END_REF]) (Figure 10). These point mutations cause aberrant expression of Shh, normally expressed in the zone of polarizing activity (ZPA) posteriorly in the limb bud, in an additional ectopic site at the anterior margin of the limb. Adapted from [START_REF] Visel | Genomic views of distant-acting enhancers[END_REF].

Structural rearrangements and duplications of enhancers can also provoke disease.

Chromosomal rearrangement of a distal GATA2 enhancer causes dysregulation of two unrelated genes, EVI1 and GATA2, with acute myeloid leukemia (AML) as the outcome.

Removal of the distal enhancer from its genomic origin caused aberrant activation of EVI1 and simultaneous transcriptional impairment of GATA2 [START_REF] Gröschel | A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in Leukemia[END_REF]. Lastly, chromosomal duplications of a T cell-specific distal MYC enhancer, named N-Me (from NOTCH MYC enhancer) located 1.47 Mb downstream from MYC TSS, are directly implicated in the pathogenesis of human T cell acute lymphoblastic leukemia (T-ALL) (Herranz et al., 2014). Together, these results underscore the essential role of enhancer elements in development and further demonstrate that disruption of a single cis-element is sufficient to cause aberrant gene expression and disease phenotypes.

Other distal regulatory elements

Enhancers are the most extensively studied example of distal regulatory elements (DREs), however other types of cis-regulatory modules (CRMs) have also been identified. Silencers can act in an orientation-independent manner, similarly to enhancers, however they downregulate, rather than activate, target gene expression [START_REF] Brand | Characterization of a “silencer” in yeast: A DNA sequence with properties opposite to those of a transcriptional enhancer[END_REF], probably via binding transcriptional repressors [START_REF] Shore | Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication[END_REF]. Although examples of silencers have been reported in the literature for more than 30 years [START_REF] Brand | Characterization of a “silencer” in yeast: A DNA sequence with properties opposite to those of a transcriptional enhancer[END_REF][START_REF] Cao | Identification of a transcriptional silencer in the 5'-flanking region of the human ε-globin gene[END_REF], their mechanism of function and characteristic sequence and chromatin features, if any, are not well understood. Recent highly parallel reporter-based assays have augmented the number of identified putative silencer elements, both in mammals and in Drosophila (Doni Jayavelu, Jajodia, Mishra, & Hawkins, 2020; [START_REF] Gisselbrecht | Transcriptional Silencers in Drosophila Serve a Dual Role as Transcriptional Enhancers in Alternate Cellular Contexts[END_REF]. Interestingly, despite the fact that enhancers and silencers are being treated as two distinct groups of CRMs, bifunctional elements that can act as both enhancers and silencers have been discovered [START_REF] Gisselbrecht | Transcriptional Silencers in Drosophila Serve a Dual Role as Transcriptional Enhancers in Alternate Cellular Contexts[END_REF][START_REF] Jiang | Conversion of a dorsal-dependent silencer into an enhancer: Evidence for dorsal corepressors[END_REF][START_REF] Rafael Galupa | A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary Molecular Cell Article A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary[END_REF]. In some cases, this behavior has been shown to depend on the cellular context [START_REF] Gisselbrecht | Transcriptional Silencers in Drosophila Serve a Dual Role as Transcriptional Enhancers in Alternate Cellular Contexts[END_REF][START_REF] Jiang | Conversion of a dorsal-dependent silencer into an enhancer: Evidence for dorsal corepressors[END_REF], while in others it is dependent on the TAD (Topologically associating domain) (see Chapter 4) in which the CRM resides [START_REF] Rafael Galupa | A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary Molecular Cell Article A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary[END_REF]. Additional studies are required to uncover the mechanistic basis of silencers, and if and to what extent they coordinate with enhancers. Another type of cis-regulatory modules are insulators which are covered in a later section of the introduction.

Enhancer -promoter communication

Studies of several loci suggest that transcription in higher eukaryotes is often regulated by remote enhancer sequences, that can be positioned from a few up to hundreds of kilobases away from promoters [START_REF] Banerji | Expression of a β-globin gene is enhanced by remote SV40 DNA sequences[END_REF][START_REF] Bender | β-globin Gene switching and DNase I sensitivity of the endogenous β-globin locus in mice do not require the locus control region[END_REF][START_REF] Benko | Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence[END_REF][START_REF] Grosveld | Positionindependent, high-level expression of the human β-globin gene in transgenic mice[END_REF][START_REF] Uslu | Long-range enhancers regulating Myc expression are required for normal facial morphogenesis[END_REF]. Thus, communication between these distal elements is requisite for transcriptional regulation. Two basic models have been put forward to describe how enhancers and promoters communicate: (A) the tracking model and (B) the looping model (Figure 11). The tracking model states that enhancers act as nucleation points for factors which move progressively towards the promoter, through the intervening DNA sequence separating the two regulatory elements. On the other hand, the looping model proposes a direct, physical interaction between distant enhancers and promoters, with the intervening DNA sequence looping out. For example, it has been shown in in vitro experimental systems that enhancers are capable of stimulating transcription in trans when brought into close proximity to a promoter-containing plasmid [START_REF] De Bruin | Telomere looping permits gene activation by a downstream UAS in yeast[END_REF][START_REF] Müller | An enhancer stimulates transcription in <em>Trans</em> when attached to the promoter via a protein bridge[END_REF] or that the GAGA factor can form a protein link when bound to separate DNA molecules in trans [START_REF] Mahmoudi | GAGA can mediate enhancer function in trans by linking two separate DNA molecules[END_REF]. Carter et al. [START_REF] Carter | Long-range chromatin regulatory interactions in vivo[END_REF] Vernimmen & Bickmore, 2015; Q. [START_REF] Wang | Spatial and temporal recruitment of androgen receptor and its coactivators involves chromosomal looping and polymerase tracking[END_REF].

Enhancer action through chromatin loops

As discussed above, strong evidence supports the idea that spatial proximity between enhancers and promoters can increase promoter activity [START_REF] Carter | Long-range chromatin regulatory interactions in vivo[END_REF][START_REF] Tolhuis | Looping and interaction between hypersensitive sites in the active β-globin locus[END_REF]. Ever since, a body of data supports loop models, using microscopy techniques, such as fluorescent in situ hybridization (FISH), or molecular biology techniques, such as proximity ligation (3Cderived) methods [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF]Bonev et al., 2017;H. Chen et al., 2018a;[START_REF] Deng | Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor[END_REF][START_REF] Deng | Reactivation of developmentally silenced globin genes by forced chromatin looping[END_REF][START_REF] Dixon | Chromatin architecture reorganization during stem cell differentiation[END_REF][START_REF] Fulco | Activity-by-Contact model of enhancer specificity from thousands of CRISPR perturbations (CRISPRi FlowFISH)[END_REF][START_REF] Gasperini | A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens[END_REF][START_REF] Ghavi-Helm | Enhancer loops appear stable during development and are associated with paused polymerase[END_REF]Palstra et al., 2003;[START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF]Zhou et al., 2014). Key developmentally regulated genes, especially those that are expressed at high levels, appear to interact with enhancers specifically in the corresponding cell type (Palstra et al., 2003;[START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF]. A representative example is the Sox2 gene where a distal enhancer region (Sox2 control region, SCR) located >100 kb downstream of Sox2 is essential for cis-regulation of Sox2 specifically in ES cells (Zhou et al., 2014). 3C and 4C analysis revealed that the distal SCR contacts the promoter through the formation of a chromatin loop (Ben Zouari, [START_REF] Ben Zouari | 4See: A Flexible Browser to Explore 4C Data[END_REF]Zhou et al., 2014). Interestingly, homozygous Cas9-mediated deletions of this SCR resulted in significant reduction in Sox2 mRNA and protein levels, loss of ES colony features, changes in gene expression on the genome-wide level and decreased neuroectodermal formation upon differentiation. Notably, 5C [START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF] and ultra-high resolution HiC (Bonev et al., 2017) data observed a loss of the ES-specific Sox2-SCR looping interaction upon neural progenitor cell (NPC) differentiation and gain of a new contact between the Sox2 promoter and a more distal (~450 kb downstream) NPC-specific enhancer.

Direct proof of the functional importance of enhancer-promoter contacts also comes from studies that manipulated the chromatin conformation at a native environment [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF][START_REF] Deng | Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor[END_REF][START_REF] Deng | Reactivation of developmentally silenced globin genes by forced chromatin looping[END_REF]. The chromatin loop between the LCR and β-globin (Hbb) proteins to engineer inducible and reversible contacts between enhancers and promoters, which led to increased gene expression (J. H. Kim et al., 2019;[START_REF] Morgan | Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping[END_REF]. Adapted from (Schoenfelder & Fraser, 2019).

Follow-up studies showed that forced chromatin looping between the LCR and a developmentally silenced embryonic globin gene (γ-globin) in adult erythroid cells leads to strong transcriptional activation at this locus by increasing the burst fraction [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF][START_REF] Deng | Reactivation of developmentally silenced globin genes by forced chromatin looping[END_REF]. Additionally, two recent strategies of forced chromatin looping direct dCas9 (catalytically dead Cas9) fusion proteins to target genomic loci, which can be brought into close proximity either by small-molecule [START_REF] Morgan | Manipulation of nuclear architecture through CRISPR-mediated chromosomal looping[END_REF] or light-induced (J. H. Kim et al., 2019) (Figure 12B) dimerization to reversibly establish chromatin loops and induce gene expression. Recently, enhancers have also been implicated in the control of transcriptional bursting parameters [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF][START_REF] Chen | Dynamic interplay between enhancer-promoter topology and gene activity[END_REF][START_REF] Fukaya | Enhancer Control of Transcriptional Bursting[END_REF][START_REF] Lim | Visualization of Transvection in Living Drosophila Embryos[END_REF]. In particular, recruitment of the LCR to a chosen β-type globin gene promoter, increased β-globin burst frequency but not burst size [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF]. In the same line, live-cell visualization of enhancer-promoter interactions in Drosophila embryos revealed that sustained enhancer-promoter association is necessary for the initiation of transcriptional bursts (H. [START_REF] Chen | Dynamic interplay between enhancer-promoter topology and gene activity[END_REF]. Moreover, other studies reported that a shared enhancer is able to simultaneously activate linked reporter genes in cis and in trans, resulting in coordinated bursting of these genes [START_REF] Fukaya | Enhancer Control of Transcriptional Bursting[END_REF][START_REF] Lim | Visualization of Transvection in Living Drosophila Embryos[END_REF]. Overall, these studies fit with a model in which physical contacts are necessary for enhancers to activate transcription.

Potential role of eRNAs in regulating gene expression via chromatin loops

Chromatin interaction studies held in several cell lines showed a much greater abundance of eRNAs at enhancers that interacted with promoters of protein-coding genes compared to those that did not (Y. C. [START_REF] Lin | Global changes in the nuclear positioning of genes and intra-and interdomain genomic interactions that orchestrate B cell fate[END_REF]Sanyal et al., 2012). In support of that, [START_REF] Li | Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation[END_REF] used both short interfering RNAs (siRNAs) and locked nucleic acid antisense oligonucleotides (LNAs) against eRNAs produced by (E2)-bound estrogen receptor α (ERα) enhancers, in order to explore their potential roles on gene expression events. Interestingly, siRNA/LNA-mediated knockdown of eRNAs caused a significant inhibition of gene expression and enhancer-promoter specific interactions induced by E2. Furthermore, knockdown of these eRNAs resulted in a decrease of cohesin recruitment to several ERαbound enhancers, while knockdown of RAD21 (a subunit of cohesin) inhibited both gene induction and the observed induced enhancer-promoter loops. These data suggest that besides cohesin, eRNAs are also likely to have important functions in the formation or the stabilization of enhancer-promoter loops.

By contrast, when MCF7 cells were treated with flavopiridol, a drug that blocks transcription elongation by inhibiting the CDK9 kinase of the P-TEFb, Hah et al. [START_REF] Hah | Enhancer transcripts mark active estrogen receptor binding sites[END_REF] found that although the production of eRNA and gene transcripts was reduced, the estradiol-induced enhancer-promoter looping was not affected. This difference could be due to the different experimental approach used (eRNA knockdown (W. [START_REF] Li | Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation[END_REF]/ inhibition of PolII elongation [START_REF] Hah | Enhancer transcripts mark active estrogen receptor binding sites[END_REF])), or it may indicate that eRNAs are functionally important for chromatin looping only at specific gene loci.

Transcription hubs

Although population-average "C" methods support a model of chromatin loops bringing individual enhancers into contact with their cognate promoter (Palstra et al., 2003), this is challenged by recent findings. Recent imaging experiments have identified, at least for some specific loci, that enhancer-promoter proximity can be decoupled from, or even negatively correlate with gene expression (Alexander et al., 2019;Benabdallah et al., 2019;[START_REF] Espinola | Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development[END_REF][START_REF] Heist | Large distances separate coregulated genes in living Drosophila embryos[END_REF]. So how do enhancers regulate specific genes from a distance? Large multi-enhancer/promoter hubs have been discovered [START_REF] Allahyar | Enhancer hubs and loop collisions identified from single-allele topologies[END_REF][START_REF] Oudelaar | Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains[END_REF], which sometimes (but not always) co-associate with nuclear foci -or clusters -of transcription factors [START_REF] Dufourt | Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs[END_REF][START_REF] Li | Single-gene imaging links genome topology, promoter-enhancer communication and transcription control[END_REF]. Binding of TFs on DNA depends on TF's affinity for target sequences, the concentration, as well as the localization of the TF in the nuclear space [START_REF] Lambert | The Human Transcription Factors[END_REF]. Such hubs have been proposed to create local microenvironments, perhaps via the formation of liquid-liquid phaseseparated (LLPS) condensates [START_REF] Sabari | Coactivator condensation at super-enhancers links phase separation and gene control[END_REF], whereby regulatory factors can be rapidly exchanged within a high local concentration without direct juxtaposition of promoters and enhancers [START_REF] Mir | Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos[END_REF][START_REF] Tsai | Nuclear microenvironments modulate transcription from low-affinity enhancers[END_REF] (Figure 13). Recent analysis of the activity of linked reporter genes using quantitative live-imaging further strengthens this revised hub model, by showing that a single enhancer is able to simultaneously coactivate two promoters linked in cis and in trans [START_REF] Fukaya | Enhancer Control of Transcriptional Bursting[END_REF][START_REF] Lim | Visualization of Transvection in Living Drosophila Embryos[END_REF]. These results oppose the classic enhancer-promoter looping model and suggest that target promoters can share common clusters of TFs and Pol II [START_REF] Lim | Enhancer-promoter communication : hubs or loops ?[END_REF]. Kim & Shendure, 2019; Z. Liu & Tjian, 2018;[START_REF] Mir | Dense bicoid hubs accentuate binding along the morphogen gradient[END_REF][START_REF] Swinstead | Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions[END_REF]. For example, Sox2 was found to spend most of its time (>95%) in a stochastic diffusive state or to be involved in non-specific DNA interactions. Only a small fraction of Sox2 molecules at any time was bound to specific recognition sites and even then dwell-times are on the scale of few (~12) seconds (J. Chen et al., 2014). Since transcription factors show high turnover, a growing view is that temporal occupancy at the target site is sustained by high local concentrationsclusters-of TFs (J. Chen et al., 2014;[START_REF] Lim | Enhancer-promoter communication : hubs or loops ?[END_REF]) (Figure 13). Such TF clusters have been identified in several systems, including Drosophila embryos (Bicoid, Zelda) [START_REF] Dufourt | Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs[END_REF][START_REF] Mir | Dense bicoid hubs accentuate binding along the morphogen gradient[END_REF][START_REF] Yamada | The Drosophila Pioneer Factor Zelda Modulates the Nuclear Microenvironment of a Dorsal Target Enhancer to Potentiate Transcriptional Output[END_REF], yeast (Mig1) [START_REF] Wollman | Transcription factor clusters regulate genes in eukaryotic cells[END_REF] and ESCs (Oct4, Sox2) [START_REF] Lim | Enhancer-promoter communication : hubs or loops ?[END_REF]Z. Liu et al., 2014).

Similarly, recent studies using super-resolution imaging on living cells revealed that RNA Pol II forms transient clusters (Figure 13), with an average lifetime of ~8 seconds [START_REF] Cho | RNA Polymerase II cluster dynamics predict mRNA output in living cells[END_REF][START_REF] Cisse | Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells[END_REF], which contradicts earlier fixed cell studies supporting the existence of Pol II clusters as statically pre-assembled architectures, named "transcription factories" [START_REF] Iborra | Active RNA polymerases are localized within discrete transcription "factories" in human nuclei[END_REF]. These dynamics were considerably faster than the period needed to complete transcription of a mammalian gene. Indeed, Pol II clusters were found to be more pronounced upon treatment with flavopiridol, a known P-TEFb inhibitor [START_REF] Chao | Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo[END_REF], suggesting a role of these clusters for transcription initiation rather than productive elongation [START_REF] Cisse | Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells[END_REF]. A follow up study demonstrated that transient Pol II clusters positively correlate with the number of nascent mRNAs subsequently synthesized [START_REF] Cho | RNA Polymerase II cluster dynamics predict mRNA output in living cells[END_REF]. Treatment of cells with DRB, another P-TEFb inhibitor [START_REF] Chodosh | 5,6-Dichloro-1-β-Dribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro[END_REF]) induced stable Pol II clusters similar to those observed upon flavopiridol treatment. Interestingly, upon removal of DRB transcriptional bursting resumed, suggesting that Pol II clusters are loaded on gene promoters to form and regulate transcriptional bursts [START_REF] Cho | RNA Polymerase II cluster dynamics predict mRNA output in living cells[END_REF]. Pol II contains an intrinsically disordered C-terminal domain (CTD) [START_REF] Portz | Structural heterogeneity in the intrinsically disordered RNA polymerase II C-terminal domain[END_REF]. In some cases, the CTD has been found to undergo liquid-liquid phase separation [START_REF] Boehning | RNA polymerase II clustering through carboxy-terminal domain phase separation[END_REF] while in others it does not [START_REF] Lu | Phaseseparation mechanism for C-terminal hyperphosphorylation of RNA polymerase II[END_REF]. However, CTD was found to interact with kinase cyclin T1, a subunit of P-TEFb, droplets [START_REF] Lu | Phaseseparation mechanism for C-terminal hyperphosphorylation of RNA polymerase II[END_REF] and the Mediator complex [START_REF] Chong | Imaging dynamic and selective low-complexity domain interactions that control gene transcription[END_REF], forming large clusters at sites of active transcription. Additionally, phosphorylation of CTD liberates Pol II from hubs and enables the transition into active elongation [START_REF] Boehning | RNA polymerase II clustering through carboxy-terminal domain phase separation[END_REF][START_REF] Lu | Phaseseparation mechanism for C-terminal hyperphosphorylation of RNA polymerase II[END_REF]. However, additional studies are necessary to get more insights into this process.

Enhancer hubs

The recently developed Multi-contact 4C (MC-4C) [START_REF] Allahyar | Enhancer hubs and loop collisions identified from single-allele topologies[END_REF] and Tri-C [START_REF] Oudelaar | Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains[END_REF] methods were used to study multi-way contacts at the β-globin locus and were able to identify interaction patterns suggestive of the formation of enhancer hubs. Highly interacting enhancers that share binding by specific sets of transcription factors have been identified in several other loci (de Wit et al., 2013;[START_REF] Denholtz | Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization[END_REF][START_REF] Petrovic | Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques[END_REF]

What are the mechanisms of clustering?

Two models have been proposed to explain the mechanism of clustering, depending on how molecules are interacting and recruited into clusters. The first model postulates formation of transcription condensates that exhibit properties of liquid-liquid phase separation (LLPS) [START_REF] Boija | Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains[END_REF][START_REF] Cho | Mediator and RNA polymerase II clusters associate in transcription-dependent condensates[END_REF][START_REF] Chong | Imaging dynamic and selective low-complexity domain interactions that control gene transcription[END_REF][START_REF] Sabari | Coactivator condensation at super-enhancers links phase separation and gene control[END_REF]) (Figure 13), as has been proposed for the formation of membraneless organelles, such as the nucleolus [START_REF] Feric | Coexisting Liquid Phases Underlie Nucleolar Subcompartments[END_REF], inside cells. Phase separation is characterized by multiple low-affinity interactions between proteins and nucleic acids that drive demixing of particular combinations of factors into different "phases" (analogous to demixing of an oil/water emulsion), such that compatible factors form distinct physical droplets or condensates (Y. Shin & Brangwynne, 2017). The activation domains of TFs are particularly enriched for low complexity intrinsically disordered regions (IDRs) [START_REF] Hnisz | A Phase Separation Model for Transcriptional Control[END_REF]S. Kim & Shendure, 2019). Recent results from in vitro and in vivo experiments, have indicated that weak and dynamically multivalent IDR-IDR interactions result in the formation of phase-separated condensates [START_REF] Chong | Imaging dynamic and selective low-complexity domain interactions that control gene transcription[END_REF][START_REF] Sabari | Coactivator condensation at super-enhancers links phase separation and gene control[END_REF]. Pol II and the co-activator Mediator have been found to colocalize in clusters, which associate with chromatin and have properties of phase-separated condensates [START_REF] Cho | Mediator and RNA polymerase II clusters associate in transcription-dependent condensates[END_REF]. Furthermore, MED1 subunit of the Mediator along with the co-factor BRD4 have been found to form nuclear bodies at super-enhancers [START_REF] Sabari | Coactivator condensation at super-enhancers links phase separation and gene control[END_REF]

Is enhancer-promoter proximity even required?

Although several studies support that enhancer control of gene expression requires direct promoter contact, recent imaging experiments have shown enhancers at large distances from activated genes (Alexander et al., 2019;Benabdallah et al., 2019;[START_REF] Lim | Visualization of Transvection in Living Drosophila Embryos[END_REF]. A case in point comes from the aforementioned ZRS-Shh communication, whereby all Shh enhancers are found within the same ~960 kb TAD [START_REF] Anderson | Long range regulation of the sonic hedgehog gene[END_REF][START_REF] Williamson | Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity[END_REF], of which one of the most distal, ZRS, resides within the intron of the unrelated Lmbr1 gene, and is specific to developing limb bud (Figure 14). In developing limb buds, the ZRS enhancer controls the expression of Shh located 850 kb away (Lettice et al., 2003). Adapted from (Schoenfelder & Fraser, 2019).

Using 3D-FISH and structured illumination microscopy, Williamson and colleagues [START_REF] Williamson | Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity[END_REF] reported close proximity between Shh and ZRS in the nucleus of all cell types examined, regardless of Shh activity status. Nevertheless, high levels of colocalization (<200 nm) between Shh and the ZRS were observed only in the zone of polarizing activity (ZPA) of the distal posterior limb bud where Shh is expressed, consistent with a specific enhancer-promoter loop contact. This finding contradicts a previously published work that identified similar rates of Shh-ZRS colocalization between the Shh-expressing ZPA and anterior limb buds, in which Shh is not expressed under normal conditions (Amano et al., 2009).

The same group used the CRISPR-Cas9 technology to delete major CTCF sites of the Shh TAD in mouse ESCs and in vivo [START_REF] Williamson | Developmentally regulated Shh expression is robust to TAD perturbations[END_REF]. Loss of individual CTCF sites (ΔCTCF1, 2, 3) at the Shh TAD boundaries disrupted chromatin structure and the spatial proximity between Shh and ZRS in both ESCs and E11.5 limb bud tissue. Strikingly, CTCFsite deletion had no effect on Shh expression pattern or development and mice carrying homozygous CTCF site deletions were viable, without any deleterious phenotypes, arguing that the previously identified loop between Shh and ZRS in the posterior limb bud is not functionally important. These results are compatible with evidence showing increased Shh neuronal enhancer-promoter distance upon transcription activation (Benabdallah et al., 2019).

In the developing mouse brain, Shh expression is driven by a set of enhancers, the Shh-Brain-Enhancers (SBEs), located from 100 up to 780 kb upstream of Shh [START_REF] Benabdallah | SBE6: A novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells[END_REF][START_REF] Jeong | A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies longrange ventral forebrain enhancers[END_REF][START_REF] Yao | Cis-regulatory architecture of a brain signaling center predates the origin of chordates[END_REF] Together, these results provide strong evidence that direct molecular contact is not essential for enhancer activation of its target gene. All these observations could be explained by the "hub", "cluster" or "condensates" hypothesis. However, a follow up study on Pou5f1 and Sox2 loci demonstrated that distal enhancers are frequently in close distance (<200 nm for Pou5f1; <180 nm for Sox2) around the transcription site. Altogether, despite remarkable progress over the past years, much requires to be understood about the mechanisms of enhancer-promoter communication.

Introduction

Genomes are more than linear sequences of DNA. Two meters of mammalian DNA is compacted by an order of ~ 10 5 to be contained within each cell nucleus, implying that chromosomes must be highly folded structures. Since early descriptions of chromatin-dense heterochromatin and more open euchromatin, it has been appreciated that chromosome structure is heterogeneous yet highly organized. Crystallographic and electron microscopy studies have given detailed structural insight into the primary folding of DNA into 10-nm nucleosome fibers, although the nature of any higher-order folding was difficult to elucidate [START_REF] Woodcock | Chromatin architecture[END_REF]. Recent advances showed that nucleosome fibers do not appear to form higher-order "30-nm filaments" in vivo; instead the different compaction states between interphase euchromatin and heterochromatin and even mitotic chromosomes are brought about by different local concentrations of 10-nm nucleosome fibers [START_REF] Fussner | Open and closed domains in the mouse genome are configured as 10-nm chromatin fibres[END_REF][START_REF] Ou | ChromEMT: visualizing 3D chromatin structure and compaction in interphase and mitotic cells[END_REF]. The advent of the chromosome conformation capture method [START_REF] Dekker | Capturing chromosome conformation[END_REF] and its higher-throughput variants [START_REF] Dostie | Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements[END_REF][START_REF] Fullwood | An oestrogen-receptor-alpha-bound human chromatin interactome[END_REF][START_REF] Hughes | Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, highthroughput experiment[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF][START_REF] Mifsud | Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C[END_REF][10][START_REF] Simonis | Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C)[END_REF] allowed higher-order chromosome folding to be inferred by identifying spatial proximity between distal genomic sequences (see Ref. [12] for overview of the technical differences between the methods). Collectively, these built up a hierarchical model of chromosome folding, whereby architectural features are present at multiple scales, each correlating with transcriptional control (Fig. 1), although questions remain as to whether chromatin topology is a driver or passenger of genome function [START_REF] Sexton | The role of chromosome domains in shaping the functional genome[END_REF]. Firstly, distal regulatory elements such as enhancers are brought into physical proximity with their cognate gene promoters via the formation of chromatin loops. Second, chromosomes appear to be organized into discretely folded modules, termed topologically associated domains (TADs), which may delimit autonomously regulated chromatin regions. Third, genes or TADs tend to form long-range interaction networks with regions of similar transcriptional activity or chromatin state, which has been proposed to allow coordinated control of gene expression programs. Lastly, chromosomes occupy distinct territories within the interphase nucleus, with nonrandom radial positions linked to gene density and activity. This chapter will give an overview of our understanding of all these scales of chromosome folding. Pluripotent stem cells (PSCs) derived from the blastocyst inner cell mass are indefinitely selfrenewing and can be differentiated into any of the three germ layers [START_REF] Evans | Establishment in culture of pluripotent cells from mouse embryos[END_REF][START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF]. All lineage-specific genes need to be capable of activation under appropriate developmental cues. As a consequence, PSCs have widespread low-level transcription across the whole genome [START_REF] Efroni | Global transcription in pluripotent embryonic stem cells[END_REF] and have hallmarks of chromatin that Hierarchical spatial organization of the genome. (A) A linear view of genome control, whereby a regulated gene (red) is located far from a regulatory enhancer (green), bound by specific transcription factors (purple and yellow spheres), with unregulated genes (black) located in the intervening sequence. (B) A three-dimensional view of the same regulated gene. First, chromatin loops bring genes and their regulatory elements into direct physical proximity, often bridged by bound transcription factors (spheres) and the engaged RNA polymerase complex (pink oval and sea green shape). These loops are organized into discrete TAD modules, believed to be in turn defined by cohesin rings (blue) and barrier elements bound by CTCF (yellow hexagons). Specific gene coassociations (e.g., between the red and green gene indicated in the figure) occur within and between TADs within compartments of chromatin sharing the same epigenetic features, denoted by blue and red compartment colors. Compartments are in turn organized into discrete chromosome territories.

95 Chromatin loops: Remote control of gene transcription is more open than in differentiated cells: a reduced amount of heterochromatin, and looser binding of architectural proteins such as linker histones [START_REF]Open chromatin in pluripotency and reprogramming[END_REF]. Importantly, establishment of this chromatin state appears to be an epigenetic barrier to all methods of reprogramming differentiated cells to induced PSCs (iPSCs) [START_REF] Papp | Epigenetics of reprogramming to induced pluripotency[END_REF]. Repressed yet rapidly inducible lineage-specific genes are kept in a poised state, characterized by a "bivalent" chromatin mark containing both trimethylation of histone H3 lysine-27 (H3K27me3), deposited by the Polycomb group (PcG) protein repressors, and trimethylation of histone H3 lysine-4 (H3K4me3), associated with active promoters. This chromatin state is not unique to PSCs, but is more prevalent in less differentiated cells, and believed to confer developmental plasticity on the transcriptional fate of marked genes (reviewed in Ref. [START_REF] Voigt | A double take on bivalent promoters[END_REF]). Despite the special chromatin configuration of PSCs, the underlying principles of their chromosome folding appear to be largely conserved. Recent studies have identified and characterized the major players in PSC chromatin organization, some constitutive factors and others unique to pluripotency, which will be discussed in more detail in this chapter.

Chromatin loops: Remote control of gene transcription

Since early transgenic studies, it has been appreciated that promoter sequences are insufficient for full activation of most developmental genes, nor to confer faithful spatiotemporal expression patterns. Most metazoan genes are also under the control of distal regulatory elements, which can act over megabase distances and reside within nonregulated genes [START_REF] Amano | Chromosomal dynamics at the Shh locus: limb bud-specific differential regulation of competence and active transcription[END_REF]. Seminal studies of the beta-globin locus demonstrated that a cluster of erythroid enhancers, the locus control region (LCR), comes into closer physical proximity with the activated globin gene promoter than the intervening sequence [21], implying the formation of chromatin loops. Such loops linking enhancers to their cognate genes were subsequently identified in many other tissues, including PSCs [START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF]. The resulting "active chromatin hub" bringing together the regulatory factors bound to enhancer and promoter sequences is proposed to facilitate transcription. Extensive epigenomic profiling of PSCs and various cell and tissue types suggested that enhancers impart most control over gene expression programs and hence cell identity, since they are much more developmentally plastic than promoters [START_REF] Epigenomics | Integrative analysis of 111 reference human epigenomes[END_REF]. Among more conventional punctate enhancers, LCR-like clusters or "super-enhancers" were found enriched near genes controlling pluripotency in PSCs [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF] and lineage-specific genes in differentiated cells [START_REF] Hnisz | Super-enhancers in the control of cell identity and disease[END_REF], and it has been proposed that they are somehow a distinct, more important class of regulatory element controlling cell identity. However, more precise genetic dissection of "super-enhancers" in PSCs or erythrocytes suggests that they are merely clusters of conventional enhancers, with additive rather than synergistic effects on transcription [26,[START_REF] Moorthy | Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes[END_REF].

Despite their genome-wide identification and importance in epigenetic regulation, it has been difficult to unambiguously assign gene targets to enhancers, as they may not regulate the closest genes on the linear sequence [START_REF] Amano | Chromosomal dynamics at the Shh locus: limb bud-specific differential regulation of competence and active transcription[END_REF]. A significant effort has been made with different 3C variants to identify all promoter-centered looping interactions and thus identify the enhancers which physically contact, and presumably regulate, each promoter [START_REF] De Wit | The pluripotent genome in three dimensions is shaped around pluripotency factors[END_REF][START_REF] Li | Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation[END_REF][START_REF] Sanyal | The long-range interaction landscape of gene promoters[END_REF]. The most extensive promoter "interactomes" have been determined with the very recently developed Promoter Capture Hi-C (PCHi-C) method, which in essence entails high-throughput sequencing of 3C interactions (as for Hi-C) after preenrichment by sequence capture of the 3C material with complementary oligonucleotides covering all promoters 96 Chapter 4 Stem cell chromosome folding of a genome [START_REF] Hughes | Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, highthroughput experiment[END_REF][START_REF] Sahlen | Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution[END_REF][START_REF] Schoenfelder | The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements[END_REF]. Collectively, all of these studies varied in resolution and coverage and were applied to different cell types but arrived at the same general conclusions. First, promoter-interacting regions are enriched in hallmarks for enhancers, such as monomethylation of histone H3 lysine-4 (H3K4me1) and acetylation of histone H3 lysine-27 (H3K27ac), and this enrichment is much more pronounced for active genes. Second, many enhancers do not interact with the genes that are closest in terms of linear sequence, demonstrating that the previously described "long-range" enhancers are not exceptional cases. Third, many promoter-enhancer interactions bypass intervening sites for the insulator protein CTCF (CCCTC-binding factor), which were believed to prevent enhancers communicating with more distal genes [START_REF] Phillips | CTCF: master weaver of the genome[END_REF]. These findings were also the case for PSC promoter interactomes [START_REF] Sahlen | Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution[END_REF][START_REF] Schoenfelder | The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements[END_REF]. Interestingly, bivalent promoters were enriched in interactions with enhancers carrying the chromatin hallmarks of "poised" enhancers (H3K4me1 and H3K27me3, instead of H3K27ac). Perturbation of PcG proteins does not cause large-scale reduction of these interactions as the bivalent state is lost; rather the interacting enhancers adopt more active chromatin states, consistent with derepression of PcG-regulated genes [START_REF] Schoenfelder | Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome[END_REF].

Despite our growing appreciation of the 3-D promoter-enhancer interaction landscape, it is still unclear how a regulatory element locates its cognate promoter. Furthermore, questions remain as to whether their interaction is sufficient for transcriptional activation, and whether transcription (re-) initiation, elongation and/or another process is regulated. The LCR does not form distal interactions in nonerythroid cells and specifically contacts the transcribed globin gene in the developmental stage where it is expressed [21], suggesting that such enhancer-promoter loops can be instructive for transcription. In support of this, it was found that overexpressed pluripotency transcription factors bound to enhancer and promoter sequences of the OCT4 locus in both human iPSCs and unreprogrammed cells, but that iPSCs uniquely had enhancer-promoter interactions and OCT4 transcription [START_REF] Zhang | Intrachromosomal looping is required for activation of endogenous pluripotency genes during reprogramming[END_REF]. Furthermore, elegant studies showed that artificial induction of chromatin loops within the beta-globin locus can stimulate transcription [START_REF] Deng | Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor[END_REF][START_REF] Deng | Reactivation of developmentally silenced globin genes by forced chromatin looping[END_REF]. However, enhancer-promoter loops have been identified in other model systems, which form prior to transcriptional activation, suggesting that chromatin topology may be permissive for gene expression, but is not always sufficient. Some of these cases are for acute responses of differentiated cells to extracellular signals [START_REF] Hakim | Diverse gene reprogramming events occur in the same spatial clusters of distal regulatory elements[END_REF][START_REF] Jin | A high-resolution map of the three-dimensional chromatin interactome in human cells[END_REF], where preformed enhancer contacts would be advantageous for a rapid transcriptional induction. However in other cases, such as Drosophila embryogenesis or iPSC reprogramming, enhancer-promoter interactions were detected cell cycles and/or days before transcriptional induction, suggesting that stable chromatin loops are not restricted to acute biological processes [START_REF] Apostolou | Genome-wide chromatin interactions at the Nanog locus in pluripotency, differentiation, and reprogramming[END_REF][START_REF] Ghavi-Helm | Enhancer loops appear stable during development and are associated with paused polymerase[END_REF][START_REF] Wei | Klf4 organizes long-range chromosomal interactions with the oct4 locus in reprogramming and pluripotency[END_REF]. Very recent PCHi-C studies of PSC [START_REF] Freire-Pritchett | Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells[END_REF] and other progenitor [START_REF] Rubin | Lineage-specific dynamic and pre-established enhancer-promoter contacts cooperate in terminal differentiation[END_REF][START_REF] Siersbaek | Dynamic rewiring of promoter-anchored chromatin loops during adipocyte differentiation[END_REF] differentiation systems identified large-scale occurrences of stable and dynamic promoter-enhancer interactions, suggesting that both instructive and permissive looping models apply to transcriptional control. It is currently unclear how these two loop types are mechanistically distinguished. The "poised" interactions reported in Drosophila were enriched at promoters loaded with paused RNA polymerase [START_REF] Ghavi-Helm | Enhancer loops appear stable during development and are associated with paused polymerase[END_REF]. Similarly, bivalent gene promoters in PSCs have PcG-dependent paused polymerase [START_REF] Stock | Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells[END_REF], and these tend to participate in permissive rather than instructive chromatin loops [START_REF] Schoenfelder | Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome[END_REF]. Coupled with independent findings that induced chromatin loops rescued transcription initiation but not elongation in the artificial globin system tested [START_REF] Deng | Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor[END_REF] and that enhancers may contact transcribed gene bodies in addition to promoters [START_REF] Lee | Dynamic enhancer-gene body contacts during transcription elongation[END_REF], it is interesting to speculate that instructive chromatin loops can stimulate transcription initiation and elongation, whereas permissive chromatin loops require extra regulatory input for efficient elongation (Fig. 2).

66 The factors in chromatin looping: Not just transcription

The factors in chromatin looping: Not just transcription

As the initial studies concentrated on enhancer interactions, the first factors to be implicated in chromatin looping were transcription factors, which cobind to both promoter and distal regulatory elements and are thus gene-and often cell type specific [START_REF] Drissen | The active spatial organization of the beta-globin locus requires the transcription factor EKLF[END_REF][START_REF] Jing | Exchange of GATA factors mediates transitions in looped chromatin organization at a developmentally regulated gene locus[END_REF]. In mouse PSCs, for example, Klf4 is required Instructive and permissive models for enhancer-promoter chromatin loops. Top: The locus containing a silent gene (gray) and a distal enhancer (green) is in a linear conformation, with no enhancer-promoter contacts. Middle: The enhancer and promoter interact, presumably via binding of specific transcription factors (red triangles), and paused RNA polymerase (pink shape) is bound, but productive transcription is unable to occur. Bottom: The elongating RNA polymerase (sea green shape) now produces transcripts (magenta spheres), with the active gene (salmon pink) in a conformation allowing interactions between promoter, enhancer and the transcribed gene body. The instructive looping model allows a direct transition between the silent and transcribed states, whereas the permissive model passes through the intermediate poised state.

98

Chapter 4 Stem cell chromosome folding for enhancer interactions within the Oct4 locus and subsequent maintenance of pluripotency [START_REF] Wei | Klf4 organizes long-range chromosomal interactions with the oct4 locus in reprogramming and pluripotency[END_REF]. In most of these studies, it was difficult to decouple the effects of factor depletion on chromatin looping and transcription, although induced looping experiments in some loci [START_REF] Deng | Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor[END_REF] and observations of interaction loss preceding transcriptional perturbation in others [START_REF] Wei | Klf4 organizes long-range chromosomal interactions with the oct4 locus in reprogramming and pluripotency[END_REF] suggests that binding of transcription factor-mediated chromatin interactions is not just a by-product of transcriptional activation. Beyond gene-specific loops the general transcriptional coactivator Mediator is frequently found at transcribed promoters and enhancers and is implicated in most chromatin loops associated with gene activation [START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF][START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF]. However, it remains unclear whether the transcription factors, Mediator, and/or some other factors are principally involved in chromatin looping, nor whether this is brought about by simple protein-protein interactions between factors at distal genomic sites, or involves a more complex process to deform the chromatin fiber or environment, such as phase-separated condensates mediated by coactivators [START_REF] Sabari | Coactivator condensation at super-enhancers links phase separation and gene control[END_REF].

Beyond promoter-centric interactions, the other factor implicated in chromatin loops by early studies was CTCF, the major insulator protein in mammals [START_REF] Phillips | CTCF: master weaver of the genome[END_REF][START_REF] Splinter | CTCF mediates long-range chromatin looping and local histone modifications in the betaglobin locus[END_REF]. Initially, these loops were considered to mediate insulation by keeping genes and distal regulatory elements physically separated [START_REF] Kurukuti | CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2[END_REF], although the finding that many enhancers can bypass CTCF sites questions the universality of this model [START_REF] Sanyal | The long-range interaction landscape of gene promoters[END_REF][START_REF] Schoenfelder | The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements[END_REF]. Homotypic interactions between CTCF sites are present in the strongest interactions detected in genome-wide Hi-C studies [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF], and although tissue-specific CTCF-mediated loops have been reported [START_REF] Hanssen | Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo[END_REF][START_REF] Hou | Cell type specificity of chromatin organization mediated by CTCF and cohesin[END_REF], the majority appear to be constitutive, longer-range interactions, indicative of a more general chromosomal architectural role [START_REF] Sanyal | The long-range interaction landscape of gene promoters[END_REF][START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF]. Although CTCF tends to be depleted from enhancer-promoter interactions directly, CTCF-mediated loops interweave with and appear to reinforce them, since deletion of specific sites can actually perturb enhancer contacts [START_REF] Ren | CTCF-mediated enhancer-promoter interaction is a critical regulator of cell-to-cell variation of gene expression[END_REF]. Interestingly, such deletions only caused modest reductions in transcription levels, but greatly increased cell-to-cell variability, suggesting that CTCF-mediated loops confer robust expression control by context-dependent architectures and not solely by classical insulator activity. Curiously, CTCF loops seem to only occur between sites in convergent orientation [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF][START_REF] Vietri Rudan | Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture[END_REF], a topological constraint which does not apply to transcription factor binding motifs at looping regions. In support, genome editing experiments to invert specific sites did not generally affect CTCF binding, but severely disrupted chromatin loops [START_REF] De Wit | CTCF binding polarity determines chromatin looping[END_REF][60][START_REF] Sanborn | Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes[END_REF]. However, the inverted sites often did not participate in de novo interactions with CTCF sites now brought into a compatible orientation, suggesting that the motif alone is not sufficient to define chromatin topology.

A factor implicated in both of these seemingly distinct "transcriptional" and "architectural" chromatin loops is cohesin, a protein complex initially discovered to tether sister chromatids together after DNA replication [START_REF] Nasmyth | Cohesin: its roles and mechanisms[END_REF]. Cohesin has been implicated in both enhancer-promoter interactions [START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF][START_REF] Zhang | Intrachromosomal looping is required for activation of endogenous pluripotency genes during reprogramming[END_REF][START_REF] Wei | Klf4 organizes long-range chromosomal interactions with the oct4 locus in reprogramming and pluripotency[END_REF][START_REF] Hadjur | Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus[END_REF] and CTCF-mediated loops [START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF][START_REF] Demare | The genomic landscape of cohesin-associated chromatin interactions[END_REF], although many interactions of both types have also been detected, which are not accompanied by bound cohesin. The cohesin complex tethers sister chromatids by the formation of a ring structure, which needs to be broken to release the chromatids at mitosis [START_REF] Nasmyth | Cohesin: its roles and mechanisms[END_REF]. Although yet to be demonstrated, it is tempting to speculate that the same ring structure can stabilize chromatin loops, particularly since cohesin binding appeared to discriminate stable, poised enhancer-promoter interactions from acute loops brought about by specific transcription factors in a model of terminal differentiation [START_REF] Rubin | Lineage-specific dynamic and pre-established enhancer-promoter contacts cooperate in terminal differentiation[END_REF]. Further, depletion of cohesin destabilized chromatin interactions required for maintenance of pluripotency in PSCs [START_REF] Zhang | Intrachromosomal looping is required for activation of endogenous pluripotency genes during reprogramming[END_REF], although cell cycle effects from abrogation of sister chromatid cohesion could not be ruled out in this study. The mechanisms of postreplicative cohesin loading and unloading have been well studied (reviewed in Ref. [START_REF] Nasmyth | Cohesin: its roles and mechanisms[END_REF]), although it is less clear how cohesin is recruited to specific genomic sites associated with chromatin loops. Cohesin proteins have 99 Topologically associated domains as units of genome regulation no sequence-specific binding activities, but have been shown to interact with CTCF [START_REF] Rubio | CTCF physically links cohesin to chromatin[END_REF], Mediator [START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF], and some transcription factors [START_REF] Wei | Klf4 organizes long-range chromosomal interactions with the oct4 locus in reprogramming and pluripotency[END_REF], sometimes in conjunction with the cohesin-loading factor Nipbl, implying that cohesin recruitment is secondary to binding of these sequence-specific binding factors. Future studies should elucidate what determines whether cohesin is brought to a chromatin interaction and whether or how this affects chromatin topology dynamics.

Overall, chromatin loops provide a first order of chromosome folding, modulating communication between gene promoters and distal regulatory elements. The core factors, such as Mediator, cohesin and CTCF are involved in presumably the same mechanisms in PSCs and differentiated cells; specific transcription factors may provide some of the looping specificity, including the pluripotency transcription factors in PSCs. Polycomb appears to play an additional role in permissive loops at bivalent gene promoters, but this may not be unique to stem cells.

Topologically associated domains as units of genome regulation

One of the first observations to come from Hi-C studies was the apparent organization of chromosomes into discretely folded modules, or TADs, of multikilobase to megabase size, whereby chromatin interactions are strong within a domain, but sharply reduced for interactions spanning a border between two TADs [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation Centre[END_REF][START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF]. TAD demarcation correlates extremely well with the organization of "linear" epigenetic marks, such as histone modifications, transcriptional activity, lamin association and DNA replication timing [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation Centre[END_REF][START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF][START_REF] Dily | Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation[END_REF][START_REF] Pope | Topologically associated domains are stable units of replication-timing regulation[END_REF], so has been proposed to reflect both a spatial and functional organization of chromosomes into autonomously regulated gene neighborhoods. TADs could facilitate regulation of gene programs in two, nonmutually exclusive ways. First, they can spatially delimit the effective operational range of distal regulatory elements such as enhancers, thus preventing their aberrant activation of neighboring genes. In support of this model, most promoter interactions detected by PCHi-C are confined to the same TAD [START_REF] Schoenfelder | The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements[END_REF], and high-throughput enhancer trap studies showed that TADs also operationally delimit reporter gene expression patterns [START_REF] Symmons | Functional and topological characteristics of mammalian regulatory domains[END_REF]. Most strikingly, genetic diseases have now been attributed to aberrant enhancer-promoter contacts caused by deletions of TAD borders, inversions or translocations of TADs, or duplication events creating new hybrid TADs [START_REF] Franke | Formation of new chromatin domains determines pathogenicity of genomic duplications[END_REF][START_REF] Groschel | A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia[END_REF][START_REF] Lupianez | Disruptions of topological chromatin domains cause pathogenic rewiring of geneenhancer interactions[END_REF], although the phenotypic consequences seem to be context dependent [START_REF] Despang | Functional dissection of the Sox9-Kcnj2 locus identifies nonessential and instructive roles of TAD architecture[END_REF] (Fig. 3). Conversely, efficient enhancer activity on target genes may be assured by limiting their three-dimensional search ranges to within TADs. In support of this, systematic genetic manipulation of the TAD containing the Shh locus showed that the limb-specific enhancer was relatively insensitive to distance from the gene, provided they were in the same domain [START_REF] Symmons | The Shh topological domain facilitates the action of remote enhancers by reducing the effects of genomic distances[END_REF]. Such a confined interaction search space has also been proposed to explain why TADs can contain coordinately regulated genes [START_REF] Sexton | The role of chromosome domains in shaping the functional genome[END_REF][START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation Centre[END_REF][START_REF] Dily | Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation[END_REF].

Comparisons between cell types, including between naïve and primed PSCs, and between PSCs and different derived lineages, revealed that the majority of TADs are invariant [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Dixon | Chromatin architecture reorganization during stem cell differentiation[END_REF][START_REF] Ji | 3D chromosome regulatory landscape of human pluripotent cells[END_REF], even at syntenic chromosomal regions across different species [START_REF] Vietri Rudan | Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture[END_REF][START_REF] Harmston | Topologically associating domains are ancient features that coincide with metazoan clusters of extreme noncoding conservation[END_REF]. Super-resolution DNA fluorescent in situ hybridization (FISH) support this structural conservation further [START_REF] Fabre | Visualizing the HoxD gene cluster at the nanoscale level[END_REF], suggesting that TADs are somehow genetically encoded and relatively insensitive to tissue-specific epigenomic profiles. At first, this view of invariant TADs appears at odds with their correlation with epigenetic marks [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF], which are in turn highly developmentally dynamic [START_REF] Epigenomics | Integrative analysis of 111 reference human epigenomes[END_REF]. More focused studies of selected loci during PSC differentiation observed that "sub-TADs" and enhancer-promoter interactions were rewired within stable larger domains [START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF], leading to the suggestion that TADs may indeed be stable, and that cell reprogramming is accompanied by finer-scale chromatin topology dynamics. Nevertheless, complete TAD remodeling has been observed during the collinear activation of Hox genes, whereby a single TAD encompassing the inactive gene cluster splits into two with a developmentally shifting border between the active and repressed Hox genes [START_REF] Noordermeer | Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci[END_REF][START_REF] Noordermeer | The dynamic architecture of Hox gene clusters[END_REF]. More detailed higher-resolution studies are required to explore to what extent the distinction between a TAD and a "sub-TAD" is semantic, and how much chromatin domains can or need to be restructured to accommodate epigenetic and transcriptional changes during differentiation [START_REF] Bonev | Multiscale 3D genome rewiring during mouse neural development[END_REF].

Despite recent intensive studies, it remains unclear exactly how TADs are formed or stabilized. Borders are enriched in active genes and hallmarks of transcription, such as RNA polymerase and H3K4me3, as well as the "architectural" proteins cohesin and CTCF [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF]. As these factors are the major players in chromatin loops, TADs could be a consequence of very strong interactions between TAD borders [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. However, TAD borders represent only a small subset of genome-wide CTCF sites and active genes, so their presence alone is insufficient to define chromatin domains. Large genetic deletions at borders can cause TADs to fuse [START_REF] Lupianez | Disruptions of topological chromatin domains cause pathogenic rewiring of geneenhancer interactions[END_REF], or for preexisting intra-TAD interactions to reinforce as new, "secondary" borders [START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation Centre[END_REF][START_REF] Giorgetti | Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription[END_REF]. Precise deletions of single CTCF sites only caused mild effects on the overall TAD structure, but could nevertheless have important functional consequences from increased inappropriate enhancer interactions [START_REF] Hnisz | Activation of proto-oncogenes by disruption of chromosome neighborhoods[END_REF][START_REF] Narendra | CTCF-mediated topological boundaries during development foster appropriate gene regulation[END_REF][START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF]. A recent study with tunable ablation of CTCF in PSCs revealed that the residual amounts of protein left after a traditional knockdown was sufficient to maintain TAD structure [START_REF] Nora | Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization[END_REF]. Complete ablation, on the other hand, severely disrupted ~ 80% of TADs, with widespread but mild misregulation of transcription. Even more dramatically, systematic TAD loss has been reported when cohesin is completely ablated, either by degradation or block of loading onto chromosomes [START_REF] Rao | Cohesin loss eliminates all loop domains[END_REF][START_REF] Schwarzer | Two independent modes of chromatin organization revealed by cohesin removal[END_REF][START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF]. Cohesin thus appears essential for generation or stability of loops and TADs, whereas CTCF plays a large but not complete role in defining the positions of TADs.

Building chromosomal domains: Looping principles extended a TAD

Since Hi-C pairwise interaction maps have been generated, physical models have attempted to explain some or all of the complex architectural features discovered [START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF][START_REF] Sanborn | Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes[END_REF][START_REF] Barbieri | Complexity of chromatin folding is captured by the strings and binders switch model[END_REF][START_REF] Jost | Modeling epigenome folding: formation and dynamics of topologically associated chromatin domains[END_REF]. The loop extrusion model, shown in Fig. 4, provides an explanation for TAD structures containing convergent CTCF elements at the borders and relatively uniform interactions within the domains [START_REF] Sanborn | Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes[END_REF]94,[START_REF] Fudenberg | Formation of chromosomal domains by loop extrusion[END_REF]. An extrusion factor or

103

Building chromosomal domains: Looping principles extended a TAD factors is proposed to bind at seemingly random sites within the genome, then physically extrudes a chromatin loop bidirectionally, with two components of the extruding factor translocating in opposite directions. An equilibrium between extrusion and factor dissociation from chromatin is set up as the extruded loop grows, particularly influenced by physical barriers to extrusion set up at TAD borders. Asymmetric barriers, such as CTCF sites, may be expected to be in a convergent orientation to appropriately meet the oncoming translocating chromatin fiber. Since cohesin forms ring structures to tether and organize sister chromatids [START_REF] Nasmyth | Disseminating the genome: joining, resolving, and separation sister chromatids during mitosis and meiosis[END_REF], it is a major candidate for a loop extrusion factor. Further, TAD maintenance appears to be an active process, since TAD maintenance requires ATP and the cohesin complex ATPase domain [START_REF] Vian | The energetics and physiological impact of cohesin extrusion[END_REF]. Chromatin immunoprecipitation studies finding frequent cooccupancy of cohesin and CTCF, particularly at TAD borders, can therefore be interpreted as more stable, stalled loop extrusion complexes. This model predicts that altering the extrusion/dissociation equilibrium, for instance, by influencing extrusion factor residence time, will alter TAD size. In support of this and of cohesin being an important extrusion factor, deletion of cohesin release factor, WAPL, or loading factors such as Nipbl, increase or shrink average chromatin loop length, respectively, with a weakening of TAD structures [START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF][START_REF] Haarhuis | The cohesin release factor WAPL restricts chromatin loop extension[END_REF]. Notably, deletion of Nipbl in a different model system, mouse liver, had a more extreme phenotype, completely destroying TADs [START_REF] Schwarzer | Two independent modes of chromatin organization revealed by cohesin removal[END_REF]. Depending on the cell type, extruding factors in addition to cohesin may operate, and/or extra factors may inefficiently load/disassemble cohesin from interphase chromosomes. The bulky RNA polymerase complex and associated coactivators could potentially form a CTCF-independent barrier to loop extrusion, explaining the prevalence of active genes at TAD borders [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF]. Cohesin-mediated enhancer-promoter interactions may thus be considered as metastable loop extrusion intermediates. However, many chromatin interactions are not associated with bound cohesin, again implying that chromatin loops may be stabilized by multiple protein-protein interactions, and/or that other factors can mediate loop extrusion.

Overall, seemingly autonomous regulatory domains are set up in PSCs and other somatic cells by largely evolutionarily and developmentally stable TADs. Their basic architectural principles, outlined in this chapter, may allow genes to be more precisely homed to their cognate regulatory elements, although major questions remain poorly addressed. For example, what is the interplay between bulk TAD organization and dynamic chromatin ultrastructures observed at the large transcriptional changes accompanying development [START_REF] Bonev | Multiscale 3D genome rewiring during mouse neural development[END_REF]? What role do specific intra-TAD interactions play in TAD stability? We have much to learn about the "fine print" of TAD "architectural blueprints" and how this organization fits within chromatin regulation and other scales of chromosome folding.

FIG. 4

The loop extrusion model for TAD formation. (A) NIPBL (yellow oval) loads cohesin rings (purple and cyan circles) on to chromatin, which then extrudes a growing loop by translocating in opposite directions. The extrusion process is stalled when the base of the loop contains two barrier elements (green hexagons), usually CTCF sites, in convergent orientation, creating metastable loops in turn building TAD structures. Cohesin rings are also unloaded from chromatin by WAPL (pink oval), with extruded loop size determined by the equilibrium of cohesin loading and disassembly. (B) The loop extrusion model explains phenotypes of NIPBL or WAPL perturbation. Defects in cohesin loading on disruption of NIPBL drastically reduces extruded loops and hence TAD structures. Impaired cohesin disassembly by WAPL disruption shifts the equilibrium towards larger extruded loops, which may bypass CTCF-mediated borders.

Chromosome compartments: From a bipolar genome to specific functional networks

Before TADs were resolved with improved sequencing depth, the first Hi-C maps identified a curious property of chromatin interactions at the megabase scale: the genome appeared to be organized into large domains, or compartments, of two different types. "A" compartments, enriched in hallmarks of transcriptionally active genes, preferentially interacted with other "A" compartments, whereas inactive "B" chromatin compartments interacted with other "B" compartments [START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]. Intermixing of the two compartments was significantly depleted, suggesting a spatial segregation of active and inactive chromatin within the nucleus. Extensive repetitive sequences are normally filtered out of Hi-C analyses, so B compartments are unlikely to be simply the classical heterochromatin observed in microscopy studies. It should also be noted that conventional Hi-C studies give the average interaction landscape of millions of fixed nuclei, so the presence of two compartment types should not be interpreted as there being two segregated "poles" of chromatin within the nucleus. Rather, FISH and single-cell Hi-C experiments suggest that a particular gene will tend to coassociate with other chromosomal regions bearing similar epigenetic marks, even though the exact interaction partner(s) can vary widely from cell to cell [START_REF] Nagano | Cell-cycle dynamics of chromosomal organization at single-cell resolution[END_REF][START_REF] Wang | Spatial organization of chromatin domains and compartments in single chromosomes[END_REF]. Closer analysis of Hi-C interaction patterns revealed subcompartments of A and B types [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF], which may even be extended to a continuous spectrum [START_REF] Nagano | Cell-cycle dynamics of chromosomal organization at single-cell resolution[END_REF], characterized by different epigenetic signatures. The major principle of genome compartmentalization thus appears that, at a large scale, genes with similar activities tend to coassociate in the nuclear space. In agreement, whereas PSC differentiation is not accompanied by large-scale TAD structural changes, many genes switch compartments to interact with other regions matching their upregulated or repressed state [START_REF] Dixon | Chromatin architecture reorganization during stem cell differentiation[END_REF].

Before elucidation of genomic compartments, specific networks of long-range interactions between coregulated genes had been identified in the context of developmental gene activation [10] or repression [START_REF] Bantignies | Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila[END_REF]. In these examples, genes spatially coassociate at nuclear foci of RNA polymerase [10,[START_REF] Osborne | Active genes dynamically colocalize to shared sites of ongoing transcription[END_REF][START_REF] Papantonis | TNFalpha signals through specialized factories where responsive coding and miRNA genes are transcribed[END_REF] or PcG proteins [START_REF] Bantignies | Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila[END_REF][START_REF] Grimaud | RNAi components are required for nuclear clustering of Polycomb group responsive elements[END_REF], presumably allowing shared access to regulatory factors and coordinate control of genes within the network. In support of this, specialized spatial networks have been identified between genes regulated by common transcription factors [10,[START_REF] Papantonis | TNFalpha signals through specialized factories where responsive coding and miRNA genes are transcribed[END_REF]. Focused studies in (i)PSCs before and after differentiation or reprogramming suggest that PcG proteins and pluripotency transcription factors form major spatial networks in the pluripotent state [START_REF] De Wit | The pluripotent genome in three dimensions is shaped around pluripotency factors[END_REF][START_REF] Schoenfelder | Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome[END_REF][START_REF] Denholtz | Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization[END_REF]. Interestingly, perturbations of one gene can affect expression of others within their specific spatial network [START_REF] Fanucchi | Chromosomal contact permits transcription between coregulated genes[END_REF]. Although the universality of such "transcription factories" within metazoan nuclei is under debate [START_REF] Zhao | Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy[END_REF], spatial gene networks would both explain and provide a functional framework for genome compartmentalization by a self-organization model [START_REF] Kang | A dynamical model reveals gene co-localizations in nucleus[END_REF][START_REF] Rajapakse | The emergence of lineage-specific chromosomal topologies from coordinate gene regulation[END_REF]. As the majority of DNA-bound factors have short residence times on their cognate sites [START_REF] Phair | High mobility of proteins in the mammalian cell nucleus[END_REF], chance encounters between two loci create an increased local concentration of any factors that are bound at both genes. When a factor dissociates, it is thus more likely to be retrapped by a spatial cluster of nearby binding sites than to diffuse to a different location, thus reinforcing a growing, self-organizing network. The genome compartments deduced from global Hi-C maps could conceivably be an amalgamation of multiple such self-organized networks, although they have yet to be reliably deconvolved.

Since Drosophila inter-TAD interaction patterns mimic compartment organization [START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF] and mammalian TADs form subdomains [START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF], it was initially proposed that compartments just represent larger-scale TADs, subject to the same organizational principles. However, TAD and compartment architectures can be decoupled in mammals: CTCF ablation disrupts TADs with minimal changes to compartments [START_REF] Nora | Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization[END_REF],

105 Breaking TADs: The exceptions to the rule and removal of cohesin severely affects loops and TADs but actually reinforces genomic compartments [START_REF] Rao | Cohesin loss eliminates all loop domains[END_REF][START_REF] Schwarzer | Two independent modes of chromatin organization revealed by cohesin removal[END_REF][START_REF] Wutz | Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins[END_REF]. Thus competing architectural principles may actually influence chromosome folding, a concept taken further in a comparative Hi-C study, which reclassified TAD borders in active genes as small A compartments perturbing the organization of flanking B compartments [START_REF] Rowley | Evolutionarily conserved principles predict 3D chromatin organization[END_REF]. A possible explanation for apparent TAD-compartment competition is that compartment self-organization allows general reinforcement of entire gene program regulation but creates too large a search space for efficient targeting of enhancers to specific genes. TAD organization may thus restrict the enhancer search space within smaller domains, perhaps ensuring transcriptional fidelity, potentially explaining why TAD disruption has widespread positive and negative, albeit minor, effects on gene expression. In summary, although the mechanistic details are likely to differ, homotypic interactions appear to organize chromosomes at different levels, from individual chromatin loops to whole megabase-sized compartments.

Chromosome territories and interchromosomal interactions

DNA FISH experiments show that interphase chromosomes predominantly occupy discrete regions of the nucleus, termed chromosome territories, but that chromosome intermingling is frequent [START_REF] Branco | Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations[END_REF]. Such a configuration presumably prevents tangling between chromosome strands and breakage during mitosis; conversely, physical models suggest that it can be the direct result of very slow equilibration after cell division [START_REF] Rosa | Structure and dynamics of interphase chromosomes[END_REF]. Interestingly, all the different physical models explaining the behavior of Hi-C data provide a means for single chromosomes to be folded and unfolded without tangling as well [START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF][START_REF] Sanborn | Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes[END_REF][START_REF] Fudenberg | Formation of chromosomal domains by loop extrusion[END_REF]. Gene activity is correlated with nuclear location away from the bulk of chromosome territories, for example, when Hox genes are activated during PSC differentiation [START_REF] Chambeyron | Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription[END_REF], where they are proposed to be decompacted and accessible to transcriptional machinery. In line with these findings, Hi-C experiments report much stronger intrachromosomal interactions, with a high enrichment for any interchromosomal interactions to be within the active A compartment [START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF][START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF]. Specific cases of interchromosomal promoter-enhancer interactions have been reported [START_REF] Lomvardas | Interchromosomal interactions and olfactory receptor choice[END_REF][START_REF] Spilianakis | Interchromosomal associations between alternatively expressed loci[END_REF] but remain rare and controversial [START_REF] Fuss | Local and cis effects of the H element on expression of odorant receptor genes in mouse[END_REF]. More likely a large repertoire of transient or infrequent interchromosomal interactions arises from the same self-organization principles proposed to build intrachromosomal compartments. An interesting corollary of this phenomenon is that the most recurrent oncogenic translocation partners correlate with preexisting interchromosomal interactions, which may be linked to their participation in spatial gene networks [START_REF] Osborne | Myc dynamically and preferentially relocates to a transcription factory occupied by Igh[END_REF][START_REF] Rocha | Close proximity to Igh is a contributing factor to AID-mediated translocation[END_REF][START_REF] Zhang | Spatial organization of the mouse genome and its role in recurrent chromosomal translocations[END_REF], although more study is needed to address whether this link is causal or correlative.

Breaking TADs: The exceptions to the rule

As the previous parts of this chapter have described, the genome is subject to the same organizational "rules" in PSCs and differentiated cells. TAD structures appear relatively stable throughout [START_REF] Dixon | Chromatin architecture reorganization during stem cell differentiation[END_REF], and spatial gene networks linked to pluripotency transcription factors in PSCs [START_REF] De Wit | The pluripotent genome in three dimensions is shaped around pluripotency factors[END_REF][START_REF] Schoenfelder | Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome[END_REF][START_REF] Denholtz | Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization[END_REF] are replaced by different ones employing cell type-specific factors but similar principles (e.g., Klf1 [10]). However, three important cases have been discovered where chromosome structure changes drastically, each of which have a relevance in maintaining or leaving a pluripotent state.

The most dramatic genomic structural changes occur when chromosomes condense, align, and segregate during mitosis, accompanied by global transcriptional shutdown and dissociation of the majority 106 Chapter 4 Stem cell chromosome folding of regulatory factors from chromatin [START_REF] Raccaud | Transcription factor retention on mitotic chromosomes: regulatory mechanisms and impact on cell fate decisions[END_REF]. In concordance, bulk Hi-C studies in sorted mitotic cells revealed a complete loss of TADs and compartments, with the chromosomes forming linear rods composed of consecutive looped domains accompanied by a drastic reduction in long-range interactions [START_REF] Naumova | Organization of the mitotic chromosome[END_REF]. Such a configuration facilitates metaphase alignment of the chromosomes but raises questions of how epigenetic information, such as that required to maintain pluripotency, is transmitted to daughter cells. Mitotic retention of "bookmarking" factors, such as Esrrb or Sox2 in PSCs [START_REF] Deluz | A role for mitotic bookmarking of SOX2 in pluripotency and differentiation[END_REF][START_REF] Festuccia | Mitotic binding of Esrrb marks key regulatory regions of the pluripotency network[END_REF], and persistent histone modifications on chromatin likely play a role, but since even "stable" enhancer-promoter loops seem to be destroyed, it is unclear how interphase chromatin architecture is reestablished at each cell cycle. TAD organization is restored very early in G1, coincident with the moment when the replication timing of chromatin domains is established [START_REF] Nagano | Cell-cycle dynamics of chromosomal organization at single-cell resolution[END_REF][START_REF] Naumova | Organization of the mitotic chromosome[END_REF][START_REF] Dileep | Topologically associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication timing program[END_REF]. Very early G1 cells also experience a brief pulse of transcriptional hyperactivity [START_REF] Hsiung | A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition[END_REF], and it is interesting to speculate that engaged RNA polymerases facilitate chromatin architectural remodeling to the interphase state via the formation of active chromatin hubs and cotranscriptional recruitment of nucleosome remodelers and histone modifications. Recently a large catalog of single-cell Hi-C profiles allowed chromosome topology to be finely mapped according to cell cycle progression [START_REF] Nagano | Cell-cycle dynamics of chromosomal organization at single-cell resolution[END_REF]. An intriguing finding was that whereas genome compartments steadily strengthened across the cell cycle, TADs were noticeably weakened at the onset of DNA replication and stayed so throughout G2. This further supports the idea that TADs and compartments are organized by fundamentally different mechanisms, and elucidating precisely what they are and how they are altered in the cell cycle will be extremely interesting. One possibility is that a limiting amount of cohesin complexes are diverted from G1 chromosome loop extrusion to G2 chromatid cohesion, but this has yet to be shown.

Before PSCs arise in the inner cell mass, major genome structural remodeling has already taken place. Sperm chromatin is uniquely compacted, with protamines replacing the majority of histones, and fundamentally different to that of the oocyte. On fertilization, these two genomes need to become more equivalent for coordinated genetic control of the zygote and subsequent developing embryo. Recent few-or single-cell Hi-C datasets have studied chromatin topology on the transitions from oocyte to zygote and early stage embryos, with some surprising results [START_REF] Du | Allelic reprogramming of 3D chromatin architecture during early mammalian development[END_REF][START_REF] Flyamer | Single-nucleus hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition[END_REF][START_REF] Ke | 3D chromatin structures of mature gametes and structural reprogramming during mammalian embryogenesis[END_REF]. TADs appear largely absent in oocytes, with weak [START_REF] Flyamer | Single-nucleus hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition[END_REF] to no [START_REF] Du | Allelic reprogramming of 3D chromatin architecture during early mammalian development[END_REF][START_REF] Ke | 3D chromatin structures of mature gametes and structural reprogramming during mammalian embryogenesis[END_REF] presence in early zygotes, and are built up slowly on progression to the eight-cell stage [START_REF] Du | Allelic reprogramming of 3D chromatin architecture during early mammalian development[END_REF][START_REF] Ke | 3D chromatin structures of mature gametes and structural reprogramming during mammalian embryogenesis[END_REF]. Curiously an appreciable depletion of interactions between the two parental genomes is observed until the eight-cell stage too, suggesting that the genomes remain spatially segregated, and that establishing their "equivalency" is somehow linked to TAD assembly. Curiously, compartments are specifically absent in the maternal genome within early zygotes [START_REF] Flyamer | Single-nucleus hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition[END_REF], but it is unclear how or why. The onset of zygotic transcription would be a likely candidate to help build up TADs, in the same manner that has been proposed just after mitosis. However, transcriptional inhibition only had minor effects on TAD maturation in both mammalian [START_REF] Du | Allelic reprogramming of 3D chromatin architecture during early mammalian development[END_REF][START_REF] Ke | 3D chromatin structures of mature gametes and structural reprogramming during mammalian embryogenesis[END_REF] and Drosophila [START_REF] Hug | Chromatin architecture emerges during zygotic genome activation independent of transcription[END_REF] early embryos. Interestingly, DNA replication inhibition did seem to have an effect [START_REF] Ke | 3D chromatin structures of mature gametes and structural reprogramming during mammalian embryogenesis[END_REF], seemingly in contrast to the finding that S phase weakens TADs in PSCs [START_REF] Nagano | Cell-cycle dynamics of chromosomal organization at single-cell resolution[END_REF]. How these unique architectures are established and remodeled remain unclear; reprogramming the epigenetic landscape in early development has been an intensive area of study for many years, and we are beginning to have the tools to include chromosome topology in future research in this exciting field.

The third major chromosomal reorganization to have been uncovered is X chromosome inactivation. Both X chromosomes within female PSCs start off equivalent, but later in development, one copy must become predominantly heterochromatic and transcriptionally silent to balance gene dosage between the sex chromosomes and the autosomes. The epigenetic changes associated with choice of which chromosome to silence and the onset and the maintenance of X inactivation are intensively studied areas [START_REF] Finestra | X chromosome inactivation: silencing, topology and reactivation[END_REF]. Comparing chromatin topologies between the active (Xa) and the inactive (Xi) X chromosome revealed that TADs and compartments are largely absent from the Xi, although they are maintained around genes that escape X inactivation [START_REF] Darrow | Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture[END_REF][START_REF] Giorgetti | 3S 5'-TGGGTGTTTGTGACTGCAAC Whole_Sox2_8C _Rev 5'-TGAGGACTTGGCCTGGACTC Whole_Sox2_117 T_For 5'-ACGCTGGGTTTGACAGCA Anchor1R2 5'-ACATTGCCTGGATCCTCGG Anchor1S4 5'-GCCGACTGCAGAGGCAATG Whole_Sox2_117 T_Rev 5'-CCTCACTGAGCCTAAGCCTCTA RT-qPCR for expression analysis mSDHA For 5'-GCTCCTGCCTCTGTGGTTGA mSDHA Rev 5'-AGCAACACCGATGAGCCTG Oct4 For 5'-TGGAAAGCAACTCAGAGGGA Oct4 Rev 5'-TTCTGCAGGGCTTTCATGTC Nanog For 5'-AGCAGAAGTACCTCAGCCTC Nanog Rev 5'-CCGCTTGCACTTCATCCTTT Sox2 For 5'-GCTCGCAGACCTACATGAAC Sox2 Rev 5'-TGGAGTGGGAGGAAGAGGTA Sox2 (129) Rev 5'-CGCCTAACGTACCACTAGAACTTT Sox2 (CAST) Rev 5'-CGCCTAACGTACCACTAGAACTTA Actb For 5'-CTAAGGCCAACCGTGAAAAGAT Actb Rev 5'-CACAGCCTGGATGGCTACGT Myc For 5'-TACAATCTGCGAGCCAGGAC Myc Rev 5'-AAGTTCACGTTGAGGGGCAT RT-qPCR for screening CRISPR deleted clones SRR111_int_129 _F 5'-GAAAGGCAGGGAGTTGCAGT SRR111_int_R 5'-TCCCTCACACCTGAAGGGTA SRR111_int_CA ST_F 5'-AGATGAAAGGCAGGGAGTAGTG References[END_REF]. Although the controversy about this topic has been described throughout the chapter, this finding suggests that ongoing transcription may have some role in TAD generation or stability. Despite the paucity of TADs, the Xi is not devoid of higherorder structure: it segregates into two "megadomains," the function of which is not entirely clear. These megadomains are dependent on the macrosatellite DXZ4 repeat, which forms the border between them, and the non-coding RNA Xist, which is essential for X inactivation onset and maintenance [START_REF] Darrow | Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture[END_REF][START_REF] Giorgetti | 3S 5'-TGGGTGTTTGTGACTGCAAC Whole_Sox2_8C _Rev 5'-TGAGGACTTGGCCTGGACTC Whole_Sox2_117 T_For 5'-ACGCTGGGTTTGACAGCA Anchor1R2 5'-ACATTGCCTGGATCCTCGG Anchor1S4 5'-GCCGACTGCAGAGGCAATG Whole_Sox2_117 T_Rev 5'-CCTCACTGAGCCTAAGCCTCTA RT-qPCR for expression analysis mSDHA For 5'-GCTCCTGCCTCTGTGGTTGA mSDHA Rev 5'-AGCAACACCGATGAGCCTG Oct4 For 5'-TGGAAAGCAACTCAGAGGGA Oct4 Rev 5'-TTCTGCAGGGCTTTCATGTC Nanog For 5'-AGCAGAAGTACCTCAGCCTC Nanog Rev 5'-CCGCTTGCACTTCATCCTTT Sox2 For 5'-GCTCGCAGACCTACATGAAC Sox2 Rev 5'-TGGAGTGGGAGGAAGAGGTA Sox2 (129) Rev 5'-CGCCTAACGTACCACTAGAACTTT Sox2 (CAST) Rev 5'-CGCCTAACGTACCACTAGAACTTA Actb For 5'-CTAAGGCCAACCGTGAAAAGAT Actb Rev 5'-CACAGCCTGGATGGCTACGT Myc For 5'-TACAATCTGCGAGCCAGGAC Myc Rev 5'-AAGTTCACGTTGAGGGGCAT RT-qPCR for screening CRISPR deleted clones SRR111_int_129 _F 5'-GAAAGGCAGGGAGTTGCAGT SRR111_int_R 5'-TCCCTCACACCTGAAGGGTA SRR111_int_CA ST_F 5'-AGATGAAAGGCAGGGAGTAGTG References[END_REF]. Future research is likely to elucidate if and how megadomain structure facilitates chromosome-wide transcriptional shutdown and if they can be induced experimentally in other conditions.

Concluding remarks

Pluripotency requires the robust transcriptional silencing of all lineage-specific genes but in a manner allowing their efficient activation in response to differentiation signals. This challenge is met by a specialized epigenomic profile and chromatin architecture, maintaining developmental genes in a poised bivalent state. Nevertheless the fundamental principles of chromosome folding apply equally to PSCs as to differentiated cells. Gene promoters are regulated by looping interactions with distal regulatory elements, some instructive, such as the enhancer-promoter contacts at pluripotency gene loci [START_REF] Kagey | Mediator and cohesin connect gene expression and chromatin architecture[END_REF], and some permissive, in particular the PcG-regulated interactions between bivalent promoters and poised promoters [START_REF] Schoenfelder | Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome[END_REF]. These loops appear to be regulated by constitutive factors, such as CTCF, cohesin, and Mediator, with specificity also conferred by pluripotency transcription factors. As for nearly all mammalian cells in interphase, PSC genomes are spatially organized into TADs, which may modulate the specificity and robustness of enhancer activity. Loop extrusion by cohesin complexes is the most likely and conserved mechanism for TAD generation and turnover [START_REF] Sanborn | Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes[END_REF][START_REF] Fudenberg | Formation of chromosomal domains by loop extrusion[END_REF]. TADs and genes are further organized into genome compartments, which may contain spatial networks of coordinately regulated genes, such as that centered around the pluripotency genes [START_REF] De Wit | The pluripotent genome in three dimensions is shaped around pluripotency factors[END_REF]. A growing list of single-cell studies supports a self-organization model of gene compartmentalization, whereby the coassociation of similarly regulated genes is reinforced, but one particular gene pair need only coassociate in a small subset of the cell population. Future research to elucidate the mechanisms regulating chromatin topology and dynamics promise to give groundbreaking insight into how gene programs are regulated and will be extremely fruitful in understanding the pluripotent epigenetic state.

A static view of chromatin architecture

Over the past decades, many studies have assessed the spatial proximity and nuclear organization of specific genomic loci, using microscopic techniques, such as fluorescent in situ hybridization (FISH) [START_REF] Giorgetti | Closing the loop: 3C versus DNA FISH[END_REF][START_REF] Mccord | Chromosome Conformation Capture and Beyond: Toward an Integrative View of Chromosome Structure and Function[END_REF], or molecular biology techniques, such as variants of chromosome conformation capture (3C) (Dekker et al., 2002), adapted to high-throughput sequencing (Hi-C) (Lieberman-aiden et al., 2009). Collectively, these studies demonstrated a correlation between chromatin topology and underlying gene activity. 3C-based approaches provide genome-wide contact maps, usually performed on populations of millions of cells, generating snapshots of the population and thus complementary methods are needed to investigate heterogeneity or variability at the single-cell level.

The 3C-based methods involve formaldehyde crosslinking, followed by ligation of the overhangs generated by digestion with a restriction enzyme and determination of pair-wise contacts using either PCR or sequencing (Denker & De Laat, 2016;[START_REF] Wit | A decade of 3C technologies-insights into nuclear organization[END_REF]. The number of detected ligation products is a measure of how often the two corresponding genomic sequences were in close physical proximity, in a cell population and at the time point of the fixation. Depending on the biological question being asked, chromosome conformation capture-based approaches can be divided into "one-to-one" (3C) (Dekker et al., 2002), "oneto-all" (circular chromosome conformation capture combined with high-throughput sequencing (4C-seq)) (Van De Werken et al., 2012), "many-to-many" (5C [START_REF] Dostie | Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements[END_REF], ChIA-PET [START_REF] Fullwood | An oestrogen-receptor-α-bound human chromatin interactome[END_REF], Capture-HiC (Hughes et al., 2014)) and "all-to-all" (highthroughput chromosome conformation capture (Hi-C) (Lieberman-aiden et al., 2009)) (Figure 15). After a reverse crosslinking step, different approaches can be used to detect chromatin interactions. For example, the 4C-seq protocol entails a second round of digestion and religation is utilized, followed by inverse PCR with specific primers to detect all of the chromatin interactions involving a specific locus of interest (the "bait") (Simonis et al., 2006;Van De Werken et al., 2012). However, the crosslinking and ligation steps used in 3C-based approaches can potentially produce biased results. For that purpose, ligation-free GAM (genome architecture mapping) [START_REF] Beagrie | Complex multi-enhancer contacts captured by genome architecture mapping[END_REF] and crosslinking-and ligation-free DamC [START_REF] Redolfi | DamC reveals principles of chromatin folding in vivo without crosslinking and ligation[END_REF] techniques have been developed, both methods showing good agreement with 4Cseq and Hi-C data. Microscopy-based methods, such as DNA FISH, have largely confirmed 3C-based findings [START_REF] Bintu | Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells[END_REF][START_REF] Cardozo Gizzi | Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms[END_REF][START_REF] Finn | Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization[END_REF][START_REF] Giorgetti | Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription[END_REF][START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation centre[END_REF]. However, DNA FISH and 3C are conceptually different techniques that provide essentially different and complementary types of information [START_REF] Giorgetti | Closing the loop: 3C versus DNA FISH[END_REF]. DNA FISH provides a powerful means to directly determine the actual positions, distribution, 3D distances between genomic loci, as well as their position relative to TF/Pol II foci (when coupled with immunostaining) inside single cells, an inaccessible information in 3C-based experiments [START_REF] Giorgetti | Closing the loop: 3C versus DNA FISH[END_REF]. Nevertheless, conventional FISH is limited to probing few candidate loci, due to the restricted number of spectrally distinguishable fluorophores that can be simultaneously imaged by standard microscopes. Additionally, FISH probes are designed for each target region of interest and can thus detect only few genomic loci in parallel. These limitations constrain the genomic coverage achievable by DNA FISH.

Although 3C-related techniques remain much superior in coverage, FISH has nonetheless uncovered many fundamental principles of genome organization, such as the preferential association between similarly regulated genes [START_REF] Schoenfelder | Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells[END_REF].

While these methods have provided major insights into 3D genome organization, recent advances using single-cell biochemistry [START_REF] Cattoni | Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions[END_REF][START_REF] Flyamer | Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition[END_REF][START_REF] Nagano | Single-cell Hi-C reveals cell-to-cell variability in chromosome structure[END_REF][START_REF] Stevens | 3D structures of individual mammalian genomes studied by single-cell Hi-C[END_REF] and super-resolution microscopy on fixed cells [START_REF] Beliveau | Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes[END_REF][START_REF] Bintu | Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells[END_REF][START_REF] Cattoni | Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions[END_REF][START_REF] Finn | Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization[END_REF][START_REF] Mateo | Visualizing DNA folding and RNA in embryos at single-cell resolution[END_REF][START_REF] Nir | Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling[END_REF][START_REF] Su | Genome-Scale Imaging of the 3D Organization and Transcriptional Activity of Chromatin[END_REF][START_REF] Szabo | TADs are 3D structural units of higher-order chromosome organization in Drosophila[END_REF]S. Wang et al., 2016), have demonstrated high variability of chromatin conformations between individual cells.

Results from these single-cell methods confirmed compartmentalization (S. [START_REF] Wang | Spatial organization ofchromatin domains and compartments in single chromosomes[END_REF] and the physical existence of TADs, initially characterized by population-average Hi-C, albeit with high spatial heterogeneity [START_REF] Bintu | Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells[END_REF][START_REF] Mateo | Visualizing DNA folding and RNA in embryos at single-cell resolution[END_REF][START_REF] Szabo | TADs are 3D structural units of higher-order chromosome organization in Drosophila[END_REF]S. Wang et al., 2016), formed by a plethora of low-frequency interactions rather than one stable chromatin loop [START_REF] Cattoni | Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions[END_REF][START_REF] Finn | Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization[END_REF][START_REF] Giorgetti | Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription[END_REF]) (Figure 16). Interestingly, the boundaries are highly variable across cells and depletion of cohesin, which ablates TADs at the population level [START_REF] Rao | Cohesin Loss Eliminates All Loop Domains[END_REF], did not diminish TAD-like structures in individual cells [START_REF] Bintu | Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells[END_REF]. The relatively low-frequency interactions in the population and the occurrence of diverse conformations of the chromatin fiber in individual cells suggest the coexistence of a wide spectrum of genome configurations across a cell population [START_REF] Finn | Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization[END_REF], in agreement with previous physical models which have demonstrated that interaction maps are the average of multiple diverse genome conformations [START_REF] Giorgetti | Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription[END_REF][START_REF] Kalhor | Genome architectures revealed by tethered chromosome conformation capture and population-based modeling[END_REF]. Overall, Hi-C and imaging studies on fixed cells have uncovered a wealth of information on chromatin architecture, but the emerging models to explain links between topology and genome control all imply a certain dynamic nature of the underlying chromatin fiber to adopt transient and metastable conformations.

Chromatin dynamics

Visualizing DNA in living eukaryotic cells Whereas chromosomal structures have been deeply characterized in the last years [START_REF] Bonev | Multiscale 3D genome rewiring during mouse neural development[END_REF][START_REF] Hsieh | Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding[END_REF][START_REF] Krietenstein | Ultrastructural Details of Mammalian Chromosome Architecture[END_REF]Rao et al., 2014;[START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF] there remains a knowledge gap in how these structures alter over time, despite growing appreciation that chromatin dynamics likely play key roles in transcriptional regulation.

Technical and analytical hurdles have, until relatively recently, limited assessment of locusspecific gene mobility. The first generation of locus-tagging methods entailed integration of multiple tandem copies of repetitive lacO sequences, bound by ectopic fluorescent-tagged lac repressor (W. F. [START_REF] Marshall | Interphase chromosomes undergo constrained diffusional motion in living cells[END_REF][START_REF] Robinett | In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition[END_REF], but these very large constructs (~10 kb) are laborious to clone and may alter the chromatin state of the tagged locus (M.

Dubarry, Loïodice, Chen, Thermes, & Taddei, 2011) (Figure 17A). Due to technical difficulties in targeting the lacO constructs to specific regions, the first chromatin labelling experiments only showed general principles of their dynamics in metazoan nuclei: constrained and anomalous movement within a restricted volume, and mobility generally reduced even further in heterochromatin, consistent with results on super-resolution studies of chromatin mobility by tagging histones [START_REF] Nozaki | Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging[END_REF]. Subsequently, several editing-free systems such as fluorescent zinc finger proteins or TALEs (transcription activator like-effectors) [START_REF] Ma | Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors[END_REF][START_REF] Miyanari | Live visualization of chromatin dynamics with fluorescent TALEs[END_REF] (Figure 17B) and CRISPR-dCas9 (catalytically dead Cas9) (B. [START_REF] Chen | Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system[END_REF][START_REF] Ma | Multicolor CRISPR labeling of chromosomal loci in human cells[END_REF] (Figure 17C) constructs were designed to directly bind the endogenous sequences of interest, but it has been very challenging to obtain robust signals outside of repetitive heterochromatic sequences.

More recent approaches have proposed alternative ways to overcome the limitation of repetitive loci. [START_REF] Wang | CRISPR-mediated live imaging of genome editing and transcription[END_REF] developed CRISPR LiveFISH, an approach that deploys Cy3-labeled sgRNAs assembled with dCas9-EGFP (enhanced GFP) ex vivo as fluorescent ribonucleoproteins (fRNPs), which are delivered to cells by electroporation and allow labelling of genomic sequences in living cells. Importantly, the signal-to-noise-ration (SNR) of gRNA was 4-fold higher than that of dCas9-EGFP and gRNA did not accumulate in the nucleolus, unlike dCas9-EGFP. While this approach has a great potential for real-time visualization of DNA and RNA transcripts in living cells, through the combination of Cas9 and Cas13 (see below), it has only been shown to work at repetitive regions so far (Figure 17C).

Chimeric array of gRNA oligonucleotides (CARGO) coupled with dCas9, is another noninvasive strategy that overcomes the issue of SNR through the use of 12 gRNAs which recruit 12 dCas9-EGFP molecules to a genomic region spanning around 2 kb [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF] (Figure 17C). Interestingly, CARGO-dCas9 allows imaging of non-repetitive loci contrary to classical CRISPR-dCas9 or CRISPR LiveFISH methods. An alternative approach for imaging non-repetitive regions in living cells is the CRISPR-Tag system (B. Chen, Zou, Xu, Liang, & Huang, 2018) in which a repeating DNA tag, with a minimal size of 250 bp, is inserted near the locus of interest. The integrated repeat sequence are binding sites for four sgRNAs (Figure 17C). Instead of the standard dCas9-EGFP, dCas9 is fused with 14 copies of GFP11 tags (dCas9-GFP 14x ), a non-fluorescent fragment of GFP that upon complementation with GFP1-10 becomes a functional fluorescent protein [START_REF] Kamiyama | Versatile protein tagging in cells with split fluorescent protein[END_REF]. CRISPR-Tag system represents the shortest available DNA tag, and thus is less likely to interfere with the structure and/or function of the target locus. Nevertheless, binding of Cas9 to the DNA implies DNA unwinding, which might affect the localization of histones [START_REF] Sternberg | DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[END_REF]. Moreover, another disadvantage of the system is the fact that GFP1-10 has to be expressed exogenously.

The ANCHOR/ParB DNA labeling system is an alternative to the previous approaches. The system is based on the kinetochore-like nucleoprotein complexes, whose function is to ensure mitotic stability of bacterial chromosomes and large plasmids (N. [START_REF] Dubarry | ParABS systems of the four replicons of Burkholderia cenocepacia: New chromosome centromeres confer partition specificity[END_REF][START_REF] Passot | Centromere binding and evolution of chromosomal partition systems in the Burkholderiales[END_REF][START_REF] Sanchez | Stochastic Self-Assembly of ParB Proteins Builds the Bacterial DNA Segregation Apparatus[END_REF]. ANCHOR entails introduction of a short (~1kb), non-repetitive DNA sequence (ANCH or parS) to which fluorescently-tagged OR (bacterial partition protein or ParB) bind site-specifically [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF]. Then, ORs self-oligomerize via N-terminal protein-protein interaction and recruit further OR dimers which bind non-specifically and relatively weakly to adjacent DNA and spread along chromatin to produce a robust signal (Figure 17A). These features, together with the small size of the binding site, allow the insertion of the ANCH sequence immediately adjacent to regulatory elements with minimal perturbation of endogenous chromatin [START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF].

Importantly, orthologous parS/ParB pairs allow for simultaneous double-label experiments [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF][START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF].

Visualizing RNA in living eukaryotic cells

Recent advances in live-imaging methods have enabled the direct visualization of nascent transcripts at single-cell resolution. The MS2-MCP system has been largely used to visualize single RNAs in real time in living cells [START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF][START_REF] Fusco | Single mRNA Molecules Demonstrate Probabilistic Movement in Living Mammalian Cells associated with localized mRNA and are required for their movements [5-8]. In yeast, ASH1 mRNPs are asso-ciated with a specific myosin motor, and their move[END_REF][START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Golding | Real-time kinetics of gene activity in individual bacteria[END_REF][START_REF] Larson | Real-time observation of transcription initiation and elongation on an endogenous yeast gene[END_REF][START_REF] Yunger | Single-allele analysis of transcription kinetics in living mammalian cells[END_REF]. Transcripts are engineered to encode a repetition of 24 19-bp RNA stem-loops that are recognized in vivo by a fluorescently-tagged bacterial coat protein MS2 [START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF][START_REF] Fusco | Single mRNA Molecules Demonstrate Probabilistic Movement in Living Mammalian Cells associated with localized mRNA and are required for their movements [5-8]. In yeast, ASH1 mRNPs are asso-ciated with a specific myosin motor, and their move[END_REF][START_REF] Yunger | Single-allele analysis of transcription kinetics in living mammalian cells[END_REF] (Figure 18A). These RNA-protein interactions have been thoroughly studied, enabling the fine-tuning of the technique for different applications. However, a common concern is whether genetic insertion of MS2 aptamers into gene loci can affect RNA structure, expression or function. Other methods using fluorogenic RNA aptamers, such as Spinach [START_REF] Paige | RNA Mimics of Green Fluorescent Protein[END_REF] and Broccoli [START_REF] Filonov | Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution[END_REF], provide background-free signals but have been mostly applied in bacteria. Molecular beacons, whose fluorescence increases after target binding, have also been used to visualize endogenous, untagged RNA transcripts (M. [START_REF] Chen | A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level[END_REF]. Nevertheless, their use is costly and requires their exogenous supply to the cells. CRISPR-Cas13 is a newly identified RNA-programmable and RNA-binding RNase protein family and catalytically inactive Cas13s (dCas13s) fused to a fluorescent protein have been used for precise RNA targeting and imaging in living mammalian cells [START_REF] Abudayyeh | RNA targeting with CRISPR-Cas13[END_REF][START_REF] Cox | RNA editing with CRISPR-Cas13[END_REF][START_REF] Yang | Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems[END_REF] (Figure 18B). dLwaCas13a has been initially used to label the abundant ACTB mRNA after stress [START_REF] Abudayyeh | RNA targeting with CRISPR-Cas13[END_REF], although a latter study proposed dPspCas13b and dPguCas13b to be the most efficient dCas13s that allow robust (comparable signals to MS2-MCP) and rapid real-time RNA tracking in living cells [START_REF] Yang | Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems[END_REF]. In the same study, application of orthogonal dCas13s or combination of dCas13 and MS2-MCP allowed simultaneous dual-color label of RNAs, while combination of dCas13 with dCas9 enabled RNA-DNA visualization in living cells. While this tool has not been yet applied in a wide range of studies, its high efficiency, robustness and the absence of genetic manipulations offer great promise.

Single-gene dynamics during transcription

Recent tagging of individual genes and enhancers has given seemingly conflicting views on the effect of transcription on chromatin dynamics. On the one hand, transcription coincided with reduced mobility and confinement to smaller nuclear volume [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Ochiai | Simultaneous live imaging of the transcription and nuclear position of specific genes[END_REF]. In particular, Germier et al. [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF] combined the ANCHOR3 DNA-labeling system with MS2-labeled mRNAs to study the motion of an estrogen-inducible CyclinD1 transgene in human cells before and after the appearance of MCP-EGFP-labeled mRNAs in the same cell. Analysis of the motion of the ANCHOR3-tagged locus before and after stimulation by E2 (17 β-estradiol) showed that while the motion of the transgene domain remained sub-diffusive in the absence of transcription, transcription initiation by RNA Pol II rapidly confined the mRNA-producing gene.

Interestingly, confinement was maintained upon treatment of the cells with the transcriptional elongation inhibitor DRB (5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole). In contrast, addition of the transcriptional initiation inhibitor, triptolide, to the transcriptionally stimulated cells released locus confinement. These results suggest that initiating but not elongating RNA Pol II induces confinement of chromatin, consistent with engagement of the gene within a restricted nuclear transcriptional hub. However, in other experiments, genes and enhancers activated upon differentiation were found to move faster, and were slowed on treatment with drugs inhibiting transcriptional initiation or elongation [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF][START_REF] Nozaki | Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging[END_REF].

Gu et al. [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF] applied the CARGO-dCas9 imaging system to monitor the dynamics of the Fgf5 enhancer and promoter in mESCs and two days after inducing differentiation to epiblast-like cells (mEpiLCs), where the enhancer is activated and Fgf5 is induced [START_REF] Buecker | Reorganization of enhancer patterns in transition from naive to primed pluripotency[END_REF]. Their results demonstrated that both enhancer and promoter mobility increases in the mEpiLC state and since the Fgf5 is activated during mESCs to mEpiLCs differentiation they proposed that transcriptional activation increases locus motion. In contrast to the results provided by Germier et al. [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF], these authors reported that acute perturbation of RNA Pol II activity by DRB and flavopiridol, to target transcriptional elongation, or triptolide, to inhibit transcriptional initiation, significantly reduced the mobility of the locus.

Collectively, the increased motion of the locus was interpreted as an increase in thermal energy via the active transcription process, which may aid gene search for regulatory sequences within the TAD, rather than the formation of stable enhancer-promoter loops. Interestingly, an earlier study has reported that chromatin dynamics tend to increase during early mESC differentiation [START_REF] Masui | Live-cell chromosome dynamics and outcome of X chromosome pairing events during ES cell differentiation[END_REF]. In order to distinguish whether these observations were linked to the activity status of the cis-regulatory elements or due to differences in global chromatin organization between mESCs and mEpiLCs, the authors [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF] performed CARGO-dCas9 imaging at the Tbx3 locus, where the gene becomes down-regulated during mESC to mEpiLC transition. Imaging of Tbx3 promoter and distal super-enhancer revealed changes in mobility that were opposite to those detected at the Fgf5 locus. Nevertheless, it has to be noted that nascent mRNA was not measured in this study [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF] , thus the locus is assumed as always being transcriptionally active at the mEpiLC state, yet without having evidence for mRNA production in the analyzed cells.

Notwithstanding, Neumann et al. [START_REF] Neumann | Targeted INO80 enhances subnuclear chromatin movement and ectopic homologous recombination[END_REF] measured the movement and the transcriptional output of tagged genomic loci in budding yeast, to which transcriptional activators were targeted. According to the study, local targeting of the VP16, but not of the Gal4 acidic domain, increased chromatin mobility and this increase in motion did not correlate with the activity of elongating RNA Pol II. The authors proposed that local chromatin remodeling and nucleosome eviction, depended on the INO80 remodeler, rather than transcriptional activity drive chromatin mobility in yeast. Together, the link between chromatin dynamics and transcription largely remains unclear, not least because so few loci have been assessed.

Global chromatin diffusion dynamics

As illustrated by single particle tracking (SPT), interphase chromatin undergoes diffusive motion within the nucleus, however this motion is constrained such that a given segment of chromatin is free to move within a small subregion of the nucleus (W. F. [START_REF] Marshall | Interphase chromosomes undergo constrained diffusional motion in living cells[END_REF].

The constrained diffusion of the chromatin fiber is functionally important, since it enables a locus to explore for potential interaction partners: for example, for an enhancer to encounter its target promoter or for multiple genes to cluster into transcriptional hubs (discussed earlier in the introduction). Generally speaking, the simplest model to describe the diffusion of microscopic systems is Brownian motion, whereby movements are caused by random collisions of any particle of interest with neighboring smaller particles within the system. However, as a bulky and extremely long polymer interacting with the crowded nuclear environment, chromatin frequently displays sub-diffusive behavior, with more limited movement than for classical Brownian motion [START_REF] Bancaud | Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin[END_REF][START_REF] Tortora | Chromosome dynamics during interphase: a biophysical perspective[END_REF]. Therefore, the mean squared displacement (MSD) of chromatin is expected to follow this relationship with time: <(r(t) -r(0)) 2 > = 2nDt α , with r(t) the n-dimensional vector representing the position of particles in time and < . > the time-average over one trajectory or the ensemble-average over many trajectories.

Two parameters thus describe the diffusion properties of chromatin: the apparent diffusion coefficient D, indicating the speed of motion of a particle in a given amount of time, and the anomalous diffusion co-efficient α, defined in the range 0< α <2, which points to the type of diffusion undertaken by the particle of interest α<1 ( sub-diffusive behavior, with smaller values indicating greater constraint); α=1 (pure Brownian diffusion); α>1 ( a more directed type of motion) (Figure 19). Nonetheless, imaging chromatin within the crowded nuclear environment might be challenging, thus novel approaches, such as Dense Flow reconstruction and Correlation (DFCC) [START_REF] Shaban | Formation of correlated chromatin domains at nanoscale dynamic resolution during transcription[END_REF] and High Diffusion Mapping (Hi-D) [START_REF] Shaban | Hi-D: Nanoscale mapping of nuclear dynamics in single living cells[END_REF], have been developed to supplement SPT for studying bulk chromatin motion, although these are not adapted for the study of specifically tagged loci.

Very recently, a collaboration between my group and the lab of Nacho Molina resulted in the development of a tool for the precise measurement of diffusive properties of such tagged chromatin loci, finding that local dynamics can significantly vary with genomic context [START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF]. However, the dynamics of promoters and enhancers relative to transcriptional status was not stud

Aims of Study

Population-average "C" methods and DNA FISH support a model of chromatin loops bringing individual enhancers into contact with target genes, but this is challenged by recent findings showing enhancers at large distances from activated genes. Furthermore, it has become clear that static views of chromatin architecture are insufficient to fully understand dynamic processes such as transcription, or even maintenance of the architectures themselves. Despite growing anecdotal evidence that chromatin mobility varies with gene locus and transcriptional activity, very little is known about the "rules" governing chromatin dynamics, nor how they may affect gene regulation.

In this thesis I aimed to visualize enhancer-promoter communication in real time, coupled to labelling of nascent RNA, and to characterize chromatin dynamics at these loci. For this reason, I have adapted the ANCHOR/ParB technology, and have optimized the system for CRISPR/Cas9 knock-in of double labels into mouse embryonic stem cells. As a model for my experiments I used the locus around the pluripotency gene Sox2 and tagged its endogenous promoter and distal (>100 kb downstream) enhancer (SCR). The real-time behavior of these components were dissected in response to a battery of different perturbations, such as deletions of critical regulatory elements within the SCR, pharmacological inhibition of transcription and cell differentiation, in order to see whether and how promoter-enhancer distances and/or diffusive properties linked to transcriptional output.

Results

ANCHOR system allows visualization of specific loci without perturbing genome function

To assess enhancer-promoter proximity in real-time, we engineered ANCHOR (parS) tags [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF] at the promoter and distal enhancer (SCR (Sox2 control region); (Zhou et al., 2014)) of Sox2 in mouse embryonic stem cells. To ensure incorporation of the two tags on the same allele, we used the M. musculus/M. castaneus hybrid F1 ESC line [START_REF] Mlynarczyk-Evans | X chromosomes alternate between two states prior to random X-inactivation[END_REF] and performed allele-specific knock-in of ANCH1 and ANCH3 tags with CRISPR/Cas9 at sites where SNPs result in the PAM only being present on the musculus allele. Due to the small size (<1 kb) of the ANCH sequences, we were able to incorporate a tag fully within the SCR, between the elements (SRR; Sox2 regulatory region) containing the hallmarks of enhancer activity (Zhou et al., 2014) S2a,b). Virtual 4C plots derived from the high-resolution Hi-C map of ESCs (Bonev et al., 2017) confirm the Sox2-SCR interaction predicted from previous 3C and 4C studies (de Wit et al., 2013;Zhou et al., 2014), and show that the control tagged regions are not expected to form interactions (Fig S2a). Introducing ectopic sequences so close to regulatory regions could potentially interfere with their normal functioning, particularly if the sequences are tightly bound by proteins (M. [START_REF] Dubarry | Tight protein-DNA interactions favor gene silencing[END_REF], although ANCHOR was previously shown to be minimally invasive in yeast [START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF]. We confirmed that the ANCHOR system has no detectable effects on promoter and/or enhancer functioning in ESCs by various assays. Firstly, qRT-PCR showed that expression of pluripotency marker genes Nanog and Pou5f1 (Oct4), as well as Sox2 itself, is unaffected by incorporation of the ANCH sequences, even when the binding OR proteins are also introduced ( Fig

Enhancer-promoter proximity is accompanied by greater constraint of chromatin regulatory elements

To assess enhancer-promoter interaction dynamics in living cells, we performed spinning disc confocal microscopy on ANCH-labelled ESCs, obtaining images every 500 ms for ~2-3 minutes. We observed a consistent proximity between enhancer and promoter, but with fluctuations in their exact spatial arrangement and direct juxtaposition ( However, we note a high heterogeneity of inter-label distances between cells, as previously reported [START_REF] Finn | Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization[END_REF], and that the control labels are also rather close, as may be expected for a genomic separation of ~115 kb.

A previous study reported much larger distances between Sox2 and its downstream enhancer (mean 339 nm, compared to mean 165 nm that we observed) when they were labelled with large repeats of tetO and cuO sequences, suggesting that promoter-enhancer proximity was not required for transcriptional activation (Alexander et al., 2019). To investigate further, we generated a new F1 ESC line with ANCH labels integrated at the exact same positions as Numbers in italics denote the q-value for pairwise comparisons within the Sox2 locus; for the HoxA loci, numbers in red indicate the q-value for pairwise comparison with Sox2 and numbers in green indicate the q-value for pairwise comparison with SCR (Wilcoxon rank sum test, with

Benjamini-Hochberg multiple testing correction). d) Violin plots for apparent diffusion speed distributions for Sox2 (shades of red), SCR (shades of green), and control loci within the Sox2 locus (light grey) or the HoxA locus (grey), exactly as for c (n.s. denotes non-significant; q > 0.05).

the tetO/cuO sites (Sox2 site 3 kb further upstream of the promoter than our insertion site; SCR site outside and a further 9 kb downstream; We propose therefore that the difference in reported inter-label distances in our results and those of the previous study are due to technical differences, perhaps as a result of using the large repetitive tet/cu operators. In particular, the previous study also reported a rather large distance (mean ~250 nm) between the two operators when their genomic separation was reduced to ~14 kb by a large deletion in the intervening region (Alexander et al., 2019), around double the average distances we observe in the wild-type state.

As well as assessing how physical distances fluctuate with time, the relatively high temporal resolution of our movies also allows us to measure the local diffusive properties of the chromatin at the different tagged loci. We previously reported that apparent diffusion speed (Dapp) and the anomalous diffusion parameter (α) can significantly vary between different chromatin loci [START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF], although the underlying principles determining these differences were unclear. We applied GP-FBM (Gaussian Processes applied to Fractional Brownian Motion; [START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF] to measure Dapp and α, and found that both the Sox2 promoter and enhancer are significantly more constrained than control regions within the locus (Fig 22c). Further, the Sox2 promoter is significantly more constrained than the SCR. The measurements of α are virtually identical between the two Sox2 ANCH integration sites and between the two SCR ANCH integration sites (Fig S4b). In a previous study, we measured the local chromatin diffusive properties of three loci near the HoxA locus, finding one locus (termed T1), which is <15 kb from a putative enhancer, to be significantly more constrained than the other two (T2 and T3) in ESCs [START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF]. The Sox2 promoter and enhancer are significantly more constrained than all of the HoxA regions, and the control regions within obvious links to underlying chromatin state. Within the Sox2 locus, the SCR had the highest diffusive speed (median 2.1 μm 2 /s) and the control region between the gene and enhancer had the lowest (median 0.9 μm 2 /s); diffusive speeds were very similar between Sox2 and the downstream control region, as well as to the control loci within the HoxA locus, in between these two extremes. Notably, whereas the intervening control region diffuses significantly more slowly than all other tested loci, the faster measured diffusion speed of the SCR is not statistically significant for all other compared loci. Further, whereas the Sox2 diffusion speeds were essentially identical for the two alternative ANCH labels, there was a very large discrepancy between measurements for the two SCR labels: the label precisely within the SCR had an overall higher diffusion speed than the other loci, whereas the label 9 kb downstream had a much slower diffusion rate (median 0.7 μm 2 /s), statistically similar to the intervening control region (Fig S4c). Overall, active chromatin elements, such as promoters and enhancers, appear to have more constrained chromatin motion than nearby, inactive regions within the same locus or topologically associated domain, and their relatively shorter separation distances reflect chromosome conformation capture data. Apparent diffusion speeds can also vary with genomic locus, but there is no obvious direct link to underlying transcriptional or regulatory activity.

Sox2 inactivation during differentiation coincides with reduced chromatin constraints

In our previous study of the HoxA locus, local chromatin diffusive parameters were altered when differentiation was induced with retinoic acid [START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF]. We similarly assessed the effects of in vitro differentiation on enhancer-promoter proximity and chromatin dynamics at the Sox2 locus. As expected, removal of LIF (leukemia inhibitory factor) and 2i

(a combination of two kinase inhibitors which maintain ESCs in a ground pluripotency state; [START_REF] Ying | The ground state of embryonic stem cell self-renewal[END_REF]) from the medium, as well as supplementing with retinoic acid, causes rapid In agreement with the 4C results, the average promoter-enhancer distances are unaltered at day These experiments will be imminently performed on the cell line with labelled control probes, to dissect effects of gene inactivation from potential consequences of cell differentiation on chromatin in general.

Promoter diffusive speed is directly modulated by transcriptional initiation

To directly assess the effect of transcription on chromatin dynamics and promoter-enhancer interactions, we performed live imaging experiments on ANCH-labelled ESCs after treatment with inhibitor drugs: triptolide, which covalently binds to XPB subunit of TFIIH and mediates proteasomal degradation of promoter-bound RNA polymerase II before initiation, and flavopiridol, which inhibits CDK9 subunit of P-TEFb and hence phosphorylation of serine-2 of the RNA polymerase II C-terminal domain and subsequent transcriptional elongation [START_REF] Bensaude | Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity?[END_REF]. We incubated the cells with either drug (or DMSO control) under conditions specifically causing large reduction in mRNA levels of short-lived species, such as Myc or Sox2, without affecting levels of longer-lived mRNA species such as Actb (Fig S5a,b; Table S1), thus ensuring as far as possible treatments that acutely and efficiently block transcription with minimal pleiotropic effects. As may be expected, inhibitor treatments of the cells labelled both promoters and enhancers [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF]. Overall, most locus-and differentiation stagedependent differences we observed in chromatin dynamics and promoter-enhancer proximity appear to only be partially explained by acute transcriptional processes. Conversely, RNA polymerase II engagement and/or promoter melting at transcriptional initiation appears to directly increase apparent diffusive speed at gene promoters.

Enhancer mutations link gene expression to local diffusive properties but not promoter proximity

Despite carefully controlled conditions, transcriptional inhibitor drugs may still have nonspecific or pleiotropic effects. To more precisely assess the specific links between Sox2 25d,e). Conversely, the Sox2 promoter has a significant slowing of diffusion speed in both deletion lines and reduced constraint, specifically in the compound deletion of SRR107 and SRR111. These results reinforce the link between local promoter diffusion speed, constraint of motion and transcription, but also show an apparent uncoupling with diffusive parameters at the spatially proximal enhancer, and that enhancers and promoters appear to remain close even when transcription is reduced.

Transcribing and non-transcribing alleles have similar chromatin dynamics

The results so far suggest that gene activity somehow has a direct but rather complex effect on local chromatin architecture and dynamics. To directly assess if and how transcribing loci differ from non-transcribing genes, we integrated 24 copies of the MS2 stem loop repeat into the 3'UTR of the musculus Sox2 allele in ANCH-labelled F1 ESCs, allowing allele-specific transcripts to be visualized by binding of mScarletI-labelled MCP (see Methods; [START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF]). Triple-label live imaging thus allowed Sox2 and SCR dynamics and proximity to be simultaneously tracked with gene transcription (Fig 26). A potential limitation of the MS2-MCP system is that the stem loops may disrupt expression via effects on transcription or downstream processes, such as mRNA stability or translation. This is a special concern for the Sox2 gene, since it lacks introns and MS2 repeats can only be inserted into the 3'UTR, linked to regulation of polyadenylation, mRNA stability and translation efficiency. Indeed, a similar MS2-MCP setup in the mouse Sox2 locus has already been reported to heavily reduce Sox2 protein levels (Alexander et al., 2019). We assayed Sox2 expression in our own F1 ESC line regardless of the MS2 tag. However, qRT-PCR and western blot results show an appreciable loss of steady-state Sox2 mRNA (both at the tagged musculus and unlabeled castaneus alleles) and protein levels (Figs S7a,b). The maintained levels of nascent transcripts determined by single-molecule RNA FISH suggests that expression effects are predominantly posttranscriptional, reducing mRNA stability and/or translation efficiency, but since Sox2 protein regulates its own expression [START_REF] Chew | Reciprocal Transcriptional Regulation of Pou5f1 and Sox2 via the Oct4/Sox2 Complex in Embryonic Stem Cells[END_REF], and untagged mRNA levels are slightly reduced (Fig S7a), a slight inhibition of transcription compared to wild-type levels is likely.

Notably, the previously published Sox2-MS2 system included a selectable marker gene (Alexander et al., 2019), which could further perturb transcription and mRNA stability. Our strategy for MS2 insertion also includes a hygromycin-resistance selection marker, but this is flanked by loxP sites and excised before imaging experiments (see Methods). We note that the line maintaining the selection marker has equivalent steady-state mRNA levels as measured by qRT-PCR, but ~50% nascent transcription as measured by single-molecule FISH and even more greatly reduced protein levels (Figs S7a-c).

We performed triple-label live imaging experiments, obtaining images every 3 s for ~10 minutes (Fig 26c). This setup provided sufficient temporal resolution for GP-FBM to robustly measure diffusive parameters, while spanning a long enough period for transcriptional bursts, which have a typical duration of a few minutes [START_REF] Tunnacliffe | What Is a Transcriptional Burst?[END_REF], to be detected. The imaging setup required for sufficiently rapid ANCHOR spot detection is incompatible with three-dimensional tracking, so MCP foci indicative of genes undergoing transcriptional bursting were frequently lost for sporadic intervals on movement out of the imaged focal plane. However, on visual inspection of frames with/without MCP detection, it was relatively easy to resolve bursting and non-transcribing events (Fig 26d). In total, ~25% of all frames had a detectable MCP signal, and of 54 imaged cells, 28 (52%) had defined bursting events, with a median duration of ~4.5 minutes. Since a large number of the bursts had already started at the onset of image acquisition, or had not yet finished when acquisition was stopped, this duration is likely an underestimate, but is within the expected range reported for other transcriptional bursting studies [START_REF] Tunnacliffe | What Is a Transcriptional Burst?[END_REF]. Although greater numbers of movies are required to improve statistical confidence, this measured bursting frequency is much higher than reported in a previous study (~2/3 cells do not demonstrate bursting within a 30 min window; the vast majority of the rest have <20% frames with MCP detection; Alexander et al., 2019), further suggesting that the labelled system within Anchor_Sox2-MS2 better represents active Sox2 loci.

We extracted subsets of movies comprising the called bursting events (19 of sufficient duration for accurate measurement of diffusive parameters) and prolonged periods of absence of transcription (24; minimum duration 4.9 minutes), and performed GP-FBM to compare promoter-enhancer proximity and local chromatin diffusive properties between transcribing and non-transcribing loci. Although insufficient movies have so far been acquired to obtain statistical significance, Sox2 and SCR are slightly closer when the gene is actively transcribing (Fig 27a). Indeed, when comparing transcribing and non-transcribing distributions to the total distribution from the double-label experiments, the transcribing subset of Sox2 loci have significantly closer Sox2-SCR distances, suggesting that at least for this locus, physical promoter-enhancer juxtaposition is linked to transcriptional activation. For the triple-labelled cell lines, both the Sox2 promoter and the SCR had less constrained movement and slower diffusive speed than for the non-MS2-tagged cell line (Figs S7d-e 24b,c).

Overall, chromatin diffusive properties is locus-specific and appears to be linked to underlying activity. Discussed in more detail in the following section, regulatory regions tend to have more constrained motion, and promoters have greater apparent diffusive speeds when active, without necessarily being coupled to changes in promoter-enhancer spatial separation. ,e.

Table S1: Half-lives of mRNA species for which qRT-PCR was performed after treatment with transcriptional inhibitor drugs. Values taken from [START_REF] Sharova | Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells[END_REF]. Scatterplots showing lack of correlation between inter-probe distance and either anomalous diffusion coefficient or apparent diffusive speed, at either Sox2 or SCR in Anchor_Sox2_SCR

ESCs, or at either control region in Anchor_Control ESCs.

Figure A3

Median inter-probe distance (μm)

Revisiting enhancer looping models

As has been discussed earlier (see Introduction), a large body of research suggests that enhancer-promoter looping is required for gene expression [START_REF] Bartman | Enhancer Regulation of Transcriptional Bursting Parameters Revealed by Forced Chromatin Looping[END_REF][START_REF] Chen | Dynamic interplay between enhancer-promoter topology and gene activity[END_REF][START_REF] Deng | Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor[END_REF][START_REF] Deng | Reactivation of developmentally silenced globin genes by forced chromatin looping[END_REF]Palstra et al., 2003). However, recent imaging experiments demonstrated, at least for some specific loci, enhancers at large distances from activated target genes (Alexander et al., 2019;Benabdallah et al., 2019;[START_REF] Lim | Visualization of Transvection in Living Drosophila Embryos[END_REF], thus decoupling enhancer-promoter proximity from gene expression. In this study, I show that Sox2-SCR distances are significantly closer (median=146 nm) than control regions of equivalent genomic separation (median=195 nm), in agreement with Hi-C data (Bonev et al., 2017). Nonetheless, both Sox2-SCR and control pairs were found in close proximity, as might be expected for genomic separations of ~115 kb. Recent high-throughput imaging on fixed cells revealed a remarkable degree of cell-to-cell heterogeneity and variability in spatial genome organization [START_REF] Bintu | Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells[END_REF][START_REF] Cattoni | Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions[END_REF][START_REF] Finn | Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization[END_REF][START_REF] Giorgetti | Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription[END_REF]. Although my current experimental setup does not allow simultaneous visualization of all four regions, it is highly likely that there would be individual cells where the control regions are closer than Sox2-SCR. Future work is needed to solidify this hypothesis. A very ambitious experiment is to perform triple-label ANCHOR in order to determine whether, and how often, Sox2-SCR are really closer than Sox2-Inter control and SCR-Inter control pairs.

Besides characterizing chromatin topology at the pluripotent state, where Sox2 is expressed, this study also revealed insights into enhancer-promoter separation distances at early stages of ESC in vitro differentiation, where Sox2 is silenced. In agreement with the 4C results, Sox2 and SCR average distances were found significantly increased (median=205 nm) three days after retinoic acid (RA)-induced differentiation of mESCs, yet the probes were still in relatively close spatial proximity, and much closer compared to the average distances separating Sox2 and SCR in mESCs proposed by a previous study (Alexander et al., 2019). To directly examine if and how chromatin topology relates with gene activation, I tagged the native Sox2 mRNA of the musculus allele of ANCHOR-labelled mESCs with MS2 stem loops [START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF], providing a direct readout of transcription from the endogenous Sox2 locus. Triple-label live imaging allowed Sox2 and SCR proximity to be simultaneously measured with gene transcription. Although imaging of a greater number of cells is required to lend greater support to these preliminary results, I find here that Sox2 and SCR are slightly closer during active transcription and that the actively transcribing subset of Sox2 loci has significantly closer Sox2-SCR distances compared to the total distribution from the doublelabel experiments. Together, these findings fit with a model in which physical enhancerpromoter proximity correlates somehow with transcriptional activation. Nevertheless, when I assessed the link between Sox2 transcription and local chromatin architecture by other means, in particular after inhibition of transcriptional initiation upon treatment with triptolide, which represses Sox2 expression to almost undetectable levels, and after deletions of critical sites within the SCR, which strongly reduced Sox2 expression (>4-fold), I did not observe any effect on Sox2-SCR proximity. Overall, these results suggest that the Sox2 gene and its cognate ESC enhancer, SCR, are frequently in close proximity, but that the exact separation distance does not necessarily correlate with transcription. In light of these and others' results (Benabdallah et al., 2019;[START_REF] Lim | Visualization of Transvection in Living Drosophila Embryos[END_REF], the simplistic looping model needs to be revised. Direct physical juxtaposition of promoters and enhancers does not seem to be required (nor sufficient) for transcriptional regulation to be conferred; instead, at least for Sox2, crosstalk across the elements appears to be possible when they are within a certain, yet-to-be-determined, radius.

Sox2 chromatin dynamics are consistent with transcriptional hubs and/or condensates

Recent research has pointed to the existence of transcriptional hubs that create local microenvironments, wherein enhancers and promoters can share common clusters of Pol II and regulatory factors [START_REF] Lim | Enhancer-promoter communication : hubs or loops ?[END_REF]. Moreover, recent imaging of regulatory factors and cis-elements at pluripotency genes, including Sox2, documented ~100-to 200-nm-sized clusters of enhancer-associated TFs, created by frequent proximity of distal enhancers to target genes (J. [START_REF] Li | Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells[END_REF][START_REF] Li | Single-gene imaging links genome topology, promoter-enhancer communication and transcription control[END_REF]. Notably, all the separation distances observed in this study, including the ones between the control probes, are within the ~100-to 200-nm range described by [START_REF] Li | Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells[END_REF][START_REF] Li | Single-gene imaging links genome topology, promoter-enhancer communication and transcription control[END_REF]. Taking into consideration my findings on enhancer-promoter separation distance and chromatin diffusion parameters, it is tempting to speculate that such a transcriptional hub could be embracing Sox2 and SCR, thus constraining their mobility relative to control regions. Since the intervening sequence is also less constrained, it may loop out of the transcriptional hub and thus be freer to explore the nucleoplasm. My results are thus globally consistent with a model whereby promoters and enhancers reside, without necessarily directly juxtaposing, in the same transcriptional hub, which appears to constrain local chromatin movement. However, my results do not support a simple model whereby hubs exclusively contain actively transcribing loci. Chromatin constraints are largely unchanged on treatment with drugs inhibiting transcription and, more tellingly, there are no obvious differences in the dynamics of transcribing or non-transcribing alleles within wild-type ESCs. The links, if any, between transcriptional hubs and apparent diffusive speeds are even more obscure.

The challenge now is to identify whether Sox2 and SCR co-localize within the proposed transcriptional hub. To understand the dynamics and spatial organization of the effector proteins within the suggested hub, I will attempt to use super-resolution microscopy to visualize and track RNA Pol II and key factors that have been previously found in clusters, such as Mediator, Sox2 and Brd4 [START_REF] Cho | Mediator and RNA polymerase II clusters associate in transcription-dependent condensates[END_REF][START_REF] Li | Single-gene imaging links genome topology, promoter-enhancer communication and transcription control[END_REF][START_REF] Sabari | Coactivator condensation at super-enhancers links phase separation and gene control[END_REF]. A very ambitious goal, whose success is uncertain but would be extremely rewarding, is to couple ANCHOR labelling of Sox2 with single-particle tracking of labeled transcription factors and directly observe the major assumption of a transcriptional hub model: that TF exchange is more rapid when a gene and an enhancer are in the same microenvironment. The three-and fourfluorophore experiments will be very challenging, and even more difficult to analyze. I anticipate that this technical difficulty will be facilitated by future technical advances.

However, technical issues aside, it is also likely that the much faster dynamics of freely diffusing transcription factors cannot be meaningfully modeled with the much slower changes to the bulky chromatin fiber. Future work should also explore whether regulatory factors and

Pol II are recruited into clusters to form transcription hubs via the formation of phase-separated condensates [START_REF] Sabari | Coactivator condensation at super-enhancers links phase separation and gene control[END_REF] or through binding to cognate DNA and chromatin binding sites [START_REF] Li | Single-gene imaging links genome topology, promoter-enhancer communication and transcription control[END_REF]. Treatment of cells with 1,6-hexanediol, which disrupts condensates [START_REF] Lin | Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers[END_REF], followed by live-imaging experiments in order to measure separation distances between Sox2 and the SCR and calculate local chromatin dynamics might be informative, although indirect effects of this solvent change could confound interpretations.

Nevertheless, 1,6-hexanediol has been very recently shown to weaken enhancer-promoter interactions and TAD insulation [START_REF] Ulianov | Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells[END_REF], thus alternative experiments might be needed to explore this interesting avenue of research. Ultimately, even if for technical reasons this experiment might not be feasible to perform, combination of triple-label ANCHOR for Sox2, internal control probe and SCR with RNA Pol II and/or TF immunolabeling, would reveal the dynamics and spatial organization of the tagged loci within the proposed transcriptional hub. Applying a live antibody-based imaging approach, such as VANIMA [START_REF] Conic | Imaging of native transcription factors and histone phosphorylation at high resolution in live cells[END_REF], and super-resolution microscopy may be required to obtain meaningful resolution of transcriptional hubs.

PolII loading and a more indirect effect of transcription on chromatin dynamics?

At first glance, my results appear to contain a large contradiction. The double-label results after various means of perturbing Sox2 expression (differentiation, treatment with transcriptional inhibitors, deletion of enhancer regions) all point to a direct link between transcription and chromatin dynamics, with perturbed expression causing a generally reduced constraint at both enhancer and promoter, and a more specific reduction of apparent diffusive speed at the promoter. However, when simultaneously labeling nascent Sox2 transcription, there are no apparent differences in average diffusive properties between transcribing and non-transcribing alleles, either at the promoter or the enhancer. One possible explanation may be that in wildtype ESCs, nearly all alleles of the highly-expressed Sox2 locus are "poised" for transcriptional bursting within the same permissive nuclear microenvironment, regardless of whether or not they are synthesizing mRNA at the moment of image acquisition. Chromatin dynamics are likely to be determined by the local environment, so may thus not be expected to differ. In support for this, the Sox2 promoter/gene start contains high levels of paused Pol II in ESCs, a known characteristic of poised, rapidly-inducible genes [START_REF] Williams | Pausing of RNA Polymerase II Regulates Mammalian Developmental Potential through Control of Signaling Networks[END_REF]

Local diffusive parameters as "measurements" of regulatory activity?

To my knowledge, this work is the first live-imaging study that describes separation distances and the diffusion properties of an enhancer-promoter pair, simultaneously with gene transcription, and in response to inhibitors of transcription. Previous studies also addressed the link between chromatin dynamics and gene expression [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF], attaining seemingly conflicting results. My results both support and contradict these previous results to some extent. Firstly, as described above for my own results, the anomalous diffusion parameter (α) appears linked to "activity", with greater constraint at active regions, in line with a recent report, where transcription was shown to coincide with confinement of a mRNAproducing gene to smaller nuclear volumes [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF].

Specifically, I also found that the Sox2 promoter, but not the SCR, diffuses more rapidly (higher D app ) on activation. This finding partially agrees with one recent report: both a gene and an enhancer activated upon differentiation were found to move faster [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF], interpreted in this study as an increase in thermal energy via the active transcription process.

An alternative explanation (see also above) is that the first steps of the transcriptional cycle, and in particular chromatin remodeling and nucleosome removal as PIC is recruited and/or the promoter melting after the assembly of the PIC, actually result in less dense chromatin which eventually can move quicker. Reduced mobility of Sox2, as shown in this study upon treatment with triptolide, a known inhibitor of the helicase activity of TFIIH [START_REF] Bensaude | Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity?[END_REF], is consistent with this hypothesis. On the other hand, opposite to Gu et al., I did not observe any link between the diffusive speed of the enhancer and gene activation. Publicly available RNAseq data from mESCs (ENCODE ENCSR000CWC; [START_REF] Dunham | An integrated encyclopedia of DNA elements in the human genome[END_REF]) suggest that the SCR is not producing any eRNA. Although we have no access to analogous data from EpiLCs, where the assessed Fgf5 enhancer is active [START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF], its transcriptional activity could explain why it behaves like the active Fgf5 and Sox2 promoters, and not the SCR. Moreover, contrary to Gu et al., I concluded that inhibition of transcriptional elongation has no effect on chromatin mobility of either regulatory element. It remains to be seen whether this can be explained by technical differences or gene-specific contexts.

Despite the clear "slowing" of promoter diffusion on gene inactivation, it should be noted that the apparent diffusive coefficient of the Sox2 promoter is significantly slower than both the SCR and control regions, which are not (or very poorly) transcribed. Thus when comparing genomic loci, transcriptional activity alone is insufficient to "predict" local diffusion speed. Interestingly, another member of the lab, Guilherme Oliveira, has recently built a polymer model introducing long-range interaction forces based on available Hi-C maps (Bonev et al., 2017), and has used it to accurately predict locus-specific D app and α values around the HoxA locus, finding good agreement with the experimental results [START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF]. Chromatin architecture therefore also appears to be an important feature in determining local chromatin dynamics. This model failed to predict the experimentally-derived values at the Sox2 locus; a more complex polymer model to also include gene activity may also be required to further comprehend these findings.

Utility of ANCHOR for probing chromatin dynamics

In this study, I adapted the minimally perturbative ANCHOR/ParB DNA labelling approach, originally developed in yeast [START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF], and optimized the system for musculus allelespecific CRISPR/Cas9 knock-in of double labels. Besides the laborious engineering of the double knock-in cell lines, allele-specificity wouldn't be so feasible with dCas9 approaches, due to the lack of enough SNPs around the regulatory elements. The small size (<1 kb) of the ANCH sequence together with the non-specific and relatively weak DNA binding of OR proteins, allowed me to position the ANCH3 sequence [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF] within the immediate vicinity of Sox2 (6.5 kb centromeric to TSS) and the ANCH1 [START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF] inside the SCR (~109 kb telomeric to TSS), in contrast with a previous study that had to integrate the large cuO repeats downstream of the SCR (Alexander et al., 2019). Importantly, in order to maintain the native context of the endogenous genomic locus, I did not include any ectopic insulator sequences as has been used in a recent study examining enhancer-promoter associations in Drosophila (H. Chen et al., 2018a). As has been previously reported in yeast [START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF] Still, although the ANCHOR approach was shown to be effective and non-invasive, it is currently limited by concerns over the relatively low brightness of the fluorophores labeling OR proteins. In this study, the duration of the movies has been adjusted (~2-3 min) to avoid photobleaching of the fluorophores and cell damage, and images have been restricted to 2D

planes by confocal microscopy. To address this problem, I tried the HaloTag labeling technology [START_REF] Los | HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis[END_REF], that allows specific fluorescent labeling of fusion (Halo-OR)

proteins, but unfortunately without obtaining a much higher fluorescent signal. In the future, it will be critical to improve these technical limitations by using brighter and more versatile Halocompatible tags and by decreasing photobleaching and phototoxicity, possibly by applying lattice light-sheet microscopy (B. C. Chen et al., 2014).

Perspectives

Although the Sox2 locus has proven a powerful tool to study enhancer control, not least because the SCR is one of the few enhancers described to be absolutely essential for target gene expression in ESCs (Zhou et al., 2014), one experimental drawback is that the gene is already active in ESCs, and one can only follow the loss of expression during perturbation experiments or on differentiation. Therefore, I would like to perform the same ANCHOR setup for a promoter-enhancer pair that is silent in ESCs but activated on differentiation to neuronal precursors (NPC). Labelling of Sox2 and the distal NPC-specific Sox2 enhancer, recently identified in 5C and Hi-C maps (Bonev et al., 2017;[START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF], could be an excellent candidate pair. Before this candidate pair is to be used for imaging experiments, I will first need to delete the NPC-specific candidate enhancer with CRISPR/Cas9 and confirm that it is required for Sox2 expression. If this condition is met, I will be in a position to engineer the ANCHOR and MS2 labels, in order to follow what happens during gain of activity.

However, in order to gain a better understanding of the interplay between chromatin mobility, transcriptional hub formation and function, it is necessary to study more enhancer-promoter pairs and see how general the Sox2 properties are. In the future, it will be crucial to further probe the links between local chromatin mobility, Pol II clustering, TF dynamics and transcription readout. Combination of multicolor live-cell super-resolution imaging and singlemolecule detection, at nanometer resolution, will be a major feat for further zooming into single genes and analyzing how transcription and chromatin are regulated.

My findings, together with others [START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF][START_REF] Gu | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements[END_REF], highlight the importance of assessing chromatin dynamics at transcription sites as a means of understanding the nuclear microenvironment and regulatory principles. Notwithstanding, D app and α are quite general diffusive parameters. For example, reduced α can be explained by multiple mechanisms, such as confinement to a reduced volume or "attraction" to specific regions, that cannot be distinguished. I look forward to advances in physical theory allowing more precise inferred diffusive parameters to be defined, which could give better insights and allow even more accurate analysis of local chromatin dynamics. Coupled with more sophisticated imaging tools, and better methods for manipulating the nucleus (e.g. acute ablation of key regulatory proteins with the auxin-degradation system (N. Q. [START_REF] Liu | WAPL maintains a cohesin loading cycle to preserve cell-typespecific distal gene regulation[END_REF][START_REF] Nora | Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Article Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization[END_REF][START_REF] Rao | Cohesin loss eliminates all loop domains[END_REF], or manipulation of liquid-liquid phase separation properties (Y. [START_REF] Shin | Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome[END_REF], comprehensive understanding of the four-dimensional aspects of transcriptional regulation is becoming more and more feasible.

antibiotic selection, GFP-positive cells were sorted using a FACS Aria Fusion and seeded into 96-well plates. Once the single colonies had grown to 70-80% confluency they were split into four identical 96-well plates. The first plate was used for DNA extraction. Cells were washed with 1xPBS and then lysed for 1h at 56˚C with 30 μl lysis buffer (0.45% Tween, 0.45% Triton-X100, 2.5mM MgCl2, 50mM KCl, 10mM Tris-HCl pH8.3, 100 μg/ml proteinase K). 2 μl of genomic DNA was used for subsequent PCR screening using Expand Long Template PCR System (Roche). The second and third plates were frozen at -80C after resuspending the clones in 90% FCS/10% DMSO freezing media, while the fourth one was kept in culture for further experiments.

Determination of double-positive clones

Heterozygous clones with the correct sequences were screened by PCR in the following manner. A first (external) PCR was performed to amplify the whole candidate integration sites (5'HA-ANCH-3'HA) using Expand Long Template PCR System (Roche), a forward primer hybridizing upstream of the 5' HA and a reverse primer downstream of the 3' HA. Two additional (internal) PCRs were performed on the candidate clones in order to verify the insertion of each ANCH sequence using one external primer (forward or reverse) and one internal (reverse or forward) hybridizing to the ANCHOR. Insertions were further confirmed by sequencing. Primer sequences are given in Table 4.

RT-qPCR

Total RNA was extracted (Macherey-Nagel) from the final candidate clones, using at least one million cells, and reverse transcription for cDNA generation was performed using random hexamer primers (Thermo Fisher) and SuperScript IV (Invitrogen). RT-qPCR was performed on 10 ng cDNA in technical triplicates using QuantitTect SYBR Green PCR kit (Qiagen) on a LC480 Light Cycler (Roche). Amplification was normalized to SDHA (Succinate dehydrogenase complex flavoprotein subunit A). Primer sequences are given in Table 4.

Microscopy experiments (see below) validated the heterozygous incorporation of the ANCH sequences (detection of one single spot per ANCH sequence per cell) within the same allele (two spots were always in close proximity).

Generation of CRISPR/Cas9-mediated monoallelic SCR deletions

Monoallelic deletion (129 allele) of the 107 and 111 elements of the SCR, screening and expression analysis were facilitated by the presence of SNPs between the two alleles of the hybrid F1 (M.Musculus 129 x M.Castaneus) ESCs and were prepared as previously described (Moorthy & Mitchell, 2016b). The vectors containing the sgRNAs, Cas9-GFP and Cas9D10A-GFP, as well as the allele-specific primers for genotyping and gene expression analysis were kindly provided by Pr. Jennifer Mitchell. In brief, four sgRNAs and Cas9D10A-GFP were used to delete SRR111 enhancer region from Anchor_Sox2-SCR ESCs. The Cas9D10A mutant cuts only one strand, so the 4 guides result in staggered cuts in the two strands on either side of the target region. Once the Anchor_ΔSRR111 129-/CAST+ ESCs were obtained (Clone 3, see Table 3),

we used two sgRNAs and Cas9-GFP to delete the SRR107 region and make the Anchor_ΔSRR107.111 129-/CAST+ ESCs (Clone 30, see Table 3). Primer and sgRNA sequences are given in Table 4. 4. To excise the LoxP-HSV_TK-T2A-Hygro R resistance cassette, 1 million cells of a correctly targeted clone (H3H1, see Table 3) were transfected with 4 μg Cre-GFP vector using The condition that we selected for all further experiments was: MCP-mScarletI medium/high, OR1-GFP low and OR3-IRFP low ("Anchor_Sox2-MS2" cell line, see Table 3).

Generation of mESCs

Preparation of cells for live-imaging

150,000 cells were plated two days prior to imaging onto laminin-511-coated (BioLamina) 35mm glass bottom petri dishes (MatTek Corporation) and transfected with 2 μg of OR1-EGFP and OR3-IRFP producing plasmids (vectors available from NeoVirTech. For my thesis, both vectors were kindly provided by Pr. Kerstin Bystricky, and modified as described above except for the inclusion of NLS). Transfection was carried out using Lipofectamine 2000 Transfection Reagent (Invitrogen), according to the manufacturer's recommendation. The medium was changed just before imaging.

To examine the impact of the status of Pol II on chromatin motion in living cells, we used chemical inhibitors to block either transcription initiation (Triptolide) [START_REF] Vispé | Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA[END_REF] or elongation (Flavopiridol) [START_REF] Chao | Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo[END_REF]. ESC fresh medium was supplemented with 500 nM Triptolide (Sigma-Aldrich) or 1 μM Flavopiridol (Sigma-Aldrich) and cells were incubated for 3 or 4 hours respectively prior to image acquisition. Cells treated with DMSO

(1:2000 dilution) for 4 hours were used as a control for each live-imaging experiment.

In order to obtain NPC-like cells (NPC, Neuronal Progenitor Cells) we modified a previously described protocol (Bibel, Richter, Lacroix, & Barde, 2007). On day 0, ESCs were passaged onto laminin-511-coated (BioLamina) 35mm glass bottom petri dishes (MatTek Corporation)

and switched to a DMEM medium (4.5 g/l glucose) supplemented with 2mM Glutamax-I, 10% FCS, 0.1 mM non-essential amino acids, 0.1 mM β -mercaptoethanol and 40 μg/ml gentamycin, without LIF or CHIR99021 and PD0325901 inhibitors (2i). The following day, the medium was changed just before imaging ("day1" condition) and retinoic acid was added for the following two days ("day 2" and "day3") of culture.

Live-cell imaging of mESCs

Imaging experiments were performed on an inverted Nikon Eclipse Ti microscope equipped with a PFS (perfect focus system), a Yokogawa CSU-X1 confocal spinning disk unit, two sCMOS Photometrics Prime 95B cameras for simultaneous dual acquisition to provide 95% quantum efficiency at 11 μm x 11 μm pixels and a Leica 100x oil objective (HC PL APO 1,[START_REF] Dekker | Capturing chromosome conformation[END_REF] oil immersion). For double-label experiments using OR1-EGFP and OR3-IRFP, we excited EGFP and IRFP with a 491-nm (~100mw) and a 635-nm laser (>28mW), respectively. We detected green and far red fluorescence with an emission filter using a 525/50 nm and a 708/75 nm detection window, respectively. A thermostated heater (Tokai Hit Stage Top Incubator) allowed for heating at 37 o C, humidity and CO 2 control (5%). Time-lapse analysis of GFP and IRFP foci was performed in 2D acquiring 241 time points at a 0.5 s time interval. For triplelabel experiments using OR1-EGFP, OR3-IRFP and MCP-mScarletI, we first imaged EGFP and IRFP as above and then, at identical z-position, we excited mScarletI with 561-nm laser and we detected red fluorescence with an emission filter using a 609/54 nm detection window.

Time-lapse analysis of GFP, IRFP and mScarletI foci was performed in 2D acquiring 181 time points at a 1 s time interval.

The system was controlled using Metamorph 7.10 software. The steps of the subsequent image analysis are depicted in Figure 29.

Þ Optimizing / Enhancing localization accuracy

Despite an excellent overall performance of Icy, we frequently found that detected points were off-centered to detected particles and false positives were present (Figure 30). Trying to mitigate their effects, we model each particle using a 2D bell curve as follows:

The reference signal level is given by B G , the spot has a maximum signal I o and center of mass represented by μ. Parameters L x and L y represent spot size in respective directions and a degree of rotation is denotated by parameter θ.

Þ Noise filtering

Using signal intensity, spot size and localization error for all the particles tracked, we determine outliers by calculating median and inter-quartile ranges (IQR) for these 3 parameters. Any detection in which any of these parameters is superior/inferior to 2 IQR above/below median were considered as outliers and removed from remaining analysis.

Þ Image alignment

The set-up using two cameras helped us with the measurement of distances and diffusive parameters. Unfortunately, it inserted non-negligible alignment discrepancies between channels. Furthermore, due to the usage of two different wavelengths, we could also observe chromatic aberration. Dealing with these inaccuracies is vital for the estimation of average distances between spots.

To correct these problems, we use a set of generic affine transformations to perform a digital post-alignment in 2 steps [START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF]. The first step handles more grotesque errors associated with the dual camera setup, whilst the second will rescale the image and correct chromatic aberration (Figure 31). More details are found in [START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF].

Þ Calculating distances between particles

Finally, we can estimate 2D distance between particles in a Pythagorean framework once coordinates are properly calibrated by the alignment matrix as earlier introduced.

Þ Defining bursting windows

For the triple-label movies, the ANCHOR labels were robustly detected in nearly all frames, whereas the MCP signal appeared to frequently be lost in the z-plane. Visual inspection of the MCP signal presence/absence across the movie frames (see Results) showed clear differences between prolonged absence of transcriptional bursting and intermittent loss of signal due to zmovement. "Bursting" movies were defined as the subsets of frames forming contiguous sections of at least 49 MCP-positive frames, with no periods of >7 missing frames; "nonbursting" movies were defined as the subsets of frames forming contiguous sections of at least 98 MCP-negative frames. These subsets were extracted for re-analysis by GPTool.

Circular chromatin conformation capture sequencing (4C-seq)

4C-seq using the SCR as a bait was performed as previously described (Van De Werken et al., 2012). Briefly, 5 million cells per biological condition were cross-linked with 2%

formaldehyde in PBS and incubated on a rocker for 10 minutes at room temperature (RT). Allele-specific 4C-seq was performed as previously described [START_REF] Splinter | The inactive X chromosome adopts a unique three-dimensional conformation that is dependent on Xist RNA[END_REF]

Western Blot

Western blots were performed using standard procedures. In brief, all whole cell extract lysates were prepared in RIPA buffer (150 mM NaCl, 1 4See: A Flexible Browser to Explore 4C Data

Yousra Ben Zouari 1,2,3,4 , Angeliki Platania 1,2,3,4 , Anne M. Molitor 1,2,3,4 and Tom Sexton It is established that transcription of many metazoan genes is regulated by distal regulatory sequences beyond the promoter. Enhancers have been identified at up to megabase distances from their regulated genes, and/or proximal to or within the introns of unregulated genes. The unambiguous identification of the target genes of newly identified regulatory elements can thus be challenging. Well-studied enhancers have been found to come into direct physical proximity with regulated genes, presumably by the formation of chromatin loops. Chromosome conformation capture (3C) derivatives that assess the frequency of proximity between different genetic elements is thus a popular method for exploring gene regulation by distal regulatory elements. For studies of chromatin loops and promoter-enhancer communication, 4C (circular chromosome conformation capture) is one of the methods of choice, optimizing cost (required sequencing depth), throughput, and resolution. For ease of visual inspection of 4C data we present 4See, a versatile and user-friendly browser. 4See allows 4C profiles from the same bait to be flexibly plotted together, allowing biological replicates to either be compared, or pooled for comparisons between different cell types or experimental conditions. 4C profiles can be integrated with gene tracks, linear epigenomic profiles, and annotated regions of interest, such as called significant interactions, allowing rapid data exploration with limited computational resources or bioinformatics expertise.

INTRODUCTION

Since early transgenic studies it has been clear that promoter sequences are insufficient to regulate the spatiotemporal expression patterns of many developmental genes. "Remote control" is additionally conferred by distal activating sequences, termed enhancers, which have been intensively studied over the last years (Schoenfelder and Fraser, 2019). Genome-wide profiling of histone modifications and protein binding sites by ChIP-seq have uncovered a general chromatin signature of enhancer regions: DNase-hypersensitive, bound by the transcriptional coactivator p300, and marked by the monomethylation of lysine-4 of histone H3 (H3K4me1) (Heintzman et al., 2009). Follow-on studies refined these findings further by identifying chromatin features that were characteristic of different enhancer properties. For example, the strongest-acting enhancers are also accompanied by acetylation of lysine-27 of histone H3 (H3K27ac) (Creyghton et al., 2010;Rada-Iglesias et al., 2011) and/or acetylation on globular histone domains (Pradeepa et al., 2016), recruit RNA polymerase II, and general transcriptional machinery [START_REF] Koch | Transcription initiation platforms and GTF recruitment at tissuespecific enhancers and promoters[END_REF], and are even transcriptionally active, producing non-coding RNA (eRNAs) (Kim et al., 2010). Enhancers lacking these extra features, and sometimes even encompassing repressive marks, such as H3K27 trimethylation (H3K27me3), are proposed to be "poised" enhancers, which may become activated at later developmental stages. Interestingly, the chromatin states at enhancer sequences vary much more across cell types than those of gene promoters (Roadmap [START_REF] Epigenomics | Integrative analysis of 111 reference human epigenomes[END_REF], suggesting that much of the regulatory potential is epigenetically carried by enhancers. However despite advances in identifying enhancers genome-wide, both through epigenomic profiling and high-throughput reporter assays (Arnold et al., 2013;[START_REF] Roadmap Epigenomics | Integrative analysis of 111 reference human epigenomes[END_REF], unambiguous identification of their target genes is still a major challenge. Important developmental enhancers have been found at megabase distances from target genes, and/or within the introns of unregulated genes (Lettice et al., 2003;Amano et al., 2009;Herranz et al., 2014); previous studies estimate that up to ~90% of enhancers may indeed skip the closest genes on the linear chromosome fiber (Sanyal et al., 2012;Schoenfelder et al., 2015).

Since the advent of the chromosome conformation capture method (3C) (Dekker et al., 2002) and its variants to measure relative spatial proximity of pairwise genomic regions, many enhancers have been found to physically interact with their target genes, often with "looping out" of the intervening chromatin (Palstra et al., 2003); the resultant "active chromatin hub" has been proposed to provide the permissive regulatory environment for transcription initiation, although the exact mechanism remains unclear. In many studied cases, looping is concomitant with transcriptional induction, whereas in others, the loop is pre-formed to poise the gene for subsequent activation (Schoenfelder and Fraser, 2019). Recent reports using microscopy methods have also been made of enhancers and promoters being well separated on gene activation (Alexander et al., 2019;Benabdallah et al., 2019), although enhancer-promoter interactions were previously reported in the studied loci, raising questions as to whether interactions may completely precede transcription and/or be very transient events. In any case, physical proximity measured by 3C-based methods is becoming a popular means of ascribing target genes to otherwise cryptic distal regulatory elements, or of identifying novel candidate regulatory regions of specific genes of interest. For example, intergenic sequence variants associated with diseases have been better characterized once their target genes were identified by 3C-based approaches (Herranz et al., 2014;Schoenfelder and Fraser, 2019).

With the advent of next-generation sequencing, several higher throughput variants of 3C have been developed to obtain genomewide chromatin interaction maps. Hi-C is an "all-to-all" method, systematically assessing all pairwise chromatin contacts (Lieberman-Aiden et al., 2009). However, due to the great complexity of the sequenced material, calling specific looping interactions requires prohibitively expensive sequencing depth (Rao et al., 2014;Bonev et al., 2017), and Hi-C loop calling algorithms have been demonstrated to not be very robust [START_REF] Forcato | Comparison of computational methods for Hi-C data analysis[END_REF]. A recent modification, Capture Hi-C, incorporates capture with a pool of thousands of oligonucleotides, allowing the complexity of sequenced Hi-C material to be reduced sufficiently to assess the chromatin looping interactions with all promoters (Hughes et al., 2014;[START_REF] Sahlen | Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution[END_REF]Schoenfelder et al., 2015). However, capture libraries can be expensive, and their design still represents a tradeoff between coverage of assessed promoters and resolution of the identified loops. For the highest resolution profiling of smaller numbers of candidate regions, the method of choice is the "one-toall" 4C (circular chromosome conformation capture), which assesses all the chromatin interactions with one specific bait of interest (Simonis et al., 2006;van de Werken et al., 2012) (Figure 1A). In brief, nuclei are fixed in their native topologies with formaldehyde, digested with a restriction enzyme and re-ligated, as for 3C, such that chimeric DNA sequences are generated between restriction fragments which may be unlinked on the linear chromosome fiber but are physically proximal at the time of fixation. The purified DNA is then circularized by digestion with a secondary restriction enzyme and re-ligation under dilute conditions, allowing an inverse PCR strategy to amplify all the chimeric DNA linked to a specific bait restriction fragment of interest. The much reduced complexity of a 4C library, compared to that of Hi-C, means that promoter interactomes can be reliably profiled with just a few million sequence reads, and ~20 baits can readily be multiplexed into a sequencing run, making it a much more cost-effective method (van de Werken et al., 2012). The major limitations of 4C are the relatively small throughput in baits that can be assessed at a time, and that the direct sequencing of PCR products confounds results with large numbers of PCR duplicates that cannot be distinguished from counts of true 3C ligation events. However, in silico approaches can minimize the impact of PCR duplicates (de Wit et al., 2015), and "unique molecular identifier" variants of 4C have also been developed [START_REF] Schwartzman | UMI-4C for quantitative and targeted chromosomal contact profiling[END_REF].

Due to the growing popularity of 4C experiments, several algorithms have been developed to call significant interactions (van de Werken et al., 2012;[START_REF] Thongjuea | r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data[END_REF][START_REF] Williams | fourSig: a method for determining chromosomal interactions in 4C-Seq data[END_REF][START_REF] Klein | FourCSeq: analysis of 4C sequencing data[END_REF][START_REF] Raviram | 4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments[END_REF][START_REF] Geeven | peakC: a flexible, non-parametric peak calling package for 4C and Capture-C data[END_REF]; recent benchmarking shows that all methods work well on simulated data, but no single method is optimum for all experimental setups [START_REF] Walter | Benchmarking of 4C-seq pipelines based on real and simulated data[END_REF]. However, whereas most of these methods has an in-built tool to plot the static results after data processing, a simple, flexible browser allowing a user to rapidly visualize their 4C results is currently lacking (see Figure 2 and summary of the different plotting options currently available in Table 1). Moreover, while some methods allowed raw and/or smoothed 4C data to be exported as files that can be opened and visualized alongside epigenomic profiles on genome browsers, they offered no flexibility in plotting the epigenomic profiles directly alongside the 4C plot while different smoothing or peak calling parameters are being trialed. We recently developed ChiCMaxima, a suite of tools to analyze Capture Hi-C data, which includes a GUI (graphical user interface) to flexibly visualize data sets alongside gene annotations and epigenomic profiles [START_REF] Ben Zouari | ChiCMaxima: a robust and simple pipeline for detection and visualization of chromatin looping in Capture Hi-C[END_REF]. Here we report 4See, the adaptation of ChiCMaxima tools for the user-friendly integrative exploration of 4C data sets. 4See provides flexibility to compare different replicates side by side, or to average them together when comparing experimental conditions, and to visualize 4C profiles at different smoothing window sizes, necessary to identify putative interactions at different distances from the bait, without the need to reload or re-process the initial data. 4See utilizes quantile normalization to allow different plotted profiles to be fairly compared during the visualization. 4See also allows 4C profiles to be easily plotted alongside gene annotations and linear epigenomic tracks, as well as for specific regions (e.g. interactions called by other algorithms) to be highlighted. We anticipate that 4See will be a useful tool to the community for quick and easy exploration of 4C data, particularly when used in conjunction with existing interaction calling tools.

MATERIALS AND METHODS

Pre-Processing

4C

J1 mouse embryonic stem (ES) cells were grown on gammairradiated mouse embryonic fibroblast cells under standard Running median scores are plotted as a line graph (5 kb resolution), with domainograms plotted underneath as a heat map for median scores at steadily increasing window sizes. Positions of the CTCF site within the SCR and the Sox2 gene are indicated by arrows. Note that the independent normalization means that the SCR-Sox2 interaction differences between the two cell types is not evident, compared to other methods. (B) r3Cseq [START_REF] Thongjuea | r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data[END_REF] results for the combined three data sets, showing panels, from top to bottom: positions of Refseq genes; restriction fragment coverage; averaged profile for the "experiment" (ES) condition, with called interactions at different confidence levels highlighted; profile for the "control" (NPC) condition, with called interactions at different confidence levels highlighted; plot of the log2-ratio of experiment vs control 4C signal. The position of the SCR bait is indicated by a red dashed line. Note that r3Cseq appears to call a very large number of (continued)

Ben Zouari et al. 4See

Frontiers in Genetics | www.frontiersin.org January 2020 | Volume 10 | Article 1372 4 conditions (4.5 g/L glucose-DMEN, 15% FCS, 0.1 mM nonessential amino acids, 0.1 mM beta-mercaptoethanol, 1 mM glutamine, 500 U/mL LIF, gentamicin), then passaged onto feeder-free 0.2% gelatin-coated plates for at least two passages to remove feeder cells. For in vitro differentiation to neural precursor cells (NPCs), F1 ES cells were cultured in the same medium supplemented with 1 µM PD03259010 and 3 µM CHIR99021 ("2i" conditions) and without feeders. The cells were then cultured for six days with medium without LIF or 2i and with 10% FCS, and with 5 µM retinoic acid for the final four days, to generate embryoid bodies (Bibel et al., 2007). J1/F1 ES or differentiated cells were detached with trypsin, then washed by centrifugation in PBS before fixation. Mouse CD4 + CD8 + doublepositive (DP) thymocytes were obtained from 4 week old mouse thymus by FACS with anti-CD4-PE and anti-CD8a-FITC antibodies (eBioScience). Both cell preparations were fixed with 2% formaldehyde in mES culture medium for 10 min at 23°C. The fixation was quenched with cold glycine at a final concentration of 125 mM, then cells were washed with PBS and permeabilized on ice for 1 h with 10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.1% NP-40, and protease inhibitors. Nuclei were resuspended in DpnII restriction buffer at 10 million nuclei/mL concentration, and 5 million nuclei aliquots were further permeabilized by treatment for either 1 h with 0.4% SDS at 37°C (ES cells), or for 20 min with 0.7% SDS at 65°C, then for 40 min at 37°C (DP cells). The SDS was then neutralized by incubating for a further 1 h with either 2.6% (ES) or 3.3% (DP) Triton-X100 at 37°C. Nuclei were digested overnight with 1000 U DpnII at 37°C, then washed twice by centrifuging and resuspending in T4 DNA ligase buffer. In situ ligation was performed in 400 mL T4 DNA ligase buffer with 20,000 U T4 DNA ligase overnight at 16°C. DNA was purified by reverse crosslinking with an overnight incubation at 65°C with proteinase K, followed by RNase A digestion, phenol/chloroform extraction, and isopropanol precipitation. The DNA was digested with 5 U/mg Csp6I at 37°C overnight, then re-purified by phenol/chloroform extraction and isopropanol precipitation. The DNA was then circularized by ligation with 200 U/mg T4 DNA ligase under dilute conditions (5 ng/mL DNA), and purified by phenol/ chloroform extraction and isopropanol precipitation. 50 ng aliquots of this DNA were used as template for PCR with baitspecific primers containing Illumina adapter termini (primer sequences and optimal PCR conditions available on request). PCR reactions were pooled, primers removed by washing with 1.8× AMPure XP beads, then quantified on a Bioanalyzer (Agilent) before sequencing with a HiSeq 4000 (Illumina).

Pre-Processing 4C Data for 4See

All bait sequence (including and downstream of the primer sequence, up to but not including the GATC DpnII site) are trimmed by the demultiplexing Sabre tool (https://github.com/ najoshi/sabre), allowing two mismatches, before mapping to the mm9 genome with Bowtie (Langmead et al., 2009) (Figure 1B). Interaction calling was done with peakC [START_REF] Geeven | peakC: a flexible, non-parametric peak calling package for 4C and Capture-C data[END_REF] with different window sizes as specified by the parameter wSize. [START_REF] Williams | fourSig: a method for determining chromosomal interactions in 4C-Seq data[END_REF] results shown independently for one ES and one NPC replicate, showing smoothed 4C plots (21fragment windows). Called interactions ("Categories" 1, 2 or 3, for different confidence levels) would be highlighted on the plot, but none were called by fourSig. (D) FourCSeq [START_REF] Klein | FourCSeq: analysis of 4C sequencing data[END_REF] results shown independently for one ES and one NPC replicate, showing normalized and processed 4C signal plots (gray line graphs and black points), alongside the positions of known genes. The green line indicates the centralized 4C value, and the dashed blue lines indicate the threshold values for a z-score difference > 2. Significant interactions would be highlighted, but were not detected by FourCSeq. Note that differences between the two cell types is not so evident, compared to most other methods. (E) peakC [START_REF] Geeven | peakC: a flexible, non-parametric peak calling package for 4C and Capture-C data[END_REF] results shown for the combined analysis of the two ES replicates, independently of the NP replicate, giving smoothed 4C plots (21-fragment windows) as histograms. The red regions indicate called interactions. (F) 4C-ker [START_REF] Raviram | 4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments[END_REF] results shown for the combined analysis, with line plots of combined ES (red) and NPC (blue) 4C signal. Note that many interactions were called by 4C-ker within the plotted window for both ES and NP, but that the documentation did not provide a means to plot them alongside the shown line plots. For previously published 4C results [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF], fastq files were downloaded from the Gene Expression Omnibus and processed just like the other data sets.

Analysis and Plotting of 4C Data Sets by Other Methods

Three 4C data sets (two replicates from ES cells; one replicate from in vitro differentiation of ES cells towards NPCs) were analyzed and plotted by 4Cseqpipe (van de Werken et al., 2012), r3Cseq [START_REF] Thongjuea | r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data[END_REF], fourSig [START_REF] Williams | fourSig: a method for determining chromosomal interactions in 4C-Seq data[END_REF], FourCSeq [START_REF] Klein | FourCSeq: analysis of 4C sequencing data[END_REF], 4C-ker [START_REF] Raviram | 4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments[END_REF], and peakC [START_REF] Geeven | peakC: a flexible, non-parametric peak calling package for 4C and Capture-C data[END_REF], using the default or recommended parameters given within the documentation accompanying the tools.

4See

System Requirements 4See is a GUI written in R (version > = 3.2), with the following packages (and their dependencies) additionally required, found on Bioconductor and/or CRAN: tcltk2, tkrplot, limma, caTools, rtracklayer. All scripts and test data are available under the terms of the GNU General Public License, version 3, on Github: https:// github.com/TomSexton00/4See. The GUI is launched by sourcing the main script, 4See.r, from within an R environment. From then on, all manipulation is performed from a windows interface, and does not require use of command prompts. A full user manual in pdf format is also found with the scripts on Github, and is provided as Supplementary Data.

Input Format

4See deals with a simple text format, which we term the "cis" format, entailing a header with three tab-delimited fields (data set name, bait chromosome, bait coordinate) followed by a twocolumn table, denoting the coordinate of the mid-point of every restriction fragment found on the same chromosome as the bait, and its corresponding number of supporting sequence reads. The cis format is generated by a perl script, coord2frag.pl, provided with 4See, which maps the genomic coordinates of 4C sequencing results into their corresponding restriction fragments and then counts the number of reads for each fragment. The perl script accepts any non-headed text format for sequences, as long as a column for chromosome, coordinate, and strand can be specified. The restriction fragment information is provided by "frag" tables, headed four-column tables, giving a unique integer identifier, the chromosome, coordinate, and fragment length for each restriction fragment. These in turn are generated by a provided perl script, makefrags.pl, requiring a user input for the sequence of the primary restriction enzyme cutting site, and a folder containing the sequences in fasta format for each chromosome of the genome assembly used. The header of the cis file provides the required information on the bait location and 4C data set name, but is also used to ensure that only 4C data sets for the same bait (with identical bait chromosome and coordinate) are treated together.

Managing Conditions and Replicates

After loading one or more cis files, 4See opens a dialog box allowing the user to determine how to handle different conditions and replicates by assigning a value to each data set (Figures 3A,C). All 4C data sets assigned a non-zero integer are quantile normalized for fairer comparison across data sets [START_REF] Ritchie | limma powers differential expression analyses for RNA-sequencing and microarray studies[END_REF]. Data sets given the same value are averaged together before plotting; those assigned zero are omitted from normalization and plotting. Additional options allow the plotting color and data label to be specified by the user, and these can be re-run via the "Conditions" drop-down menu. Thus a user can rapidly compare different replicates side by side, or average them into one plot for comparison with different cell types or conditions, without needing to reload the data (Figures 3B,D).

Plot Settings

4C profiles and the chromatin interactions they uncover differ with bait and experimental condition. In particular, the ease of distinguishing peaks of 4C signal above background depends on the distance of the interaction, since background signal of random chromosome collisions is much higher at shorter ranges (Dekker et al., 2002;Lieberman-Aiden et al., 2009). Other factors, such as whether the interaction is sharp with a single regulatory element, or is broadened across larger regions, such as "super-enhancers", or the extent to which very short-range contacts dominate the plot and hide longer-range loops (which may be a consequence of the 4C digestion efficiency and/or relative compaction of the assessed locus), mean that features of chromatin topology are often overlooked with one fixed plot setting. The control panel of 4See includes options for the user to alter the region plotted, up to ± 1.5 Mb of the bait position, and to set a maximum plotted value on the y-axis (4C signal), to better visualize certain aspects of the 4C profile (Figure 4A). However a major confounding factor in visualizing 4C data is the need to smooth the plots, since "spikes" from spurious PCR duplicates make them appear very noisy at single restriction fragment resolution. Most analytical approaches counter this by taking running means (or medians) of sliding windows, but the results can be heavily influenced by the choice of window size. Reflecting this challenge, some 4C analytical tools adopt a "domainogram" approach, whereby averages are taken over many sliding windows of many different sizes, and the results are pooled together in a heat map (de [START_REF] De Wit | Global chromatin domain organization of the Drosophila genome[END_REF]van de Werken et al., 2012; see also Figure 2A), although the visual interpretation of these results is often challenging. To aid user choice in setting appropriate parameters for their particular 4C profile, 4See allows the window size (in numbers of restriction fragments) to be altered, and the appropriate running mean is calculated on the quantile normalized (and averaged, if replicates are pooled) data before plotting. In this manner, different aspects of chromosome topology can be readily explored (Figures 4B,C).

Annotations

To put the 4C profiles into a wider biological context, 4See supports the inclusion of three different types of annotations: 224 genes, linear epigenomic profiles (termed "tracks") and called interactions. Gene information is provided as a tab-delimited headed text file with the following fields: Name, Chr (with the prefix "chr"), Start, End, Strand (as "+" or "-"). When selected, the gene track is plotted in blue directly underneath the 4C profile. Only one gene track can be loaded at a time. Management of epigenomic profiles is more flexible. Any format supported by the import function of the rtracklayer package can be supported, but for running time efficiency we recommend loading bigWig files. As for the 4C profiles, the color and plotting level for each individual track can be altered by the user in an automatically loaded dialog box. As before, the plotting levels can be 0 (not plotted), or consecutive, positive integers. When tracks have the same level, their plots are autoscaled to the maximum value of all of the included data sets within the plotted window. This feature allows fairer comparison for the same epigenetic mark across different conditions/tissue types (Figure 5). Technically, the numbers of tracks that can be loaded is only limited by system memory, although the plots become difficult to visually interpret after more than four tracks are loaded at a time.

To better highlight interactions called by existing peak-calling methods, or indeed to test how different methods and/or their parameters perform on specific 4C profiles, interactions (as bed The main control panel of 4See, including options to set the x-axis ("start coordinate," "end coordinate," and "plot window") and y-axis ("max y plot") plot limits, to choose a bait name for the plot title ("bait name"), and to set the running mean window size ("smooth window") in number of restriction fragments. (B) 4See plots for a 4C data set in DP thymocytes with the Satb1 gene promoter as bait. Two instances of the same x-and y-axis limits are shown, with a running mean window of 21 (left) or 55 (right) fragments. The position of the Satb1 bait is denoted by a black vertical line, and gene position (blue arrows) and the DP H3K27ac ChIP-seq profile (black) is shown underneath the 4C plots. Pink rectangles denote regions called as interacting by peakC for the equivalent window size as the plot. For the long-range interaction, the smaller window size appears to have more spurious called interactions, less evidently linked to H3K27ac peaks; the link is better seen with greater smoothing from a larger window size. (C) As for (B), but with the Ikzf1 gene promoter as bait. In this case, the smaller window size (17 fragments) seems to give better resolution of specific interactions with distinct putative enhancers, which are merged into one at larger window sizes. files or similar, with headed "chr", "start," and "end" columns) can also be loaded, and these are represented as rectangles flanking the relevant region on the 4C profile (Figures 4B,C). A dialog box allows the user to alter the color of the annotation and to check whether or not it is plotted. The latter feature is useful since simultaneous plotting of more than one interactions set, which often overlap, can be difficult to visually interpret. Note that whereas these are labeled "Interactions" by 4See, any region described by a bed file can be highlighted in this manner. The user can thus use this setting to highlight any feature of interest, such as called differential interactions between two 4C data sets, or the presence of specific sequence motifs (e.g. CTCF) that may be expected to be enriched at interactions.

Exporting 4See Results

Once the user settings have been finalized, a pull-down menu option allows the plot to be saved in.eps format, where it can be further processed in preparation of a figure for publication or presentation. Alternatively, the data that are actually plotted in the current 4See window (one or more quantile-normalized 4C profiles with a running mean of a specified window size applied) can be exported as bedGraph files, ready for integration into other browsers, such as local instances of UCSC [START_REF] Kent | The human genome browser at UCSC[END_REF] or IGV [START_REF] Robinson | Integrative genomics viewer[END_REF].

RESULTS

We demonstrate the usefulness of 4See on different original and previously published 4C data sets. First, we investigate the interaction between the mouse Sox2 gene and an established cluster of enhancers (the "SCR", or Sox2 control region), which has been shown to be essential for Sox2 expression in pluripotent cells (Zhou et al., 2014). Using bait primers at the SCR, we generated two biological replicates for ES cells and one after in vitro differentiation ("NPC"; Figure 3). As expected, we observed a strong interaction with the Sox2 gene which is greatly reduced on differentiation. After loading the three data sets into the 4See browser, only changing the options within one dialog box is required to switch the view from plotting the two ES biological replicates side by side (omitting the differentiated data set) to confirm that they have consistent profiles, to comparing the averaged ES profile with the differentiated one.

Second, we explored different distance ranges of promoterenhancer interactions at key developmental genes in mouse CD4 + /CD8 + (double-positive, DP) thymocytes, namely the distal (~500 kb) enhancer cluster for Satb1, and the shorterrange (~50 kb) enhancer for Ikzf1 (Figure 4). Comparing 4C plots at different running mean window sizes, it is apparent that different insights can be gained, and that no one window size is optimum for all profiles. For Satb1, shorter window sizes create what appear to be noisy profiles at the large genomic span assessed, and specific interactions are harder to discern. When the running mean window size is increased, the profile becomes smoother, and apparent peaks line up well with putative enhancers, as denoted by the presence of H3K27ac.

Conversely, at the shorter distances assessed at the Ikzf1 locus, a smaller window size allows interactions with specific enhancers to be resolved, whereas they merge into one large peak at larger window sizes. In support of this observation, we called interactions using the peakC algorithm [START_REF] Geeven | peakC: a flexible, non-parametric peak calling package for 4C and Capture-C data[END_REF] at different window sizes, and found a good visual corroboration between discernible peaks and called interactions. 4See allows rapid re-plotting of 4C profiles with different window sizes, and also has the functionalities for adding the epigenomic profile and highlighting called interactions directly on the plot.

Third, we compared the same Satb1 promoter-enhancer interaction between DP thymocytes, where the gene is highly expressed, and ES cells, where the gene is silent (Figure 5). As expected, the gene does not make any specific contacts with the thymocyte enhancer in ES cells. This locus is largely devoid of H3K27ac in ES cells, but a common problem with some browsers is that an automatic scaling creates some apparent peaks from noise on a small range of the y-axis (Figure 5B). 4See counters this by providing flexibility with how the epigenomic tracks are handled. By coercing the two tracks to the same scale, the difference between the two tissues is much more evident (Figure 5C).

Finally, we used 4See to re-analyze published 4C data, namely comparing profiles from the Hoxa5 gene between wild-type ES cells and those where one or more key CTCF insulator sites have been deleted [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF]. In this study, the authors reported that CTCF site loss caused topological defects during differentiation to neurons, with inappropriate spreading of H3K27me3. However, their analyses concluded that the topology of the Hoxa locus was largely unchanged in pluripotent cells (Figure 6A). Plotting the same data with 4See, it appears that ectopic looping interactions are formed between Hoxa5 and more caudal regions of the locus (Figure 6B). Different loop calling algorithms with different parameter choices were inconsistent in calling these apparent interactions as "significant", and only one biological replicate was available, so the importance of this observation is yet to be confirmed. In any case, the CTCF site deletion did not alter H3K27me3 patterning or Hoxa gene expression within undifferentiated ES cells [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF], so any potential topological changes do not appear to be borne out in other phenotypes. However, we wish to use this example to highlight how the use of a flexible browser like 4See facilitates exploration of the data, potentially identifying new features that "one size fits all" algorithms may overlook.

DISCUSSION

Using novel and previously published data sets for demonstration, we have shown the flexibility and utility of 4See in exploring 4C data. With limited processing of sequencing results, and one line of code in the R prompt, a user-friendly windows-based interface is available for a broader community to explore chromatin interaction profiles. As a consequence, we envisage that 4See will be of great use to the chromatin field. The input cis files are not very large (~4 MB for mouse or human), so the browser can be run on most desktop computers and laptops. The major systems limitation comes from the importing of epigenomic tracks (which can be >500 MB) with the rtracklayer package, which is the slowest step and may overload some standalone computers if too many tracks are imported at once. If the user is interested in only a specific set of baits, the system load can be reduced by restricting imports to chromosome-specific tracks. Due to the reliance of 4See plotting on quantile normalization, which is confounded by an excessive number of zeros or very small values, 4See is not an appropriate tool for visualizing very long-range (>1.5 Mb) or interchromosomal interactions; although their built-in graphical capabilities are more limited, the tools linked to algorithms such as fourSig should be used instead [START_REF] Williams | fourSig: a method for determining chromosomal interactions in 4C-Seq data[END_REF][START_REF] Walter | Benchmarking of 4C-seq pipelines based on real and simulated data[END_REF]. It should also be noted that 4See does not replace the existing suite of interaction calling algorithms [START_REF] Walter | Benchmarking of 4C-seq pipelines based on real and simulated data[END_REF]. Indeed, due to its capacity to incorporate these algorithms' results into the plots, 4See should be viewed as a complementary tool for comprehensive 4C analysis, whereby the results of the algorithms can be readily visualized and compared to epigenomic tracks for validation and obtaining biological insight. Overall, 4See, in conjunction with other analytical tools, promises to facilitate chromatin interaction exploration, and will thus be of use to the epigenetics community.

Figure 1 :

 1 Figure 1: Compaction of DNA into chromatin within the interphase nucleus. .............................................

Figure 2 :

 2 Figure 2: Schematic illustration of the transcription cycle. ..............................................................................

Figure 3 :

 3 Figure 3: Function of chromatin remodelers in nucleosome dynamics. ..........................................................

Figure 4 :

 4 Figure 4: Description of bursting parameters. ...................................................................................................

Figure 5 :

 5 Figure 5 : Schematic representation of enhancers located distally from their target gene and upregulating its expression. ........................................................................................................................................................

Figure 6 :

 6 Figure 6 : Chromatin accessibility and histone marks at enhancers ...............................................................

Figure 7 :

 7 Figure 7 : Genomic methods for predicting enhancer elements. ......................................................................

Figure 8 :

 8 Figure 8 : Schematic representation of a typical vs. super-enhancer. .............................................................

Figure 9 :

 9 Figure 9 : Gene expression regulation by multiple enhancers. ........................................................................

Figure 10 :

 10 Figure 10: Deletion and mutation of the limb enhancer of Shh (ZRS) lead to limb malformations. ...........

Figure 11 :

 11 Figure 11: Models of enhancer-promoter communication. ..............................................................................

Figure 12 :

 12 Figure 12: Enhancer-promoter looping in transcriptional control. .................................................................

Figure 13 :

 13 Figure 13: Formation of transcription hub as a possible mechanism for robust regulation of gene expression. .............................................................................................................................................................

Figure 14 :

 14 Figure 14: Genomic organization of the mouse Shh locus. ...............................................................................

Figure 15 :

 15 Figure 15: 3C-based methods to study chromatin architecture. ......................................................................

Figure 16 :

 16 Figure 16 : Chromatin structure in single cells. .................................................................................................

Figure 17 :

 17 Figure 17: Systems to fluorescently tag genomic loci in living cells. ................................................................

Figure 18 :

 18 Figure 18: Systems to fluorescently visualize RNA in living cells. ...................................................................

Figure 19 :

 19 Figure 19: Comparison between different values of α and the expected MSD behavior. ..............................

Figure 20 :

 20 Figure 20: ANCHOR is non-invasive in mouse ESCs. ....................................................................................

Figure 21

 21 Figure 21: ANCHOR does not alter chromatin topology. ..............................................................................

Figure 22 :

 22 Figure 22: Close proximity and constrained motion of Sox2 promoter and enhancer. ...............................

Figure 23 :

 23 Figure 23: Local regulatory element dynamic changes on ESC differentiation. ..........................................

Figure 24 :

 24 Figure 24 : Inhibition of transcriptional initiation slows apparent promoter diffusion speed. ...................

Figure 25 :

 25 Figure 25 : Promoter-specific local dynamics changes on enhancer disruption. ..........................................

Figure 26 :

 26 Figure 26: Simultaneous visualization of promoter-enhancer dynamics and transcriptional bursting. ....

Figure 27 :

 27 Figure 27: No apparent difference in local chromatin dynamics between bursting and non-bursting alleles. ..............................................................................................................................................................................Figure S1: Detailed positions of ANCHOR labels and deletions. ...................................................................Figure S2: Validation of ANCHOR setup. .......................................................................................................Figure S3: ANCHOR does not alter chromatin topology. ..............................................................................

Figure S4 :

 S4 Figure S4: Comparison of alternative Sox2/SCR label locations. ..................................................................

Figure S5 :

 S5 Figure S5: Transcriptional inhibition does not alter dynamics of control regions. ......................................

Figure S6 :

 S6 Figure S6: Heterozygous SCR mutations do not affect pluripotency. ...........................................................

Figure

  Figure S7: Transcriptional and post-transcriptional perturbation of Sox2 expression on incorporation of MS2 tag. ...............................................................................................................................................................

Figure A1 :

 A1 Figure A1: Two populations of promoter-enhancer distances in Anchor_Sox2-SCR ESCs. ......................

Figure A2 :

 A2 Figure A2: Proximity does not correlate with local diffusive parameters in individual movies. ................

Figure A3 :

 A3 Figure A3: Comparison of DMSO-treated and untreated Anchor_Sox2-SCR proximities and local dynamics. .............................................................................................................................................................

Figure 28 :

 28 Figure 28: Schematic illustration of the reporter gene used to measure transcribed Sox2 in living mESCs. ..............................................................................................................................................................................

Figure 29 :

 29 Figure 29: Steps of image processing and data analysis. ................................................................................

Figure 30 :

 30 Figure 30: Comparison between results obtained using Icy and further optimized with aforementioned method. ................................................................................................................................................................

Figure 31 :

 31 Figure 31: Image alignment using the GP-Tool program. ..............................................................................

Table 1 : 5 :

 15 Improper enhancer-promoter wiring causes disease. .Reagents. ...............................................................................................................................................

Figure 1 :

 1 Figure 1: Compaction of DNA into chromatin within the interphase nucleus. Adapted

Figure 2 :

 2 Figure 2: Schematic illustration of the transcription cycle. (A) Promoter opening at the core promoter (B) Pre-initiation complex assembly. (C) Promoter-proximal pausing. (D) Pause release. (E) Productive elongation and filling of paused region. Adapted from (Adelman & Lis, 2012).

Figure 3 :

 3 Figure 3: Function of chromatin remodelers in nucleosome dynamics. Remodelers use the energy from ATP hydrolysis to alter promoter architecture and expose binding sites in the promoter. Most remodelers in the ISWI family help conduct chromatin assembly and nucleosome organization which often promotes repression. SWI/SNF-family remodelers can slide and eject nucleosomes and positively stimulate transcription. Remodelers of the SWR1 family reconstruct nucleosomes by replacing canonical H2A with the histone variant H2A.Z.

Figure 5 :

 5 Figure 5 : Schematic representation of enhancers located distally from their target gene

Figure 6 :

 6 Figure 6 : Chromatin accessibility and histone marks at enhancers. (A) Active enhancers often bear the H3K27ac and H3K4me1 marks. (B) Inactive or closed enhancer regions are often marked by the active H3K4me1 and the repressive H3K27me3 marks. (C) Primed

Figure 7 :

 7 Figure 7 : Genomic methods for predicting enhancer elements. (A) ChIP-seq using antibodies against transcription factors (TF) is used in order to determine TF binding sites throughout the genome. However, TF binding sites do not always correspond to functional enhancers. (B) DNase-seq is utilized to identify active enhancers based on DNA accessibility after nucleosome removal and independently of TF binding. (C) Active enhancers are flanked by nucleosomes bearing characteristic histone marks that can be detected by ChIP-seq.

Figure 8 :

 8 Figure 8 : Schematic representation of a typical vs. super-enhancer. The higher accumulation of activators at super-enhancers is believed to contribute to higher transcriptional activity (TA). Adapted from (Hnisz, Shrinivas, Young, Chakraborty, & Sharp, 2017).

Figure 9 :

 9 Figure 9 : Gene expression regulation by multiple enhancers. (A) Multiple enhancers cooperating in one tissue to increase the expression of a shared gene. (B) Combinations of two or more enhancers (depicted by different colors) control the expression of a shared key

Figure 10 :

 10 Figure 10: Deletion and mutation of the limb enhancer of Shh (ZRS) lead to limb malformations. (A) Mice with deletion of the ZRS have gravely truncated limbs[START_REF] Sagai | Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb[END_REF]. (B-C) Hands of two patients with point mutations in the ZRS(Lettice et al., 2003).

Figure 11 :

 11 Figure 11: Models of enhancer-promoter communication. (A) Tracking and (B) Looping model. Adapted from[START_REF] Vernimmen | The Hierarchy of Transcriptional Activation: From Enhancer to Promoter[END_REF].

  developed an in situ technique termed, RNA TRAP (tagging and recovery of associated proteins), to tag and recover chromatin in the close vicinity of a transcriptionally active gene. By applying this method at the Hbb locus (encoding βglobin) in mouse erythroid fetal liver cells, Carter and colleagues demonstrated that the LCR of the β-globin gene cluster is in close physical proximity to the actively transcribing β-globin gene, located over 50 kb away, that it regulates in vivo. These results represented the first direct evidence of long-range enhancer-promoter communication and provided support for the looping model. Shortly afterwards, Tolhuis et al.[START_REF] Tolhuis | Looping and interaction between hypersensitive sites in the active β-globin locus[END_REF] used 3C (Capturing chromosome conformation) analysis(Dekker, Rippe, Dekker, & Kleckner, 2002) to demonstrate that this LCR comes in close spatial proximity with the active β-globin genes in the expressing erythroid cells, while a linear type of structure was found in non-expressing brain cells. It is worth mentioning that the communication between distal enhancers and promoters through the formation of chromatin loops does not exclude a scenario in which enhancers are communicating through a tracking mechanism[START_REF] Hatzis | Dynamics of enhancer-promoter communication during differentiation-induced gene activation[END_REF]

  promoter requires the erythroid-specific TF GATA1 and the adaptor protein Ldb1 (LIM domain binding protein 1), which is recruited to DNA via GATA1 and the transcription factors TAL1, LMO2 and E2A. In a seminal study, Deng et al.[START_REF] Deng | Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor[END_REF] used GATA1-null G1E erythroid cells in which an LCR-globin loop is absent and globin genes are inactive. In the absence of GATA1, Ldb1 is no longer recruited to the β-globin promoter but it can still bind to the LCR through the other TFs. Artificial zinc finger (ZF) tethering of the selfassociation domain (SA) of Ldb1 to the β-globin promoter in G1E cells induced a chromatin loop which was sufficient to induce robust transcriptional activation of β-globin, even in the absence of GATA1, directly demonstrating that enhancer-promoter juxtaposition can activate transcription (Figure12A).

Figure 12 :

 12 Figure 12: Enhancer-promoter looping in transcriptional control. (A) Forced looping between the β-globin (Hbb) promoter and its enhancer (LCR) via zinc finger (ZF) tethering of Ldb1 (LIM domain binding protein 1) to the Hbb promoter was sufficient to increase β-globin expression, even in the absence of the erythroid-specific transcription factor GATA1 (Deng et al., 2012). (B) Variants of forced chromatin looping use catalytically dead Cas9 (dCas9) fusion

Figure 13 :

 13 Figure 13: Formation of transcription hub as a possible mechanism for robust regulation of gene expression. TFs, Pol II, Mediators and other regulatory factors form clusters, perhaps via liquid-liquid phase-separation (LLPS), that facilitate enhancer-promoter interactions for transcriptional activation without direct juxtaposition of enhancers and promoters. Adapted from (Lim & Levine, 2021).

Figure 14 :

 14 Figure 14: Genomic organization of the mouse Shh locus. In developing limb buds, the ZRS

  FIG.1

  FIG.2

70 101

 70 FIG. 3See figure legend on opposite page.

FIG. 3

 3 FIG. 3Genetic diseases can be mediated by pathological alterations to TAD structures. (A) Schematic TAD architecture around a typical wild-type locus, whereby two TADs (triangles of different blue shades) are demarcated by a boundary (green hexagon). The left TAD contains an inactive gene (black rectangle), and the right TAD contains an active gene under the control of an enhancer (red oval). (B) Deletion of the TAD boundary causes the unification of the two TADs, with aberrant activation of gene 1, which is now contained within the same domain as the enhancer. (C) Duplication of a genomic locus containing a gene, a TAD boundary, and an enhancer can create a new TAD structure. Although the initial copy of gene 1 is still repressed, the duplicated gene may be aberrantly activated if it shares the new TAD with the duplicated enhancer. (D) Inversion or translocation events may move enhancers to different TADs, potentially causing aberrant expression of gene 1 and reduced expression of gene 2.

  FIG. 4See figure legend on opposite page.

104 Chapter 4

 1044 Stem cell chromosome folding

Figure 15 :

 15 Figure 15: 3C-based methods to study chromatin architecture. Adapted from (Bonev & Cavalli, 2016).

Figure 16 :

 16 Figure 16 : Chromatin structure in single cells. Single-cell Hi-C, polymer simulations and super-resolution DNA FISH (Bintu et al., 2018) demonstrate that population-averaged chromosome conformations arise from highly variable conformations of the chromatin fiber that occur simultaneously in single cells. Adapted from (McCord et al., 2020).

Figure 17 :

 17 Figure 17: Systems to fluorescently tag genomic loci in living cells. (A) LacO/TetO systems require the insertion of large (~10 kb) repeat binding sequences into the genome in order to visualize the locus. The ANCHOR/ParB DNA system entails introduction of a small (~1kb), non-repetitive DNA sequence to which fluorescently-tagged OR bind and spread over the surrounding chromatin. (B) Fluorescently-labeled TALEs and zinc finger proteins (ZFs) are designed to bind to a specific endogenous locus of interest. (C) CRISPR-Cas9 based approaches to visualize genomic loci in living cells using the original fluorescently tagged dCas9. Adapted from[START_REF] Shaban | Monitoring the spatio-temporal organization and dynamics of the genome[END_REF].

Figure 18 :

 18 Figure 18: Systems to fluorescently visualize RNA in living cells. (A) GFP-tagged protein MS2 (MCP) binds an array of hairpin-forming sequences engineered into the RNA of interest.Adapted from[START_REF] Wissink | Nascent RNA analyses: tracking transcription and its regulation[END_REF]. (B) Fluorescently-tagged dCas13 fused binds target RNAs in the presence of a gRNA. Adapted from[START_REF] Pichon | A Growing Toolbox to Image Gene Expression in Single Cells: Sensitive Approaches for Demanding Challenges[END_REF].

Figure 19 :

 19 Figure 19: Comparison between different values of α and the expected MSD behavior.

  (Fig 20a; Fig S1). PCR screens, sequencing and visualization of the labels by microscopy all confirmed the allelespecific and unique incorporation of the ANCH tags into their expected sites (Fig S2). As a control, we also generated an ESC F1 line with musculus allele-specific incorporation of ANCH tags at sites shifted ~60 kb, labeling presumably non-regulatory regions with maintained genomic separation (Fig 20a, b; Figs S1,

  20b). Allele-specific qRT-PCR also demonstrated that Sox2 expression from either musculus (ANCH-labeled) or castaneus (unmodified) alleles are unaltered (Fig

  Figure 20

Figure 20 :

 20 Figure 20: ANCHOR is non-invasive in mouse ESCs. a) Schematic of the different ESC lines

  Figure 21

Figure 21 :

 21 Figure 21: ANCHOR does not alter chromatin topology. Allele (musculus)-specific 4C results, with the

  Fig 22a). We did not identify large-scale spatial chromatin rearrangements within the timeframe of individual movies (median interquartile range for distances is 95 nm; maximum interquartile range for distances within one movie is 153 nm; Fig S4a). With our current setup, large nuclear contractions indicative of phototoxicity became apparent with longer imaging times, so we were unable to reliably track enhancer-promoter interactions over longer periods. However, comparisons of distance distributions between movies, while showing extensive cell-to-cell heterogeneity, revealed that the majority of cells maintain close proximity of promoter and enhancer (Fig S4a; see also Appendix 1). The average Sox2-SCR distance was significantly shorter than the distance between the control regions (median 146 nm for Sox2-SCR, 195 nm for control; Fig 22b), in agreement with the interaction differences suggested by Hi-C.

Figure 22 :

 22 Figure 22

  Fig 20a; Figs S1-2). These sites are similarly expected to participate in chromatin interactions based on Hi-C data (Fig S2a), and total and allele-specific Sox2 expression is also unaltered in this cell line (Fig S2c). In line with our ANCHOR results and contrary to what was previously reported, these labels are also frequently proximal, with a median distance (113 nm) actually slightly smaller than in the original ANCHOR line, and significantly smaller than the control label distance (Fig 22b).

the

  Sox2 locus have similar α to the other inactive regions around the HoxA locus (T2 and T3) (Fig 22c). Apparent diffusion speeds also differed across loci (Fig 22d), albeit with less

  silencing of pluripotency factor expression, including Sox2, in ANCH-labelled ESCs, with steady transcriptional shutdown almost completed by the third day (Fig 23a). Allele-specific 4C shows a concomitant reduction in interaction between the ANCH-tagged Sox2 promoter and SCR, although this is only apparent by day three of differentiation (Fig 23b). We thus performed live imaging experiments during the first three days of in vitro differentiation, whereSox2 silencing has been achieved, and cells have not completely aggregated to prevent their imaging. Cells and nuclei at day 1 of the protocol had abnormal morphology, presumably due to insufficient recovery after transfection of the OR vectors, so this timepoint was not analyzed.

Figure 23 :

 23 Figure 23

  Figure[START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF]

Figure 24 :

 24 Figure 24: Inhibition of transcriptional initiation slows apparent promoter diffusion speed. a) Violin plots for median inter-probe distance distributions, exactly as for Fig 22b, for Anchor_Sox2-SCR ESCs after treatment with different drugs. b,c) Violin plots for b) anomalous diffusion coefficient and c) apparent diffusion speed distributions of Sox2 and SCR in Anchor_Sox2-SCR ESCs after treatment with different drugs, exactly as for Figs 23d,e.

  Figure 25

Figure 25 :

 25 Figure 25: Promoter-specific local dynamics changes on enhancer disruption. a) Allelespecific qRT-PCR for Sox2 expression, normalized to SDHA housekeeping gene, in F1, Anchor_Sox2-SCR, Anchor_ΔSRR111 and Anchor_ΔSRR107,111 ESCs. Error bars represent SEM. Significant differences in gene expression were determined by t-test: p < 0.05, ** p < 0.01, *** p < 0.001. b) Allele-specific 4C results, with a region flanking near the deleted SCR region as bait, for Anchor_Sox2-SCR, Anchor_ΔSRR111 and Anchor_ΔSRR107,111 ESCs, with the same annotations as in Fig 21. The castaneus results from the unmodified allele serve as an internal control. c) Violin plots for median inter-probe distance distributions, exactly as for Fig 22b, for Anchor_Sox2-SCR, Anchor_ΔSRR111 and Anchor_ΔSRR107,111 ESCs. d,e) Violin plots for d) anomalous diffusion coefficient and e) apparent diffusion speed distributions of Sox2 and SCR in Anchor_Sox2-SCR, Anchor_ΔSRR111 and Anchor_ΔSRR107,111 ESCs, exactly as for Figs 23d,e.

(Figure 26 :

 26 Figure 26

  ). Since the MS2 tag causes reduced expression, this finding is in line with the other perturbation experiments showing that gene inactivation causes an overall reduction in Dapp and increase of α (e.g. compare with the effects of SRR deletions on Sox2 dynamics; Figs 25d,e). However perhaps surprisingly, there were no differences in constraint (Fig 27b) or apparent diffusive speed (Fig 27c) between transcribing and non-transcribing loci. Tracking of a greater number of cells will be required to lend greater support to these preliminary findings. Sox2 contains high levels of paused RNA polymerase in mouse ESCs (Williams et al., 2015) (Fig S1). It therefore appears that paused and actively transcribing genes reside in a sufficiently similar nuclear microenvironment to have essentially the same local chromatin diffusive properties, further supported by unchanged dynamics on treatment with transcriptional elongation inhibitor, flavopiridol (Figs

Figure 27 :

 27 Figure 27

Figure S1 :

 S1 Figure S1: Detailed positions of ANCHOR labels and deletions. ChIP-seq tracks for H3K27ac, CTCF, RNA polymerase II and total RNA (GEO: GSE49847) are shown around the Sox2 locus alongside the positions of the different ANCHOR labels (black dashed lines) and Sox2 gene (blue).(i-iv) denote zooms at 25 kb regions, encompassing (i) Sox2, (ii) the intervening control region, (iii) the SCR, and (iv) the downstream control region. Red dashed lines delimit the SRRs that are deleted on the musculus allele in Anchor_ΔSRR111 and Anchor_ΔSRR107,111 ESCs.

Figure S2 :

 S2 Figure S2: Validation of ANCHOR setup. a) Virtual 4C plots derived from ESC Hi-C data (Bonev et al., 2017), using the Sox2 promoter (top) or intervening control region location (bottom) as bait (filled black line). The location of the "interacting" region where ANCHOR labels are incorporated are denoted with dashed black lines. b) Representative double-label images for Anchor_Sox2-SCR, Anchor_Control and Anchor_Alexander ESCs, showing unique ANCH3 (red) and ANCH1 (green) spots at proximal sites in the nucleus, indicating that both tags have been inserted in the same allele. Scale bar: 2 μm. c) Allele-specific qRT-PCR for Sox2 expression, normalized to SDHA housekeeping gene, for F1, Anchor_Sox2-SCR (with or without transfection of OR proteins) and Anchor_Control ESCs. Error bars represent SEM. Significant differences in gene expression were determined by t-test: p < 0.05, ** p < 0.01, *** p < 0.001.

Figure S3 :Figure S4 :

 S3S4 Figure S3: ANCHOR does not alter chromatin topology. Non-allele-specific 4C results, with the SCR as bait, for F1 and Anchor_Sox2-SCR (with or without transfection of OR proteins) ESCs. Position of Sox2 gene is shown underneath in blue, and ChIP-seq tracks for H3K27ac and CTCF (GEO: GSE49847) are also shown below.

Figure S5 :

 S5 Figure S5: Transcriptional inhibition does not alter dynamics of control regions. a) qRT-PCR results, normalized to SDHA housekeeping gene, for Anchor_Sox2-SCR ESCs after treatment with different drugs. Error bars represent SEM. Significant differences in gene expression were determined by t-test: p < 0.05, ** p < 0.01, *** p < 0.001. b) Exactly as for a, but in Anchor_Control cells. c) Violin plots for median inter-probe distance distributions, exactly as for Fig 22b, for Anchor_Control ESCs after treatment with different drugs. d,e) Violin plots for d) anomalous diffusion coefficient and e) apparent diffusion speed distributions of control loci in Anchor_Control ESCs after treatment with different drugs, exactly as for Figs 23d,e.

Figure S6 :Figure S7 :

 S6S7 Figure S6: Heterozygous SCR mutations do not affect pluripotency. qRT-PCR results, normalized to SDHA housekeeping gene, for F1, Anchor_Sox2-SCR, Anchor_ΔSRR111 and Anchor_ΔSRR107,111 ESCs. Error bars represent SEM. Significant differences in gene expression were determined by t-test: p < 0.05, ** p < 0.01, *** p < 0.001.

Figure A1 :

 A1 Figure A1

Figure A2 :

 A2 Figure A2

Figure A3 :

 A3 Figure A3: Comparison of DMSO-treated and untreated Anchor_Sox2-SCR proximities and local dynamics. Violin plots for median inter-probe distance (top), anomalous diffusion coefficient of Sox2, SCR and control regions (middle), and apparent diffusive speed of Sox2, SCR and control regions (bottom), in untreated or DMSO (drug control)-treated Anchor_Sox2-SCR or Anchor_Control ESCs. The diffusive properties of the Sox2 promoter are also shown for the Sox2_Alexander line for comparison.

  , the ANCHOR/ParB system does not interfere with nucleosome formation, nor create fragile sites. Similarly (and beyond), I demonstrated that insertion of ANCH sequences and their binding with OR proteins show no change in overall Sox2 expression, neither affect markers of pluripotency despite genetic modification of the locus. Furthermore, by performing allele-specific and canonical 4C from the SCR bait I showed that Sox2-SCR interactions are maintained in wild-type (F1) and ANCHOR (Anchor_Sox2-SCR) cells, regardless of whether OR proteins are expressed. Collectively, these results highlight the noninvasive nature of ANCHOR compared to large and repetitive lac or tet operators, which can silence surrounding chromatin and create fragile sites (M.[START_REF] Dubarry | Tight protein-DNA interactions favor gene silencing[END_REF][START_REF] Jacome | Lac operator repeats generate a traceable fragile site in mammalian cells[END_REF] or dCas9 binding which can repress transcription of a target gene by interfering with RNA Pol II binding, transcription elongation, or TF binding[START_REF] Qi | Repurposing CRISPR as an RNA-γuided platform for sequence-specific control of gene expression[END_REF]. As it has been discussed previously, the correct insertion of the ANCH sequences for each locus in the musculus (129) allele of F1 ESCs was verified using PCR with several pairs of primers that span the unique homology arms. Nevertheless, it would be interesting to combine ANCHOR with DNA FISH, using probes against Sox2 and SCR, or ImmunoFISH, for simultaneous visualization of OR proteins bound to DNA, as another means to show how accurately the ANCH sequences represent Sox2 and the SCR.

with 24 ×

 24 MS2 loops at the Sox2 3′ UTR and stable expression of MCP-mScarletI, OR1-EGFP and OR3-IRFPThe targeting vector containing the 24xMS2 cassette (5'HA-T2A-LoxP-HSV_TK-T2A-Hygro R -LoxP-24XMS2-3'HA) (Figure28), as well as the vector containing Cas9 and a scaffold to encode the sgRNA targeting the region upstream of the endogenous Sox2 stop codon were designed and assembled by Dr. Tineke Lenstra. For introducing 24XMS2 loops in the Sox2 locus, 1 million Anchor_Sox2-SCR ESCs were transfected with 2 μg sgRNA-Cas9 vector and 2 μg targeting Sox2-MS2 vector using Lipofectamine 2000 Transfection Reagent (Invitrogen). Cells were given three days to recover and were then subjected to 200 µg/ml hygromycin selection for nine days. Once a stable cell population was obtained we performed single cell sorting and seeded cells into 96-well plates. Once individual colonies were formed, clones were screened by PCR and heterozygous clones with a correctly targeted 129 allele were further controlled by sequencing to confirm the correct insertion of the 24xMS2 array. the targeting vector. In this way, two bands were expected after heterozygous insertion of the loops (197 and 82 bp length), while only one band (155 bp) was present in the wt allele. Additional PCRs with allele-specific primers spanning the whole engineered region and single-molecule FISH (smFISH) (see below) were performed in order to confirm the insertion of the 24xMS2 loops in 129 allele. Primer sequences are given in Table

Figure 28 :

 28 Figure 28: Schematic illustration of the reporter gene used to measure transcribed Sox2 in living mESCs.

  Lipofectamine 2000 Transfection Reagent (Invitrogen). After incubation for three days, cells were subjected to selection with 6 μM gancyclovir for ten days. Cells containing thymidine kinase (labeled as HSV-TK, Figure28) did not survive after addition of gancyclovir and only the cells with successful LoxP removal survived. Once a stable cell population was obtained we performed single cell sorting and let individual clones grow for approximately ten days. To confirm the removal of the resistance marker (Hygro), smFISH was performed (see below) and PCR over the insertion was used to select cells based on fragment length. A single clone (H3H1A1, see Table3) with fully excised LoxP-HSV_TK-T2A-Hygro R resistance cassette was used for all further experiments.To generate cells stably expressing OR1, OR3 and MCP (MS2-coat protein) fluorescent fusion proteins, 1 million H3H1A1 cells, containing a 24 × MS2 cassette integrated in the 3′ UTR of 129 allele, were transfected with 1 μg ePiggyBac transposase expression plasmid (System Biosciences) and 1 μg epB-MCP-mScarletI, epB-EF1α-NLS-OR1-GFP, epB-EF1α-NLS-OR3-IRFP plasmids (OR1-EGFP and OR3-IRFP producing vectors available from NeoVirTech; were modified from the original source by changing the fluorescent protein at the C-terminus, introducing a Kozak sequence before the translation start site, introducing an Nterminal nuclear localization signal (NLS), and replacing the CMV promoter with EF-1α. For my thesis, both vectors were kindly provided by Pr. Kerstin Bystricky) using Lipofectamine 2000 (Invitrogen). Nine days after transfection, fluorescent cells were sorted in bins (low, medium and high) based on the expression of the fluorescent proteins. Imaging analysis was done for all bins to determine the optimal expression of the three fluorescent fusion proteins.

Figure 29 :

 29 Figure 29: Steps of image processing and data analysis.

Figure 30 :

 30 Figure 30: Comparison between results obtained using Icy and further optimized with aforementioned method. 500 particles were simulated to reproduce particles observed in microscopy sessions[START_REF] Oliveira | Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes[END_REF].

Figure 31 :

 31 Figure 31: Image alignment using the GP-Tool program. White arrows point to regions with pronounced effects caused by chromatic aberrations. After alignment algorithm is deployed, both channels are properly superposed.

  Nuclei were isolated in Lysis buffer (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% NP-40/Igepal CA-630, 1X protease inhibitor). Purified nuclei were digested overnight (O/N) at 37˚C with the first restriction enzyme (DpnII, 1500U final; New England Biolabs) and posteriorly subjected to an overnight proximity ligation at 16 ˚C with 20,000 U of T4 DNA Ligase (New England Biolabs). After ligation, chromatin was de-crosslinked and purified after proteinase K and RNAse A treatment. Circularized DNA was then linearized by a second digestion with 5 U of Csp6I (Thermo Fisher) per μg of DNA, O/N at 37˚C, followed by final O/N ligation (5ng/μl final DNA concentration) at 16˚C with T4 DNA Ligase (200 U per μg of DNA) and DNA purification using phenol/chloroform extraction.

  (red); Index (blue); Sequence specific primer (black)

FIGURE 1 |

 1 FIGURE 1 | Overview of the 4C method and analysis. (A) The 4C method entails chromatin fixation, restriction digestion and re-ligation to generate hybrid sequences between fragments that were physically proximal during fixation. The DNA is purified, digested with a secondary restriction enzyme and re-ligated under dilute conditions to generate DNA circles. Chimeric products linked to a specific bait fragment of interest (orange) are amplified by inverse PCR with bait-specific primers (orange arrows) flanked by Illumina sequencing adapters (black overhangs). The PCR products are then directly loaded onto Illumina flow-cells for highthroughput sequencing. (B) Pre-processing steps before 4See; tools denoted in bold accompany this manuscript. The fastq sequences are first trimmed to remove bait restriction fragment sequence (orange), leaving just the prey DNA sequence (green) for mapping to the reference genome with Bowtie. The mapped genomic coordinates are converted to restriction fragment space by a custom perl script, which counts the total number of reads mapping to each fragment on the same chromosome as the bait. This "cis" file can then be directly input into 4See.

FIGURE 2 |

 2 FIGURE 2 | Overview of graphing options from existing 4C analysis methods. All methods have been applied to two ES replicates and one NPC 4C data set for the Sox2 SCR bait (see also Figure3). (A) 4Cseqpipe (van deWerken et al., 2012) results shown independently for one ES replicate and the NPC data set. Running median scores are plotted as a line graph (5 kb resolution), with domainograms plotted underneath as a heat map for median scores at steadily increasing window sizes. Positions of the CTCF site within the SCR and the Sox2 gene are indicated by arrows. Note that the independent normalization means that the SCR-Sox2 interaction differences between the two cell types is not evident, compared to other methods. (B) r3Cseq[START_REF] Thongjuea | r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data[END_REF] results for the combined three data sets, showing panels, from top to bottom: positions of Refseq genes; restriction fragment coverage; averaged profile for the "experiment" (ES) condition, with called interactions at different confidence levels highlighted; profile for the "control" (NPC) condition, with called interactions at different confidence levels highlighted; plot of the log2-ratio of experiment vs control 4C signal. The position of the SCR bait is indicated by a red dashed line. Note that r3Cseq appears to call a very large number of (continued)

FIGURE 2 |

 2 FIGURE 2 | interactions. (C) fourSig[START_REF] Williams | fourSig: a method for determining chromosomal interactions in 4C-Seq data[END_REF] results shown independently for one ES and one NPC replicate, showing smoothed 4C plots(21fragment windows). Called interactions ("Categories" 1, 2 or 3, for different confidence levels) would be highlighted on the plot, but none were called by fourSig. (D) FourCSeq[START_REF] Klein | FourCSeq: analysis of 4C sequencing data[END_REF] results shown independently for one ES and one NPC replicate, showing normalized and processed 4C signal plots (gray line graphs and black points), alongside the positions of known genes. The green line indicates the centralized 4C value, and the dashed blue lines indicate the threshold values for a z-score difference > 2. Significant interactions would be highlighted, but were not detected by FourCSeq. Note that differences between the two cell types is not so evident, compared to most other methods. (E) peakC[START_REF] Geeven | peakC: a flexible, non-parametric peak calling package for 4C and Capture-C data[END_REF] results shown for the combined analysis of the two ES replicates, independently of the NP replicate, giving smoothed 4C plots (21-fragment windows) as histograms. The red regions indicate called interactions. (F) 4C-ker[START_REF] Raviram | 4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments[END_REF] results shown for the combined analysis, with line plots of combined ES (red) and NPC (blue) 4C signal. Note that many interactions were called by 4C-ker within the plotted window for both ES and NP, but that the documentation did not provide a means to plot them alongside the shown line plots.

FIGURE 3 |

 3 FIGURE 3 | 4See provides flexibility in handling multiple replicates and/or experimental conditions. (A) The 4See dialog box for conditions settings automatically opens when cis files are first loaded, in this instance two ES replicates and one NPC 4C data set for the Sox2 SCR bait. The two ES replicates have been assigned different integers to be treated independently, and the NPC data set has been omitted by assigning it 0. (B) The resultant 4See plot from the conditions set in (A), whereby the two ES 4C replicates are quantile normalized and plotted separately, one in black, the other gray. The plot has normalized 4C signal as the y-axis and genomic coordinate of the interacting fragment as the x-axis. The position of the SCR bait is denoted by a black vertical line, and gene position (blue arrows) and the ES H3K27ac ChIP-seq profile (black) is shown underneath the 4C plot. The profiles are highly consistent between replicates, with a strong interaction peak centered on the Sox2 gene; note that both the gene and enhancer have a strong enrichment for H3K27ac. (C) As for (A), but in this case the two ES replicates are given the same value to be averaged together, and the NPC data set is included as a different integer to the ES data sets. (D) As for (B), but with the settings conditions of (C), and the redundant gene and H3K27ac tracks omitted. The averaged ES 4C plot is given in black and the NPC 4C plot in red, showing a strong perturbation of the SCR-Sox2 interaction on differentiation.

FIGURE 4 |

 4 FIGURE 4 | 4See provides flexibility in running mean window sizes. (A) The main control panel of 4See, including options to set the x-axis ("start coordinate," "end coordinate," and "plot window") and y-axis ("max y plot") plot limits, to choose a bait name for the plot title ("bait name"), and to set the running mean window size ("smooth window") in number of restriction fragments. (B) 4See plots for a 4C data set in DP thymocytes with the Satb1 gene promoter as bait. Two instances of the same x-and y-axis limits are shown, with a running mean window of 21 (left) or 55 (right) fragments. The position of the Satb1 bait is denoted by a black vertical line, and gene position (blue arrows) and the DP H3K27ac ChIP-seq profile (black) is shown underneath the 4C plots. Pink rectangles denote regions called as interacting by peakC for the equivalent window size as the plot. For the long-range interaction, the smaller window size appears to have more spurious called interactions, less evidently linked to H3K27ac peaks; the link is better seen with greater smoothing from a larger window size. (C) As for (B), but with the Ikzf1 gene promoter as bait. In this case, the smaller window size (17 fragments) seems to give better resolution of specific interactions with distinct putative enhancers, which are merged into one at larger window sizes.

Ben

  Zouari et al. 4See Frontiers in Genetics | www.frontiersin.org January 2020 | Volume 10 | Article 1372 8

FIGURE 5 |

 5 FIGURE 5 | 4See provides flexibility in handling epigenomic track scales. (A) The same 4C profile as Figure 4B (55-fragment window size; black) is plotted alongside the 4C profile for ES cells, where the locus is silent, and thymocyte enhancer interactions are not evident. (B) The 4See dialog box for managing epigenomic tracks defines how the different tracks are scaled. In this case, the ChIP-seq tracks for H3K27ac in ES and DP cells are treated independently, so the autoscaling of the ES track creates some spurious peaks from noise above background. (C) As for (B), but this time the two H3K27ac tracks have been set the same integer, making the lower ES signal much more visually apparent in the plot.

FIGURE 6 |

 6 FIGURE 6 | 4See exploration can uncover previously overlooked features of chromatin topology. (A) Reproduced Supplemental Fig 7A from Narendra et al. (2015), used with permission. 4C profiles from the Hoxa5 bait are shown as domainogram heat maps for wild-type ES cells (top), as well as lines that have had deletions of one CTCF site (middle; site of deletion denoted by red asterisk) or two (bottom; additional deletion site denoted by green asterisk). The CTCF ChIP-seq profile is shown above the 4C sets.No chromatin topology differences are apparent between these cell lines, and the original study concluded that spatial phenotypes only occurred on cell differentiation[START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF]. (B) The same data, processed and plotted using 4See. The position of the Hoxa5 bait is denoted by a black vertical line, and gene position (blue arrows) and the ES CTCF ChIP-seq profile (black) is shown underneath the 4C plots. Red and green asterisks denote the positions of the single and double CTCF site deletions, as for (A). In this plot, CTCF-dependent restriction of interactions between Hoxa5 and more caudal regions (e.g. the gene body of Hoxa10) seems apparent.

La dynamique chromatinienne de Sox2 est cohérente avec les hubs et/ou condensats transcriptionnelles

  Sox2 est réduit au silence. En accord avec les résultats de l'étude 4C, les distances moyennes entre Sox2 et SCR ont augmenté de manière significative (médiane=205 nm) trois jours après la différenciation des CSE induite par l'acide rétinoïque (AR), mais les sondes étaient encore relativement proches dans l'espace, et beaucoup plus proches que les distances moyennes séparant Sox2 et SCR dans les CSE proposées par une étude précédente(Alexander et al., 2019). Pour examiner directement si et comment la topologie de la chromatine est liée à l'activation des gènes, j'ai marqué l'ARNm Sox2 natif de l'allèle musculus des CSE marquées ANCHOR avec des boucles MS2[START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF], fournissant une lecture directe de la transcription du locus Sox2 endogène. L'imagerie en direct par triple marquage a permis de mesurer simultanément la proximité de Sox2 et de SCR et la transcription du gène. En tenant compte de mes résultats sur la distance de séparation enhancer-promoteur et les paramètres de diffusion de la chromatine, il est tentant de spéculer qu'un tel hub transcriptionnel pourrait englober Sox2 et SCR, contraignant ainsi leur mobilité par rapport aux régions de contrôle. Puisque la séquence contrôle est également moins contrainte, elle peut

	Des recherches récentes ont mis en évidence l'existence de hubs transcriptionnelles qui créent
	des microenvironnements locaux, dans lesquels les enhancers et les promoteurs peuvent
	partager des clusters communs de facteurs Pol II et de facteurs de régulation (Lim & Levine,
	2021).

. Bien que ma configuration expérimentale actuelle ne permette pas la visualisation simultanée des quatre régions, il est fort probable qu'il y ait des cellules individuelles où les régions témoins sont plus proches que Sox2-SCR. Des travaux futurs sont nécessaires pour solidifier cette hypothèse.

Une expérience très ambitieuse consiste à réaliser un ANCHOR à triple marquage afin de déterminer si, et à quelle fréquence, les paires Sox2-SCR sont réellement plus proches que les paires Sox2-Inter contrôle et SCR-Inter contrôle.

Outre la caractérisation de la topologie de la chromatine à l'état pluripotent, où Sox2 est exprimé, cette étude a également permis de mieux comprendre les distances de séparation entre les promoteurs et les enhancers aux premiers stades de la différenciation in vitro des CSE, où Bien que l'imagerie d'un plus grand nombre de cellules soit nécessaire pour confirmer ces résultats préliminaires, je constate ici que Sox2 et SCR sont légèrement plus proches pendant la transcription active et que le sous-ensemble de loci Sox2 en transcription active présente des distances Sox2-SCR significativement plus proches par rapport à la distribution totale des expériences en double marquage. Ensemble, ces résultats correspondent à un modèle dans lequel la proximité physique entre l'enhancer et le promoteur est en quelque sorte corrélée à l'activation de la transcription. Néanmoins, lorsque j'ai évalué le lien entre la transcription de Sox2 et l'architecture locale de la chromatine par d'autres moyens, en particulier après l'inhibition de l'initiation de la transcription lors du traitement au triptolide, qui réprime l'expression de Sox2 à des niveaux presque indétectables, et après les délétions de sites critiques au sein du SCR, qui réduisent fortement l'expression de Sox2 (>4 fois), je n'ai observé aucun effet sur la proximité Sox2-SCR. Dans l'ensemble, ces résultats suggèrent que le gène Sox2 et son enhancer, sont fréquemment à proximité, mais que la distance de séparation exacte n'est pas nécessairement en corrélation avec la transcription. À la lumière de ces résultats et de ceux d'autres chercheurs

(Benabdallah et al., 2019 ;[START_REF] Lim | Visualization of Transvection in Living Drosophila Embryos[END_REF]

, le modèle classique de bouclage doit être révisé. La juxtaposition physique directe des promoteurs et des enhancers ne semble pas être requise (ni suffisante) pour que la régulation transcriptionnelle se déroule; au contraire, au moins pour Sox2, la communication entre les éléments semble être possible lorsqu'ils se trouvent dans un certain rayon, dont la taille reste à déterminer. sortir du hub transcriptionnel et ainsi être plus libre d'explorer le nucléoplasme. Mes résultats sont donc globalement cohérents avec un modèle dans lequel les promoteurs et les enhancers résident, sans nécessairement se juxtaposer directement, dans le même hub transcriptionnel, qui semble contraindre le mouvement local de la chromatine. Cependant, mes résultats ne soutiennent pas un modèle simple selon lequel les hubs contiennent exclusivement des loci transcriptionnellement actifs. Les contraintes chromatiniennes sont largement inchangées lors du traitement par des médicaments inhibant la transcription et, ce qui est plus révélateur, il n'y a pas de différences évidentes dans la dynamique des allèles transcriptionnellement actifs ou inactifs au sein des CSE de type sauvage. Les liens, s'ils existent, entre les hubs transcriptionnelles et les vitesses de diffusion apparentes semblent encore plus obscurs.

Le chargement de la Pol II et un effet plus indirect de la transcription sur la dynamique de la chromatine ?

  Une explication possible est que dans les CSE de type sauvage, presque tous les allèles du locus Sox2 fortement exprimé sont "prêts" à être activés transcriptionnellement dans le même microenvironnement nucléaire permissif, qu'ils synthétisent ou non l'ARNm au moment de l'acquisition de l'image. La dynamique de la chromatine est probablement déterminée par l'environnement local et ne devrait donc pas différer. Pour preuve, le promoteur de Sox2 et le début du gène contiennent des niveaux élevés de Pol II en pause dans les CSE, une caractéristique connue des gènes en pause et rapidement inductibles

À première vue, mes résultats semblent contenir une grande contradiction. Les résultats du double marquage après divers moyens de perturber l'expression de Sox2 (différenciation, traitement avec des inhibiteurs de transcription, délétion des régions de l'enhancer) indiquent tous un lien direct entre la transcription et la dynamique de la chromatine, l'expression perturbée entraînant une réduction générale de la contrainte au niveau de l'enhancer et du promoteur, et une réduction plus spécifique de la vitesse apparente de diffusion au niveau du promoteur. Cependant, lors du marquage simultané de la transcription naissante de Sox2, il n'y a pas de différences apparentes dans les propriétés diffusives moyennes entre les allèles transcriptionnellement actifs ou inactifs, que ce soit au niveau du promoteur ou de l'enhancer.

Table 1 : Improper enhancer-promoter wiring causes disease.

 1

  , colocalizing in hubs that present high occupancy by those factors (e.g., Oct4-Nanog) (deWit et al., 2013). How is gene regulation achieved by the local environment? It is tempting to speculate that, inside these hubs specialized nuclear foci -or clusters-with a characteristic

	uncovered frequent proximity of enhancers to the target gene within ~100-to 200-nm-sized
	clusters, composed of ~10-30 RF molecules, within the vicinity of the TSS, thus linking multi-
	enhancer clusters with RF accumulation and transcriptional activation: Enhancer hubs locally
	concentrate RNA Pol II RFs and create local microenvironments that can be sampled by
	multiple gene promoters (J. Li et al., 2019; Jieru Li et al., 2020). Surprisingly, when another
	imaging-based technique, called Hi-M (Cardozo Gizzi et al., 2019), was applied to two clusters

composition of regulatory and transcription factors are formed, whereby individual cisregulatory elements aggregate and switch on multiple genes simultaneously, in a cooperative rather than competitive manner, to establish robust gene expression patterns. Whether enhancer hubs represent actively transcribing regions remains largely obscure, but colocalization between enhancers, regulatory factors (RFs), Pol II and nascent transcription would be consistent with such a scenario (S.

Kim & Shendure, 2019;[START_REF] Lim | Enhancer-promoter communication : hubs or loops ?[END_REF]

. Initial supportive evidence comes from single-molecule live imaging of clustering of the TF Bicoid in early Drosophila embryos

[START_REF] Mir | Dense bicoid hubs accentuate binding along the morphogen gradient[END_REF]

, however such ideas have mostly been hypothetical since clustering could not be assigned to specific genes. More direct evidence comes from single-gene and single-molecule optical nanoscopy techniques which enabled simultaneous imaging and tracking of regulatory factors (e.g., Sox2, Brd4 and Mediator), Pol II, nascent transcription and cis-regulatory elements at target key pluripotency genes (Pou5f1, Sox2 and Nanog) in mESCs (J.

[START_REF] Li | Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells[END_REF][START_REF] Li | Single-gene imaging links genome topology, promoter-enhancer communication and transcription control[END_REF]

. These imaging experiments of key developmental genes in Drosophila embryos, multi-enhancer hubs were found to frequently arise, independently of transcriptional activity, and with rare engagement of promoters

[START_REF] Espinola | Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development[END_REF]

. Formation of these developmental hubs is partly dictated by the pioneer TF Zelda. Further studies will be required to see whether multi-enhancer hubs are ubiquitous features of transcriptional control, and how they coordinate with promoters to regulate gene expression.

  [START_REF] Li | Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells[END_REF][START_REF] Li | Single-gene imaging links genome topology, promoter-enhancer communication and transcription control[END_REF]. Evidence for this model comes from systematic characterization of the effects of mutations that abolish specific recognition of DNA and chromatin targets or IDR-IDR interactions of Sox2 and Brd4, at specific active-gene loci (Jieru[START_REF] Li | Single-gene imaging links genome topology, promoter-enhancer communication and transcription control[END_REF]. While IDR-deletion mutants incorporated into clusters, with similar

	efficiencies as those of the respective wild-type proteins, Sox2 with deleted or mutated HMG
	DNA-binding domain and Brd4 with mutated bromodomains involved in acetyl-lysine
	recognition did not. These results demonstrate that Sox2 and Brd4 clustering in mESCs nuclei
	relies on specific recognition of multiple cognate DNA and chromatin binding sites, rather than
	IDR-IDR interactions, indicating spatial clustering of distal cis-regulatory elements and target
	genes.
	. Interestingly,
	disruption of MED1 and BRD4 condensates upon 1,6-hexanediol treatment, which is known
	to disrupt liquid-like condensates (Yi Lin et al., 2016), was accompanied by reduced MED1
	and BRD4 occupancy at super-enhancers and loss of RNA Pol II occupancy at SE-associated
	genes, implying that loss of these condensates can impact transcription. In the context of
	transcription, IDR-IDR interactions between different molecules of the transcriptional
	machinery -including Pol II-might occur, thus facilitating the loading of the cluster on specific
	genomic loci.

The second model proposes that clustering is dominated by binding of multiple regulatory factors to a scaffold of cognate DNA and chromatin binding sites (S.

Kim & Shendure, 2019;

J.

Together, these results point out that different mechanisms may regulate enhancer enhancerpromoter communications. Whether phase separation is essential for 3D genome organization is under intense debate, however increasing evidence supports a role for these condensates (S.

Kim & Shendure, 2019;[START_REF] Lim | Enhancer-promoter communication : hubs or loops ?[END_REF]

. Nevertheless, tools to study phase separation in vivo are still in their infancy, and have been primarily focused on live imaging experiments on tagged protein components; the dynamics of specific gene loci relative to their residence in nuclear microenvironments is largely unknown. Elegant optogenetic clustering of IDRs can modulate phase separation (Y.

Shin et al., 2017)

, and this has been combined with dCas9targeting (CasDrop) to repetitive sequences to induce droplets consistent with transcriptional activation to heterochromatin (Y.

[START_REF] Shin | Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome[END_REF]

but such experiments have yet to be realized on single-copy gene loci, let alone determination of their effects on specific gene expression.

  . Using super-resolution 3D-FISH and 5C assays,

	Benabdallah et al. observed greater rather than decreased separation between Shh and its
	enhancers SBE6, SBE4, and SBE2/3 during differentiation of ESCs to neural progenitors and
	in vivo. Activation from a distance, using TALE-VP16 targeting either the Shh promoter or the
	distal enhancers, recapitulated the decreased enhancer-promoter proximity identified during
	ESC differentiation.

Consistently, interrogation of the spatial organization and the activity of Sox2 and its distal enhancer (SCR) in living mouse ESCs, showed no association between Sox2-SCR spatial proximity and Sox2 transcription

(Alexander et al., 2019)

. Sox2-SCR separation distances were ranging between 200-400 nm (mean ~340 nm) in the nucleus of mouse ESCs, while the same label pairs displayed greater proximity in differentiated cells, where the SCR is inactivated, compared to ESCs. Incorporation of an MS2 reporter

[START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF]

into Sox2, for direct visualization of nascent transcription in real-time, showed no correlation between Sox2-SCR distances and transcriptional activity, neither prior to nor during transcriptional bursts. This locus is the focus of my thesis work, and direct comparison and discussion of my results relative to this study (which was published during my thesis work) is detailed in later sections (see Results and Discussion).

  2, where Sox2 expression is still more than 50% of ESC levels (median 157 nm), but are increased by day 3 (median 205 nm) (Fig23c). The difference is currently not statistically significant (p = 0.09; Wilcoxon rank sum test) because only 15 movies have been obtained for day three; more data acquisition is ongoing to obtain a sufficient sample size. We also observe changes in Sox2 chromatin dynamics on differentiation: both the gene promoter and enhancer become less constrained (Figs 23d), in line with an apparent greater constraint at active elements (Fig22c;[START_REF] Germier | Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement[END_REF]. Notably, the relaxation of the promoter had already occurred by day 2, before any obvious loss of enhancer proximity, as assessed by ANCHOR or 4C. Interestingly, diffusion speed was reduced significantly at the Sox2 promoter, again a

day before detectable enhancer proximity differences, with only a weak, non-significant reduction observed for the SCR (Fig

23e

). Overall, these results suggest that promoterenhancer proximity correlates somewhat with transcriptional output, but that local chromatin dynamics precede these changes. Promoters and enhancers appear to be more constrained when active, whereas the Sox2 promoter but not the SCR has increased diffusive speed on activation.

  However despite the large discrepancy in distance measurements, both studies put the classical enhancer-promoter looping model into question.Whereas I observed greater separation of Sox2 and SCR during very early stages of in vitro ESC differentiation,Alexander et al. reported greater proximity between Sox2-SCR after full differentiation to neuronal precursors. Additionally, increased separation distances were observed between Shh and Shh brain enhancers (SBEs) during neuronal differentiation, where Shh is active(Benabdallah et al., 2019). While the latter could be attributed to the different genomic context, Sox2-SCR opposing results could arise from the different stages of differentiation monitored in each study.Alexander et al. proposed that the entire Sox2 region adopts a more compact conformation upon ESC differentiation. Longer differentiation experiments of my Anchor_Sox2-SCR mESCs are required to confirm whether the Sox2 locus is configured into a compact structure, and if and how this affects promoter-enhancer proximity and local dynamics.

	labelling systems.
	. Perturbations
	which convert poised Sox2 loci to a more refractory state may do so by altering the nuclear
	microenvironment, with subsequent effects on chromatin dynamics. For example,
	differentiation causes bulk changes in Pol II loading at the promoter, as well as loss of TF
	binding at both promoter and the SCR; these changes may directly reduce local chromatin
	mobility constraints, or indirectly via dissociation from transcriptional hubs. Triple-label
	experiments performed over much longer time periods may also uncover if any wild-type loci
	transition between the proposed "refractory" and "poised" states, determined via longer-term
	alterations in local diffusive parameters. Interestingly, chromatin dynamics were largely
	unaltered on treatment with the transcription elongation inhibitor, flavopiridol, but promoter
	dynamics were sensitive to treatment with the transcription initiation inhibitor, triptolide,
	suggesting that Pol II recruitment and/or promoter melting, but not processive elongation, are

  with slight modifications. The procedure is based on the presence of SNPs creating allele-specific restriction sites on one of the two alleles of hybrid F1 ESCs, thereby enabling amplification and direct analysis of either the 129 or the CAST allele. Instead of replacing the second restriction enzyme (Csp6I) with the "new" allele-specific restriction enzyme, we performed a normal 4C (DpnII and Csp6I digestion) and before the PCR amplification step we digested the 4C template with the appropriate restriction enzyme and purified the DNA using phenol/chloroform extraction. For the "Distal SCR" bait, AvaII restriction enzyme was used for 129-specific 4C-seq. A second viewpoint ("Proximal SCR") was used for the Anchor_ΔSRR111 129-/CAST+ cell line, since the "Distal SCR" region was deleted from the genome. For the "Proximal SCR" bait, Alw26I restriction enzyme was used for 129-specific 4C-seq and BveI for CAST-specific 4C-seq. removed from EtOH and washed with 1-2 ml freshly prepared pre-warmed wash buffer (40 ml of Ultrapure nuclease-free H 2 O, 5 ml of 20x SSC, 5 ml of deionized formamide) for 5 minutes at 37 o C. Wash buffer was aspirated and coverslips were let dry for few minutes. 50 μl of resuspended probe hybridization mix was applied on a parafilm (placed inside a Petri dish) and coverslips were gently placed on top of each drop (cells facing the drop) to avoid bubbles.A humid kimwipe was placed next to the coverslips, the Petri dish was sealed with parafilm and placed O/N at 37 o C. After the overnight incubation, coverslips were moved into a new 12well containing 2 ml of pre-warmed wash buffer and incubated for 30 minutes at 37 o C. The washing step was repeated for three times in total, each wash at 37 o C for 30 minutes. Coverslips were then rinsed with 2xSSC and washed with 1xPBS for 5 minutes at RT. Samples were mounted in Prolong Gold with DAPI (Invitrogen) on glass slides (VWR), let to dry for 24 hours in the dark, and then transferred to -20 o C or directly imaged.

	Imaging was performed on Zeiss AxioObserver 7 inverted wide-field fluorescence microscope
	with LED illumination (SpectraX, Lumencor) and sCMOS ORCA Flash 4.0 V3 717
	(Hamamatsu). A 40x oil objective lens (NA 1.4) with 1.6x Optovar was used. 27 z-stacks were
	imaged from -4 to 4 μM with 0.3 μM steps and 1x1 binning. An exposure time of 500ms was
	used for Cy3 (100% LED power), 750ms for Cy5 (100% LED power) and 25ms for DAPI
	Typically, 600 ng of the resultant 4C DNA template were used to generate 4C-seq libraries by (10% LED power). Micromanager 1.4 software was used. Image quantification was carried out
	performing a PCR using Expand Long Template PCR System (Roche) with target-specific using a custom Python pipeline. The scripts are available upon request to Lenstra Lab. Images
	designed reading and non-reading primers (see Table 4) containing Illumina sequencer were compressed to 3D image z stacks -4 to 4 µM. Cell and nuclear masks were determined
	adapters. The 4C template was PCR amplified for 27 cycles and 6 reactions were pooled using a custom Python algorithm. Spots corresponding to Sox2, MS2v6 and Hygromycin
	together. Optimal PCR conditions were found using a control template 4C material, comprising transcripts were then counted for cells and nuclei.
	genomic DNA that is digested with the secondary enzyme and re-digested to make circularized
	4C templates containing only the contiguous genomic sequence linked to the bait (Karasu &
	Sexton, 2021). Then, generated 4C-seq libraries were purified with SPRI select beads
	(Beckman Coulter) to discard primer dimer DNA products and 4C-seq DNA template were
	quantified using Bioanalyzer and pooled equimolarly for sequencing on Hiseq 4000 sequencer
	(IGBMC Genomeast platform) using single-end 50 bp reads (100 bp for the "Proximal SCR"
	bait).

were

Table 3 :

 3 Cell lines.

	Cell Line	Clone	Parental line	Description	gRNA sequence	Location (mm9)	System used
	Anchor_Sox2-SCR	36	F1 ESCs	CCCCTGGACAGCAAC AGCAG Sox2-109.4T ANCH1/+ CCCAGCCTACCTCGA Sox2-5.5C ANCH3/+ ACTCA	chr3:34,542,624 -34,544,520 chr3:34,657,473-34,659,303	CRISPR-Cas9
	Anchor_Control	18	F1 ESCs	Sox2-55C ANCH3/+ Sox2-170T ANCH1/+	GTTCAAAAACTAGA AACA CCTTGCAAGCACAAG GACGC	chr3:34,603,689-34,605,222 chr3:34,718,340-34,720,139	CRISPR-Cas9
	Anchor_Alexande r	B56	F1 ESCs	Sox2-8C ANCH3/+ Sox2-117T ANCH1/+	TATGTTAAGCTAGTT TCTGA GTAAGCTATCTCATT GCCCG	chr3:34,539,890-34,541,689 chr3:34,665,107-34,666,618	CRISPR-Cas9
							TAGCATCTGGCCAAG
	Anchor_ΔSRR11 1	3	Anchor_Sox2 -SCR	ΔSRR111 129/CAST+	GAATG CCCAACGTACATGTT	chr3:34,659,726-34,661,602	CRISPR-Cas9
							TTTGT
							AGCACAAAATAAAA
							TTTAAG
							CTATGCACATGCTGG
	Anchor_ΔSRR10 7.111	30	R111 Anchor_ΔSR	AST+ ΔSRR107.111 129/C	GACCA	34,658,026 chr3:34,656,540-	nickase Cas9
							ACTAGAGCTCAACCT
							TGGCC
							GGTTAGTTCTCTTCA
							GCAAG
	Anchor+MS2+Hy groR	H3H1	Anchor_Sox2 -SCR	24xMS2 loops introduced in the Sox2 locus	GCCAGCCCTCACATG TGCGAC	chr3:34,549,639-34,550,797	CRISPR-Cas9
	Anchor+ HygroR	MS2-	H3H1A1	Anchor+MS2 +HygroR	Removed resistance (Hygro R ) marker		Cre recombinas e
					Cells	stably
					express
					PiggyBac
	Anchor_Sox2-MS2		H3H1A1	vectors MCP-mScarletI; epB-epB-EF1α-NLS-		PiggyBac transposon system
					OR1-GFP; epB-
					EF1α-NLS-
					OR3-IRFP

Table 4 :

 4 Primers.

	Primer Name	Sequence
		"Anchor_Sox2-SCR" PCR screening

  1,2,3,4 * 1 Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France, 2 CNRS UMR7104, Illkirch, France, 3 INSERM U1258, Illkirch, France, 4 University of Strasbourg, Illkirch, France

TABLE 1 |

 1 Comparison of graphing options from existing 4C analysis methods. 4Cseqpipe No Several custom scripts to convert fastq files to multiple formats and intermediate files. These failed for test data and intermediate files had to be made manually. No Requires bed file of restriction fragments and bedgraph of 4C coverage per observed fragment. Custom scripts to generate from sam files failed and input files had to be made manually.

	Method	GUI Preprocessing	Handles conditions/replicates	Plotting options	Annotations
			No	Can alter trendline resolution and	Limited:
				plotting window (coordinate	manually
				space); domainogram parameters	curated bed file
				fixed	gives arrows on
					plot
	r3Cseq	No Processes bam files directly	Yes, but restricted to pairwise	Can only alter plotting window	RefSeq genes
			comparison of "experiment" and
			"control" conditions
	fourSig	No Custom script converts bam to input format	No	Can only alter plotting window	None
	FourCSeq No Need to set up metadata table in R, which points to	Handled in one combined object,	Can only alter plotting window	Positions of
		processed bam files	but default is to plot each data set		genes from
			individually, and documentation		transcriptome
			does not say how to do otherwise.		(unlabeled)
	4C-ker		Yes	In principle, many settings can be	None
				changed in the R command
				prompt (ggplot2 call settings), but
				is not documented or user-friendly
	peakC	No Essentially the same as this manuscript, but utility	Handles replicates but not different	Can only alter plotting window	None
		scripts not provided	conditions

4See Frontiers in Genetics | www.frontiersin.org January 2020 | Volume 10 | Article 1372 5

Acknowledgments

ACKNOWLEDGMENTS

Sequencing was performed by the IGBMC GenomEast platform, a member of the France Genomique consortium (ANR-10-INBS-0009). We thank Claudine Ebel and Muriel Philipps for their support within the flow cytometry platform.

FUNDING

This study was supported by funds from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Starting Grant 678624-CHROMTOPOLOGY), the ATIP-Avenir program, and the grant ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d'Avenir ANR-10-IDEX-0002-02. YBZ is supported by funds from LabEX INRT, la Region Grand Est and the ERC. AP is supported by funds from LabEX INRT, the ERC, and the Ligue Nationale Contre le Cancer. AM is supported by funds from IDEX (University of Strasbourg) and the Institut National du Cancer. TS is supported by INSERM.

DATA AVAILABILITY STATEMENT

Raw 4C data and the processed cis files first described here are available on GEO (GSE137417); previously published 4C results [START_REF] Narendra | CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation[END_REF] are available as data set GSE60240. ChIPseq tracks were obtained from GEO for mouse ES (Shen et al., 2012) (CTCF and H3K27ac; GSE29218) and DP cells [START_REF] Vanhille | High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq[END_REF] (H3K27ac; GSE63732).

Abbreviations

3C

Chromosome conformation capture 4C

Chromatin architecture and topology

During my thesis I wrote a book chapter (Platania & Sexton, 2020), giving an overview of how chromatin folding is linked to cell fate decisions via transcriptional regulation, focusing on pluripotent and somatic cells, as well as particular cases (e.g. mitosis) where chromatin folding appears very different.

ES CTCF

ES H3K27ac

Normalized 4C signal

Appendix 1

Batch effects and variability within Anchor_Sox2-SCR line

The Anchor_Sox2-SCR line was the first to be generated, and has been used for experiments over a longer time period than other lines (Anchor_Control or Anchor-Alexander) or its derivatives (Anchor_ΔSRR111, Anchor_ΔSRR107,111 and Anchor_Sox2-MS2), undergoing more passages. When the inter-probe distance distributions of all of the accumulated doublelabel movies for Anchor_Sox2-SCR are processed together, the cells strikingly group into two populations, a population where the SCR and Sox2 promoter are very close (median distance ~110 nm), and a group where they are noticeably farther apart (median distance ~225 nm). In the violin plots this creates a clear bimodal distribution, with not much density around the population median. Although it may have been interesting to speculate that these different distances could represent two functional states of the locus, such as transcriptionally active or competent versus silent/refractory, this pattern is not observed for any other cell line or in fact the DMSO-treated Sox2 results are nearly identical to that obtained in the alternative Sox2 probe site used in the Anchor_Alexander cell line. Although the reasons for the discrepancies in the untreated Anchor_Sox2-SCR results are unclear, there is sufficient evidence to suggest that the specific batches of results with DMSO treatment on the same cell line are more reliable representations of physiological Sox2 loci. The DMSO-treated Anchor_Sox2-SCR and DMSO-treated Anchor_Control datasets were thus used throughout the analyses in favor of the untreated ones. Further batches of experiments will be required to attain more robust measurements from untreated Anchor_Sox2-SCR cells, perhaps with stable integration of OR proteins instead of transient transfections, which may generate batch-tobatch variation.

Discussion

In this study, with a combination of genome editing and multi-color live-cell imaging, I was able to extract some very interesting features about the separation distances and the local chromatin dynamics of regulatory elements, an aspect that has been overlooked in most previous studies of genome functions. Overall, I found that the Sox2 gene and its cognate ES enhancer are frequently in close spatial proximity, but that the exact distance does not necessarily correlate with transcriptional firing. Instead, transcriptional activity is generally associated with alterations in local chromatin dynamics, whereby regulatory regions are generally more constrained than "neutral" sequences, and apparent diffusive speed is increased at promoters when genes are activated. Alterations in chromatin dynamics correlate with gene activity (summarized in Table 2), but loci participating in active transcriptional bursts do not have notably different chromatin dynamics to poised loci.

Comparison to previous findings

During my thesis another laboratory published its work, in which they interrogated the spatial organization and activity of Sox2 and SCR in living F1 mESCs (Alexander et al., 2019). By utilizing cuO and tetO arrays to label the promoter and enhancer regions at the 129 allele, the authors demonstrated that Sox2 and SCR are separated by much greater distances (mean ~340 nm), compared to the ones reported in my study, in the nucleus of mESCs. I directly assessed this discrepancy by introducing ANCHOR into the exact same genomic locations of this study, finding that these probes were also frequently proximal. I propose therefore that the large separation distances reported by the authors (Alexander et al., 2019) are due to technical differences, possibly caused by the large repetitive tet/cu operators. Notably, the authors included a control line, where a 111 kb fragment has been deleted between the cuO and tetO pairs, leaving a 14 kb tether between them (Alexander et al., 2019). Curiously, the mean separation distance observed was ~250 nm and never below 100 nm, as has been previously

reported for DNA FISH probe pairs separated by few tens (~30-60) of kilobases [START_REF] Giorgetti | Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription[END_REF]. I assume that the large separation distances reported by Alexander et al. (Alexander et al., 2019) are caused by the integration of multiple copies of repetitive operator sequences and the stable binding of the fluorescent-tagged repressors.

Furthermore, in this study, by using a Sox2-MS2 system, we measured much higher bursting frequencies compared to what was previously reported (Alexander et al., 2019). These differences might be caused by the resistance selection marker that was maintained in the locus in the other study (excised from my Anchor_Sox2-MS2 cells), and could further perturb Sox2 transcription and mRNA stability. Indeed, I observed that the line maintaining the selectable marker gene, exhibited reduced nascent mRNA and protein levels, as measured by smFISH and western blot. These results confer an additional value to my ANCHOR and MS2-MCP

Materials & Methods

ESC culture

The mouse F1 (M.Musculus

Generation of stable cell lines containing ANCH1 and ANCH3 sequences

The presence of a single nucleotide polymorphism (SNP) in the PAM sequence of each sgRNA target on the M. Castaneus (CAST) allele caused preferential insertion of the ANCH sequences on the M.Musculus 129 (129) allele (Zhou et al., 2014). The three ("Anchor_Sox2-SCR", "Anchor_Control" and "Anchor_Alexander", see Table 3) ANCHOR transgenic lines were generated by allele-specific ( 129) CRISPR/Cas9-mediated knock-in experiments in the following way. First, flanking homology arms (HA) (see Table 3) were amplified from a bacterial artificial chromosome (BAC RP23-274P9) accommodating the Sox2 locus using the Q5 High-Fidelity DNA Polymerase (New England Biolabs) and introduced into a vector containing either ANCH1 [START_REF] Saad | DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living[END_REF] according to the supplier's recommendation. Transfected cells were allowed to recover for two days, cultured for 24h with 3 μg/ml puromycin and then for 48h with 1 μg/ml puromycin. After

4C-seq data analysis

Fastq sequencing files were demultiplexed and the bait sequence, up to but not including the DpnII site, were trimmed with the Sabre tool (https://github.com/najoshi/sabre) before mapping to the mm9 genome with Bowtie (Langmead, Trapnell, Pop, & Salzberg, 2009). The mapped reads were then processed and visualized by the 4See tool [START_REF] Ben Zouari | 4See: A Flexible Browser to Explore 4C Data[END_REF].

Virtual 4C plots

All fastq files from ES Hi-C data (Bonev et al., 2017)were downloaded and re-mapped to the mm9 genome assembly and normalized using FAN-C [START_REF] Kruse | FAN-C: a feature-rich framework for the analysis and visualisation of chromosome conformation capture data[END_REF]. The Angeliki PLATANIA

Résumé

Le lien entre la dynamique de la chromatine et la transcription reste flou, notamment parce que très peu de loci ont été évalués. Dans cette étude, en utilisant le locus autour du gène de la pluripotence Sox2 comme modèle, j'ai réalisé un double marquage pour suivre la communication et la mobilité du promoteur et de l'enhancer, couplé à un marquage de l'ARN naissant de Sox2 et étudié la dynamique de la machinerie protéique transcriptionnelle.

Contrairement à une étude précédente, j'ai observé que Sox2 est très proche de son enhancer mais aussi, que leur distance de séparation n'est pas totalement corrélée avec l'activation de la transcription. La perturbation de l'expression de Sox2 lors de la différenciation des cellules, des délétions de parties de l'enhancer et de l'inhibition de l'initiation transcriptionnelle, ont montré que la mobilité est augmentée spécifiquement au niveau du promoteur de Sox2 en même temps que l'activation de la transcription. Cependant, les loci participant à l'activation de la transcription ne présentent pas une dynamique chromatinienne sensiblement différente lorsqu'ils sont transcriptionnellement inactifs.

Résumé en anglais

The link between chromatin dynamics and transcription remains largely unclear, not least because so few loci have been assessed. In this study, using the locus around the pluripotency gene Sox2 as a model, I performed double-label ANCHOR to track promoter and enhancer communication and mobility, coupled to labelling of nascent Sox2 RNA and studies of the dynamics of the transcriptional protein machinery. Contrary to a previous study, I observed that Sox2 is in very close proximity with its enhancer, but also that the exact separation distance does not exactly correlate with transcriptional bursting. Perturbation of Sox2 expression upon differentiation, deletions within the enhancer and inhibition of transcriptional initiation, showed that the apparent diffusive speed is increased specifically at Sox2 promoter together with transcriptional activation. However, loci participating in transcriptional bursts do not have notably different chromatin dynamics to poised loci.

Key words: Sox2, chromatin dynamics, live-imaging, transcription, RNA Pol II

Assessing the links between enhancer-promoter proximity, local chromatin dynamics and gene activity