Liz

Aatgacttacccatggatcc Liz

Agtatgcaggcggtcgctcc Liz

Cttccttctactgctgcagg Liz

Acaactggagcaagtcattt Liz

Gtgtcactttgatagtccct Liz

Caaaggccaagtctcattcc Liz

Tggccttaaatggtttacgt Liz

Gtagaacctgaagacagcac Liz

Catgcttccttttgtcaagg Liz

Gtttcctttggcaaagctag Liz

Ctccctaaacacagggcgta Liz

Agcaaacgtgagaattctcc Liz

Cggcaagatgagtgccctac Liz

Tcctttaaggtgtcctgtgg Liz

Tggaacctacagggaagaag Liz

Ctgcttgctggagtctgatg Liz

Gaaaagttcctgctgcccag Liz

Gtgctacatagtacaagcca Liz

Ggtgtgacatgctttaattc Liz

Gccctgaaagacactagtac Liz

Tgaataactgaattggccca Liz

Agggaactcatcaaattgct Liz

Agcacagtataggagttctt Liz

Ggccattgtgagctacataa Liz

Tacacaggtatagggcagaa Liz

Tctgtgttgtatacacaggt Liz

Cagcgttgacatctgagtca Liz

Ggctttcttaaatagtagac Liz

Agctatactccatggtcaca Liz

Aacatcagtagagcatggta Liz

Tggcaataaagttgccactt Liz

Agtgtgacagcatgaaactt Liz

Ggaccagtaattgcttggaa Liz

Ggatccagctcgatagtact Liz

Aaattgtagcctgtgctagg Liz

Gtgcagtggataacacatcg Liz

Tgctgtaattacagagctgc Liz

Cacgtttgaaaaatatagtc Liz

Tgggaataataaccagctca Liz

Cgtgctttctggatctcatg Liz

Ctccatgcactgtgtaagtc Liz

Agagatcaacttgcaattct Liz

Attgagggcttttggaccac Liz

Atgctataaatttgcgtgca Liz

Ggcaggaaaggaaatcaaac

TRANSIENT TRANSCRIPTION IN THE EARLY EMBRYO SETS AN EPIGENETIC STATE THAT PROGRAMS POSTNATAL GROWTH

Keywords: méthylation de l'ADN, empreinte parentale, développement embryonnaire, génétique de la souris, croissance DNA methylation, genomic imprinting, embryo development, mouse genetic, growth. RESULTS

L'empreinte parentale est un mécanisme de régulation épigénétique qui réduit l'expression d'environ 120 gènes à une seule dose parentale. L'expression monoallélique dépend de marques différentielles de méthylation de l'ADN, établies dans l'ovocyte et le spermatozoïde et maintenues après fécondation dans l'individu en développement. Chez les mammifères, les gènes soumis à empreinte sont essentiels au développement embryonnaire et à certaines fonctions comportementales et physiologiques après la naissance. Définir les mécanismes de régulation et la fonction des gènes soumis à empreinte est une question cruciale en biologie du développement et en pathologie. Mon travail de thèse a concerné l'étude fonctionnelle du locus Zdbf2 chez la souris. Zdbf2 est un gène exprimé paternellement, conservé chez l'humain, mais dont la fonction était inconnue. J'ai pu démontrer que l'activation de Zdbf2 dans le cerveau de souris pré-pubères dépend d'un signal épigénétique indélébile mis en place dès les premiers jours de développement embryonnaire. Cette programmation précoce de Zdbf2 assure une croissance normale du nouveau-né. Mes résultats indiquent de plus que la dose, mais pas l'origine parentale de Zdbf2 est essentielle. Ces découvertes ont été possibles par la création de divers modèles mutants avec des variations de la dose de Zdbf2 dans l'axe hypothalamo-hypophysaire. Ce travail met en lumière la fonction cruciale d'un gène soumis à empreinte, de sa régulation dans l'embryon précoce à son rôle sur la physiologie adulte.

I am grateful to all the members of my jury who accepted to evaluate my PhD work: Pr. Anne Ferguson-Smith and Dr. Andrew Pospisilik, reviewers, Pr. Anthony Isles and Dr. Michel Cohen-Tannoudji, examiners, Dr. Frederique Peronnet, president of the jury. It is a great honor to be evaluated by such inspiring scientists and I hope they will appreciate reading my work. I thank them in advance for spending some of their time on my PhD work.

I also thank the members of my PhD advisory committee who helped me and gave me great advices in the course of my PhD: Edith Heard, my tutor; Claire Rougeulle, my scientific expert and Stephane Ronsseray, the doctoral school supervisor.

It is kind of crazy for me to realize it is already the end of my PhD… and I finally got all the failure I was dreaming of … ;) I have joined Deborah's lab more than 4 years ago and this period has probably been the most exciting of my life. Being a PhD student in this lab has been a wonderful experience and I feel I grew up so much scientifically and personally. This has been possible only because I have been extremely lucky to be surrounded by the most inspiring people and I have constantly learnt from them. I would like to spend some time thanking everyone who contributed to this adventure.

Deborah has been an incredibly supportive PhD supervisor. To honor you, I can promise you I will try to do my best to make the least French spelling errors that I can! I would like to thank her for the scientific training she gave me and particularly for trusting me. I accomplished things I had never thought I would be capable of and that's only because she constantly encouraged me to do them. This to me is the most important quality of a PhD supervisor. Deborah is not only a very smart, talented and inspiring scientist but she is someone is pleasant to work with: she knows how to create a tight-knit team.

Working in the Bourc'his team has been an everyday happiness and I seriously cannot think about a better group of people to work with, they are just the coolest scientists I have ever met and we all became true friends. I would like to particularly thank Max, my second mentor. Max is a real encyclopedia of science and it has been a real pleasure to team up with him on the first part of my PhD project. A huge part of my work would not have been possible without his input. Moreover, even if he is the worst secret keeper in the world, he is definitely the coolest American I have ever met! To complete the crew of our bay area, I would like to sincerely thank Joan and Raquel, for helping me during all my panic attacks in the lab, for their precious technical help, their constant support and all the rest, how would I have survived in the lab without you? Jojo, you saved my first Mendeley crisis, so a promise is a promise, here is the first citation of this PhD manuscript, a breakthrough paper of 2016: [START_REF] Barau | The DNA methyltransferase DNMT3C protects male germ cells from transposon activity[END_REF]. Tomek, you not only deserve the title of the weirdo of the lab but also the prize of the cleanest experiments and the nicest co-worker (exequo with Raquel though). Julian, you saved me more than once during the path of my PhD with your precious technical support from embryology to informatics! Aurelie, you are the little bioinformatic angel of the lab, thank you for being always so available. Finally, Mathilde, the latest addition to the team and the only one who accepted to be the new organization master in-chief. I am more than happy to handover you the PhD crown, I am sure you will make a good use of it! But of course I cannot wrap up this PhD without a thought for my two first models PhD students in the lab (each in their own way!), Marius and Soso, vous m'avez tant manqué au labo! Marius, may be see you in SF!! I would like also to thanks Mattia, the most sociable person I have ever met, for his precious help with the study of the sDMR-KO mice.

Among the many things I learnt this past few years, the importance of a scientific and sociable environment to succeed is one of them. The BDD is without a doubt the best Department, worldwide. I sincerely thank all the people that I have interacted with during my PhD, they all have been extremely inspiring. I would like to especially thank Edith Heard, the director of our Unit who has been incredibly supportive, having a model of such a talented scientific woman around gave me a daily motivation. I thank all the members of Edith's team, for their scientific interaction, their help and their friendship. Particularly, a huge MERCI to Lucile who is the only colleague capable of staying in the lab with you until 5am to support you writing your PhD. BDD is not only synonymous with an exceptional place to do science but also with the best party environment! Thank you to everyone who made these PhD years so much fun, they are numerous and I will never forget them. However, what happened in BDD, stays in BDD!! The Xmas party has always been my favorite one and you can meet special people… J. Finally, thank you to the WoS !!!!!!!! I also thank Colin, the true hero of this PhD! Nothing would have been possible without his precious help and organization on our mouse colony. Travailler avec toi a été un immense plaisir. I also thank all the animal house facility people and Fatima for great skills on the transgenesis; she is also an incredibly nice person.

Finally, I want to thank all the people who contributed, before, to my decision of making a PhD.

Pour finir, je ne peux pas mettre un point final à l'écriture de cette thèse sans remercier la deuxième partie de ma vie, tous ceux qui ne sont pas au labo mais sans qui ces 4 dernières années n'auraient pas été aussi fun! J'ai une bande d'amis tout simplement extraordinaire et je remercie chacun d'entre eux (vraiment tous, même les pires ;)) pour m'avoir supportée, pour être les plus drôles, les plus fous et les plus imprévisibles. La « bande à Basile » m'a fait rire au quotidien durant ces dernières années au labo. Les savoir tous loin de moi sera définitivement la chose la plus difficile lors de mon départ en post-doc… Un grand merci tout particulier à tous les colocs du 16, et aussi à cet appartement, signe de mon indépendance parisienne, qui en a vu de toutes les couleurs! Chloé, Momo, Justine vous êtes les meilleures colocs et notre amitié peut aujourd'hui survivre à tout. Merci à Andreï pour la rendre si heureuse. Justine, ma fan numéro 1, tu es devenue une des personnes les plus importantes de ma vie ces dernières années et tu es sans aucun doute de celles qui me manqueront le plus, je ne suis pas sure de pouvoir me débrouiller en liberté dans un autre pays sans toi ! Une pensée toute particulière à Isma, qui est si petite mais qui prend tant de place dans mon coeur ;).

Mais aussi un GRAND merci à Momo, Junior (oui tu as gagné ta place ici !) et David pour le soutien, l'aide et le réconfort intense de ces derniers jours. David, merci d'être devenu un mec sans principe; on se suit scientifiquement depuis maintenant 8 ans, alors tu viens avec moi où que j'aille en post doc ? Une pensée aussi à Juliette, pour toutes ces petites vacances salvatrices à Bordeaux et à Sarah pour que nos folies de lycéennes continuent encore longtemps ! Enfin, merci à Toto, fidèle à son poste, qui est là depuis si longtemps.

Je remercie ma soeur pour son extrême soutien depuis le premier jour où j'ai décidé d'étudier la Biologie. Mais plus particulièrement, merci à Mumu et Franck, d'une part pour avoir mis au monde deux personnes qui ont soudainement pris une place immense dans ma vie et d'autre part pour être mon modèle de vie. Vous m'avez apporté bien plus que ce que vous pouvez imaginer.

Enfin, merci à mes parents, leur soutien, leur confiance et leur amour sont le moteur de ma vie. Maman, ton courage et ta volonté me donnent la force pour tout. Papa, tu es mon inspiration pour chaque décision de ma vie, merci. J'espère aujourd'hui pouvoir vous rendre un peu plus fiers.

Résumé des travaux

Le génome des mammifères contient plusieurs milliers de gènes, présents en deux copies provenant des deux génomes parentaux. Alors que la plupart sont exprimés de façon égale à partir des deux copies parentales, une centaine de gènes dits « soumis à empreinte parentale » ont la particularité de n'être exprimés qu'en une seule dose parentale. Ce phénomène d'empreinte se met en place lors du développement embryonnaire, via des mécanismes de régulation épigénétique. Notamment, la méthylation de l'ADN joue un rôle crucial dans le contrôle de l'expression des gènes soumis à empreinte parentale de par ses fonctions inhibitrices de la transcription. Les génomes du sperme et de l'ovocyte arborent des profils de méthylation de l'ADN très différents. Cependant, alors que ces différences sont globalement effacées dans l'embryon après fécondation, la vingtaine de régions de contrôle de l'empreinte parentale (ICR, imprinting control region) sont capables de maintenir ces marques de méthylation différentielles héritées des gamètes. Ces régions génomiques sont responsables de la régulation mono-allélique de l'expression des gènes soumis à empreinte parentale. Chez les mammifères, l'expression mono-dose de ces gènes dans les tissues embryonnaires et somatiques a été montrée comme essentielle, d'une part pour le développement de l'embryon et d'autre part pour des fonctions physiologiques et neurologiques après la naissance. Chez l'humain, plusieurs maladies neuro-développementale telles que les syndromes de Prader-Willi ou d'Angelman sont associées à des dérégulations de gènes soumis à empreinte parentale.

Mes travaux de thèse ont concerné l'étude fonctionnelle du locus soumis à empreinte parentale Liz/Zdbf2. Ils ont permis d'apporter de nouvelles pierres à l'édifice des connaissances dans le domaine de l'empreinte parentale, en adressant deux problématiques majeures sur : 1) le rôle d'une nouvelle forme d'empreinte restreinte aux premiers jours du développement embryonnaire et 2) l'importance du contrôle de la dose dans la fonction d'un gène soumis à empreinte parentale.

1) Quelle est le rôle de l'empreinte transitoire chez les mammifères ?

Alors que l'empreinte parentale classique existe tout au long de la vie post-natale, la découverte d'une forme d'empreinte existant seulement pendant les premiers jours du développement de l'embryon de souris est particulièrement intrigante (Proudhon et al., 2012). Cette empreinte transitoire a-t-elle un rôle restreint à l'embryon précoce ou permet-elle de mettre en place une mémoire cellulaire à long terme ? Quel pourraitêtre le rôle de gènes soumis à empreinte parentale transitoire dont l'expression monoallélique est restreinte à l'embryon pluripotent ? Afin d'élucider le rôle de l'empreinte transitoire chez la souris, la première partie de mon projet de thèse s'est centrée sur l'étude du locus Liz/Zdbf2, localisé sur le chromosome 1 murin et conservé sur le chromosome 2 humain [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF].

Liz (long isoform of Zdbf2) est un transcrit alternatif du gène Zdbf2 (zinc finger DBFtype containing 2) qui prend naissance en amont de son promoteur canonique. Dans l'embryon après fécondation, alors que Zdbf2 est réprimé, Liz est exprimé exclusivement à partir de l'allèle paternel à cause de marques de méthylation de l'ADN au niveau de son promoteur sur le chromosome maternel. Cependant, au moment de l'implantation de l'embryon (à environ 4 jours et demi de développement chez la souris), le promoteur de Liz gagne de la méthylation sur l'allèle paternel, ne permettant plus l'initiation de la transcription de Liz qui ne sera plus jamais exprimé après implantation et est donc restreint à l'embryon précoce. En utilisant un modèle de souris de perte de fonction de Liz dans l'embryon, généré par la méthode CRISPR/Cas9, mes travaux ont démontré, in vivo, le rôle de l'expression transitoire de Liz. Alors que l'expression de Liz dans l'embryon précoce est dispensable pour le développement embryonnaire, elle est essentielle pour l'activation de Zdbf2 dans le cerveau après la naissance. La transcription de Liz à partir de l'allèle paternel induit des modifications de la chromatine : le remplacement de marques d'histones inhibitrices H3K27me3 par de la méthylation de l'ADN en amont du promoteur canonique de Zdbf2 favorise son activation à partir du même allèle. De façon importante, mes résultats montrent que les souris mutantes pour Liz présentent un défaut de croissance post-natal, démontrant pour la première fois le rôle de l'empreinte transitoire dans la programmation d'un phénotype adulte (Greenberg et al., 2017).

2) Quelle est la fonction biologique de Zdbf2 dans le cerveau adulte ?

Lorsque j'ai commencé mon travail de thèse, très peu d'éléments était connus quant à la fonction du produit de Zdbf2, hormis la présence d'un domaine de type zinc-finger, homologue à la protéine DBF4 chez la levure. Mes résultats sur les mutants Liz indiquent qu'une perte d'expression de Zdbf2 induit un défaut de croissance post-natal.

Afin de caractériser d'avantage la fonction du gène Zdbf2 chez la souris adulte, j'ai généré un mutant génétique de Zdbf2 conduisant à une perte de fonction de la protéine. L'étude de cette lignée de souris m'a permis d'une part de confirmer le rôle de Zdbf2 dans la croissance post-natale et d'autre part, de caractériser de façon précise le phénotype résultant d'une perte d'expression de Zdbf2 : la moitié des souris meurent rapidement après la naissance, tandis que l'autre moitié présentent un défaut de croissance général très tôt après la naissance, culminant à l'âge de 2 semaines et persistant de façon stable chez l'adulte. L'expression de Zdbf2 dans le cerveau adulte étant majoritaire au niveau de l'axe hypothalamo-hypophysaire au moment de la puberté, nous pensons que Zdbf2 pourrait agir sur la sécrétion d'hormones hypophysaires contrôlant la croissance après la naissance.

Une question majeure dans le domaine de l'empreinte parentale est de comprendre pourquoi les gènes soumis à empreinte parentale doivent être exprimés de façon mono-allélique et parent-spécifique pour exercer leurs fonctions. Est-ce la dose ou l'origine parentale qui importe ? Bien que de nombreuses études sur des gènes soumis à empreinte parentale aient montré que des modulations de la dose influencent les phénotypes, des travaux adressant l'importance de l'origine parentale manque. Zdbf2 est exprimé uniquement à partir de l'allèle paternel. Afin d'adresser cette question au locus Zdbf2, nous avons généré deux types des mutants supplémentaires : l'un exprimant Zdbf2 de façon bi-allélique à la fois à partir des allèles paternel et maternel, et l'autre n'exprimant Zdbf2 qu'en une seule dose, comparable aux souris sauvage, mais à partir de l'allèle maternel et non paternel. Alors que les souris arborant une augmentation de Zdbf2 présentent une croissance augmentée, les souris exprimant Zdbf2 maternellement ont un poids similaire aux souris sauvages.

Ceci m'a permis de démontrer formellement que, dans le cas du gène Zdbf2, seule la dose et non pas l'origine parentale importe pour sa fonction sur la croissance postnatale.

Preface

Mendelian inheritance is probably one of the most famous principles in Biology. Gregor

Mendel proposed these fundamental laws in 1865, following more than four years of genetic experiments with pea plants. Mendel notably reported that genes come in pairs and are inherited equally from each parent. One of the rare exception to this dogma of heredity is genomic imprinting. Conversely to what Mendel predicted, the expression of a subset of genes, the so-called "imprinted genes", is not equally transmitted from both parents. This phenomenon, which escapes the "traditional" rules of genetic transmission, has fascinated me since the first time I have heard about it, which I am pretty sure was during one of my university class, given by Dr. Deborah Bourc'his.

In the following Introduction, I will first describe the history of genomic imprinting from what has been reported initially in insects and plants and then focus on mammalian genomic imprinting, by addressing the major discoveries from the pioneers in the field. In a second part, I will describe the epigenetic mechanisms that direct the regulation of this parent-of-origin gene-silencing phenomenon. Imprinted genes have been well recognized as essential for mammalian development and physiological functions after birth. I will address in a third part the evolution and functions of imprinted genes in mammals. Finally, during the course of my PhD, while characterizing one of those imprinted genes, I was confronted to an endocrine phenotype regulated by the hypothalamo-pituitary axis in mice. I will thus dedicate a fourth part to basic notions regarding the development and function of this major axis.

I would like to particularly acknowledge the reviews that were used as starting material to write this Introduction: [START_REF] Bartolomei | Mammalian genomic imprinting[END_REF]Ferguson-Smith, 2011;[START_REF] Messerschmidt | DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos[END_REF]Peters, 2014;[START_REF] Plasschaert | Genomic imprinting in development, growth, behavior and stem cells[END_REF][START_REF] Rizzoti | Genetic regulation of murine pituitary development[END_REF][START_REF] Sanmiguel | DNA methylation dynamics of genomic imprinting in mouse development †[END_REF]Seah and Messerschmidt, 2018;[START_REF] Efstratiadis | Genetics of mouse growth[END_REF] 1. The discovery of genomic imprinting: an historical overview

Genomic imprinting is defined as the non-equivalence of the two parental genomes in diploid species. This inequality is due to an asymmetric mark that controls the differential behavior of the maternal and paternal genomes and causes parent-of-origin gene expression. The first observation of a parent-of-origin phenomenon was made almost a century ago [START_REF] Metz | Chromosomes and sex in Sciara[END_REF] but the term "imprinting" has been coined for the first time in 1960, to describe the sex determination mechanism adopted by Sciaridae flies (also known as fungus gnats) [START_REF] Crouse | The controlling element in sex chromosome behavior in Sciara[END_REF]. In the 1970`s, some cases of parent-of-origin effects were phenotypically observed in insects, plants and mice [START_REF] Johnson | Hairpin-tail: a case of post-reductional gene action in the mouse egg[END_REF][START_REF] Kermicle | Dependence of the R-mottled aleurone phenotype in maize on mode of sexual transmission[END_REF][START_REF] Khosla | Genomic imprinting in the mealybugs[END_REF]. However, since this phenomenon did not fully obey to Mendel's laws of heredity, it took some time for the scientific community to recognize the existence of genomic imprinting. In the field of mammalian biology, the first experimental proofs of the existence of parental genomic imprinting were made by [START_REF] Surani | Imprinting and the initiation of gene silencing in the germ line[END_REF]Solter in 1984 (Barton et al., 1984;McGrath and Solter, 1984a;[START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF]. Since these discoveries, huge efforts have been put toward the description of the process of imprinting and how it controls the monoallelic and parent-of-origin expression of imprinted genes in mammals. In this part, I will develop the major discoveries that have been made since the first use of the term "imprinting". I will briefly mention the parent-of-origin effects described in insects and plants to place the field in its context of the first observations that have been reported;

I will then focus on the origins of the concept in mammals.

Parent-of-origin effect in insects

1.1.a First observations

In 1925, studies by Metz on meiosis in Sciara males, a genus of the Sciaridae flies (Figure 1A), described a phenomenon of elimination of a complete haploid set of paternal chromosomes during meiosis I (Metz, 1925, Figure 1B). Sex determination in this species involves the loss of sex chromosomes during embryonic divisions (Figure 1C). Zygotes receive one X chromosome from the female gamete and a set of two X's from the sperm. During embryonic cleavage, female embryos lose one paternal X chromosome, which results in a fully diploid genome, whereas male embryos eliminate the two paternal X chromosomes, which make them somatically hemizygous for the X.

This phenomenon is among the first observation of the existence of a differential behavior between the parental genomes and has been described as "impressed" by Metz [START_REF] Metz | Chromosome Behavior, Inheritance and Sex Determination in Sciara[END_REF]. It is 20 years later, in 1960, that the cytogeneticist Helen Crouse will use for the first time the term "imprinting" to qualify this phenomenon [START_REF] Crouse | The controlling element in sex chromosome behavior in Sciara[END_REF]. She probably used this term after its meaning in ethology and psychology where it is defined as "any kind of phase-sensitive learning that is rapid and apparently independent of the consequences of behavior".

1.1.b Other examples

In insects, what has been coined as "imprinting" reflects the silencing of whole parental chromosomes rather than a specific genomic locus. In Sciara, silencing is achieved by the elimination of parental sex chromosomes in the embryo. However, two other systems of parental genome exclusion have been described in insects. On the one hand, the germline exclusion system is defined by active and imprint-dependent elimination of the paternal genome in the germline, which leads to a mature sperm carrying only maternal genomic material. On the other hand, a widespread genome exclusion system has been reported in social insects, including the Hymenopterans group (ants, bees, wasps and relatives). In those species, females arise from a diploid fertilized egg but males develop from unfertilized haploid eggs and thus lack the entire paternal genome at all stages of their development. This system is known as the haplodiploidy sex determination and leads to an exclusive transmission of the maternal genome [START_REF] Queller | Theory of genomic imprinting conflict in social insects[END_REF]. (A) Picture of a Sciara fly (fungus gnat) (B) Formation of male gametes during Sciara development. In the gastrula, one paternal X chromosome (Xp) is eliminated, then meiosis occurs in the pupa. During meiosis I, the paternal genome is eliminated. The maternal set of autosomes (Am) is then eliminated during meiosis II and the surviving maternal genome will be inherited as paternal in the sperm.

(C) After fertilization, in the seventh cleavage, all somatic precursors lose two Xps in males or one Xp in females.

Drawing adapted from «Imprinting and Paternal Genome Elimination in Insects » book chapter, Glenn Herrick and Jon Seger, 1999 Another example of imprinting of sex determination in insects has been reported for coccid insects, which do not have sex chromosomes and use a paternal exclusioninstead of elimination-system. The sex in this family of insects is determined by the silencing of an entire set of chromosomes through heterochromatinization [START_REF] Khosla | Genomic imprinting in the mealybugs[END_REF], in a pattern reminiscent to X chromosome inactivation in mammals [START_REF] Heard | X-Chromosome Inactivation in Mammals[END_REF]. Finally, the case of the well-known model organism Drosophila melanogaster is a bit more complicated because conflicting results have been obtained. Indeed, experimental proofs showed the existence of parent-of-origin gene expression in Drosophila, suggesting that they are capable of imprinting [START_REF] Joanis | Genomic imprinting in Drosophila is maintained by the products of Suppressor of variegation and trithorax group, but not Polycomb group, genes[END_REF][START_REF] Lloyd | Genomic imprinting and position-effect variegation in Drosophila melanogaster[END_REF]. However, more recent studies analyzing those genes could not find any evidence of imprinting but rather intrinsic noise causing differential expression. Moreover, they showed that gynogenetic (maternal-only) and androgenetic (paternal-only) embryos are viable in flies [START_REF] Coolon | Genomic imprinting absent in drosophila melanogaster adult females[END_REF][START_REF] Wittkopp | Parent-of-origin effects on mRNA expression in Drosophila melanogaster not caused by genomic imprinting[END_REF]. Genomic imprinting in Drosophila is thus an unclear part of the field and although it might exist at some loci located on the mini-X chromosome, it does not appear to be a widespread, nor an essential phenomenon. In conclusion, in insects, the precise mechanisms that govern parent-specific silencing of entire chromosomes are not clear and the role of imprinting seems to be restricted to sex determination. In general, parent-of-origin effects in insects diverge from what have been shown in plants and mammals, which raises the question of the evolutionary origin of genomic imprinting, likely through independent events.

Genomic imprinting in Plants

1.2.a Imprinting in the endosperm

The identification of the first imprinted gene was made in plants, in 1970, when

Kermicle described the parental effect of the R allele transmission on maize pigmentation [START_REF] Kermicle | Dependence of the R-mottled aleurone phenotype in maize on mode of sexual transmission[END_REF]. The concept of imprinting in plants is more similar to the one in mammals compared to insect imprinting, primarily as parent-of-origin silencing in plants is a locus-specific rather than chromosome-wide process.

Reproduction in flowering plants, also known as angiosperms, is characterized by a double fertilization process [START_REF] Russell | Double Fertilization[END_REF]. Male gametogenesis produces two identical haploid sperm cells. In females, gametogenesis results in two types of mature reproductive cells: one egg, which corresponds to the female gamete, and two haploid central cells. The two sperm cells fertilize both the egg and the two central cells to give rise respectively to a diploid embryo (2n) and a triploid endosperm (3n) (Figure 2A).

The endosperm is the extra-embryonic tissue of the plant required for nutrient transfer to the embryo, similarly to the placenta in mammals. Interestingly, imprinted genes have been identified exclusively in the extra-embryonic tissues in Arabidopsis thaliana and maize and accordingly, imprinting has been shown to play crucial role in seed development [START_REF] Gehring | Imprinting and Seed Development[END_REF][START_REF] Scott | Parent-of-origin effects on seed development in Arabidopsis thaliana[END_REF].

For a long time, the R locus in maize was the first one described as imprinted in the endosperm. The R gene is responsible for the accumulation of red pigmentation in the outer layer of the endosperm. When RR females (red color) are crossed with rr males (colorless), all the resulting kernels (RR/r) are fully colored, but in the reciprocal crosses, the obtained kernels (rr/R) exhibit a mottled pigmentation (Figure 2B and 2C).

Importantly, this parent-of-origin effect of the R gene transmission is not observed in other plant organs from the diploid embryo. A logical explanation for this phenomenon could imply a dosage effect associated with the triploid endosperm: two doses of R confer red color, whereas one dose of R leads to a mottled pattern. However, Kermicle furnished an elegant experimental proof that parental imprinting at the R locus was responsible of the differential pigmentation upon paternal or maternal crosses: by introducing another copy of the R gene from the pollen, he showed that rr/RR kernels had the same pigmentation as the rr/R genotype. Conversely, the introduction of an r allele trough the pollen, generating RR/rr kernels, resulted in a colored outer layer of the endosperm [START_REF] Kermicle | Dependence of the R-mottled aleurone phenotype in maize on mode of sexual transmission[END_REF]. Thus rr/RR and RR/rr kernels, although having the same genotype, have different phenotypes: this is the ultimate conceptual evidence of the existence of another layer of regulation than the DNA sequence itself. At that time, Kermicle described this parent-dependent behavior of the gene as "paragenetic", term that precedes the concept of epigenetics to define this imprinting mechanism [START_REF] Kermicle | Gametic imprinting in maize in relation to the angiosperm life cycle[END_REF].

1.2.b Mechanisms of genomic imprinting in plants

The most well-documented mechanism underlying imprinted expression in plants is DNA methylation, as it is the case in mammals. The establishment and control of genomic imprinting in mammals will be extensively addressed in the next chapter.

The evidence that DNA can be subject to the addition of a methyl group on cytosine nucleotides in a broad range of eukaryotes, is very ancient [START_REF] Johnson | Researches on pyrimidines. C111. The discovery of 5-methyl-cytosine in tuberculinic acid, the nucleic acid of the tubercle bacillus[END_REF][START_REF] Wyatt | Occurrence of 5-methyl-cytosine in nucleic acids[END_REF]. In plants, cytosine methylation can be found in three different sequence contexts: CG, CHG or CHH (where H correspond to A,T or C). Cytosine modification is recognized as an epigenetic mechanism involved in gene and transposon silencing [START_REF] Martienssen | DNA methylation and epigenetic inheritance in plants and filamentous fungi[END_REF]. Evidence that DNA methylation is a key player for genomic imprinting control in plants comes from genetic experiments in Arabidopsis thaliana. Among several gene products that have been shown to mediate the addition and the maintenance of DNA methylation in Arabidopsis, the MET-1 DNA methyltransferase maintenance enzyme has been functionally characterized as essential for DNA methylation in the plant genome [START_REF] Ronemus | Demethylation-induced developmental pleiotropy in Arabidopsis[END_REF]. In met-1 loss-of-function plants, the fwa and fis2 imprinted genes exhibit imprint loss accompanied by bi-allelic expression, showing the requirement of maintenance of DNA methylation for the imprint (Jullien et al., 2006a). Further studies have confirmed the main role of cytosine methylation in silencing the un-expressed allele of imprinted genes. However, some imprinted regions are not affected by the loss of DNA methylation in met-1 mutants, suggesting that DNA methylation is not the only mechanism at play. Indeed, other epigenetic processes have been reported to correlate with imprinted gene silencing in plants. Notably, the tri-methylation chromatin silencing mark at the lysine 27 of histone 3 (H3K27me3) inhibits the expression of the imprinted Mea gene in vegetative tissues and pollen (Jullien et al., 2006b).

Interestingly, we will see later that those epigenetic mechanisms of imprinting regulation in plants exhibit a lot of similarities with the mammalian system, suggesting a case for convergent evolution [START_REF] Feil | Convergent evolution of genomic imprinting in plants and mammals[END_REF]. However, while genomic imprinting in plants and mammals share some mechanistic and functional properties, it remains clear that imprinting does not have the same importance for embryogenesis in both species.

Genomic imprinting in mammals

1.3.a Parent-of-origin effects

When the field of mouse genetics started to burst during the first decades of the 20th century, several cases of spontaneously appearing short-tailed and tailless mice were reported in the house mouse (Paul, 1935). These "T (tail) mutations" (or "T alleles")

were on the same linkage group on chromosome 17 and showed multiple effects on embryonic development, genetic recombination, male fertility and Mendelian segregation in mice [START_REF] Lyon | Investigations of the nature of t-alleles in the mouse[END_REF]. In 1974, Johnson put some effort in the characterization of one of these mutations that arose in the Jackson laboratory colony, the hairpin-tail allele (T hp ) [START_REF] Johnson | Hairpin-tail: a case of post-reductional gene action in the mouse egg[END_REF], associated with a short tailed phenotype with a frequent acute bend near the tip. The unique characteristic of this allele was its unusual parental transmission. The first surprising observation came from mouse mating: there was almost a 2-fold difference in the litter size upon maternal compared to paternal transmission of the T hp allele (an average of respectively 4.1 and 7.3). Interestingly, heterozygote embryos showed two different phenotypes depending on whether the mutation was transmitted by the father or the mother: +/+ females crossed with T hp /+ males led to varying degrees of shortness tail, whereas T hp /+ females mated with +/+ males resulted in oedematous embryos with short and kinky tail and polydactyly (Figure 3). The extreme embryonic phenotype when T hp is maternally transmitted induced in utero death, while paternal transmission of T hp gave birth to viable pups. Johnson first concluded that the T hp mutation was subject to maternal effect, so that the embryonic phenotype of heterozygote embryos was exacerbated when the mother was heterozygote as well. However, when crossing female and male heterozygotes, the two types of phenotypes segregated within the embryos [START_REF] Johnson | Hairpin-tail: a case of post-reductional gene action in the mouse egg[END_REF]. The transmission of the T alleles thus appeared as an exception of the dogma that heterozygous mutations are phenotypically equal, independently of the sex of the contributing gamete. Altogether, these observations of the T-associated maternal effect (Tme) mutation, were the first report of what we know today as a classical imprinting-related inheritance and constitute the basis of the discovery of the first imprinted gene in the mouse (see below). The following year, another case of genomic imprinting was observed in mice: the paternal X chromosome was shown to be preferentially silenced in the extra-embryonic tissues of XX female embryos [START_REF] Takagi | Preferential expression the paternally derived X chromosome in the extraembryonal membranes of the mouse[END_REF].

1.3.b Embryology experiments

Parthenogenetic embryos are maternal-only products, resulting from eggs stimulated to develop in the absence of a male contribution. The use of these embryos could have major applications both in the field of mouse developmental biology and in animal breeding, to identify recessive genes. However, although mouse parthenogenetic embryos implant in the maternal uterus, it was known since the 1970s' that they cannot develop past mid-gestation (embryonic day 9, E9, see Figure 4 for mouse embryo development) [START_REF] Graham | Parthenogenetic mouse blastocysts[END_REF][START_REF] Kaufman | Normal postimplantation development of mouse parthenogenetic embryos to the forelimb bud stage[END_REF][START_REF] Surani | Development of gynogenetic eggs in the mouse: Implications for parthenogenetic embryos[END_REF].

Subsequently, Surani and colleagues attempted to decipher the cause of the nonviability of parthenogenetic embryos by manipulating fertilized diploid and triploid eggs to generate androgenetic (two male pronuclei) and gynogenetic (two female pronuclei) embryos (Figure 5) [START_REF] Surani | Development of gynogenetic eggs in the mouse: Implications for parthenogenetic embryos[END_REF]). Among the five types of modified embryos they generated and transferred to pseudo-pregnant females, only the triploid (Figure 5D) and the reconstituted biparental diploid embryos (Figure 5E) were able to develop past day 9 of pregnancy. Uniparental diploid embryos containing two copies of female or male pronuclei (Figure 5B, C and F), albeit some of them were able to implant, developed poorly and died before E9. From these results, they concluded that the lethality of parthenogenetic conceptuses is probably due to nuclear deficiencies caused by homozygosity for lethal alleles. However, they could not completely exclude the role of cytoplasmic factors in the developmental failure of androgenetic and gynogenic embryos: upon pronucleus aspiration, some cytosol was necessarily removed, while control embryos were intact. [START_REF] Mcgrath | Nuclear transplantation in the mouse embryo by microsurgery and cell fusion[END_REF], they published a series of articles providing proofs for the requirement of both maternal and paternal genomes for normal development (Barton et al., 1984;[START_REF] Mcgrath | Completion of mouse embryogenesis requires both the maternal and paternal genomes[END_REF]Solter, 1984a, 1984b;[START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF]. Compared to previous experiments, biparental and uniparental conceptuses were both generated by pronuclear transplantation (Figure 6). Using zygotes where the maternal pronucleus was removed, they generated androgenetic embryos by transplantation of a second paternal pronucleus, and biparental embryos by transfer of a maternal pronucleus (Figure 6A). Inversely, after removal of the paternal pronucleus, they generated gynogenetic embryos by transplanting a maternal pronucleus, and biparental embryos through the transfer of a paternal pronucleus (Figure 6B). All reconstructed embryo types reached the blastocyst stage, but both androgenetic and gynogenetic conceptuses stopped embryogenesis shortly after implantation. Here, they could formally demonstrate that the lethality of uniparental embryos did not involve cytoplasmic but rather nuclear defects due to the lack of biparental contribution rather than homozygosity of deleterious alleles (McGrath and Solter, 1984a). But, most importantly, the comparison of the embryonic defects in the two types of uniparental conceptuses revealed a differential and mirror contribution of the male and female genomes to embryogenesis. While the maternal genome seemed essential for the development of embryonic tissues [START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF], the paternal genome was required for the development of the extra-embryonic tissues (Barton et al., 1984) (Figure 6). Altogether, these experiments proved the functional nonequivalence of the parental genomes in mammals, and that biparental reproduction is therefore required for development. Moreover, this parental imprint was obviously nongenetic, as all conceptuses were genetically equivalent. Of note, genomic imprinting in plants appears to have a less strict requirement than in mammals since plant parthenogenetic embryos can form viable progeny.

These fundamental discoveries prompted the emergence of an exciting new area of research in the field of mammalian developmental biology. Surani and Solter were both recognized earlier this year by the award of the prestigious Gaidner international prize for the « discovery of mammalian genomic imprinting that causes parent-of-origin specific gene expression and its consequences for development and disease ». Image from [START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF]. Biparental diploid Control

1.3.c Uniparental disomy experiments

Following the Surani and Solter discovery, it was accepted that, although the genetic contribution of the sperm and egg to the zygote is equal, their information is not functionally equivalent. Independently, mice carrying chromosomal translocations and non-disjunctions were used in genetic studies [START_REF] Lyon | Factors affecting the observed number of young resulting from adjacent-2 disjunction in mice carrying a translocation[END_REF][START_REF] Lyon | A genetic method for measuring non-disjunction in mice with Robertsonian translocations[END_REF][START_REF] Snell | An analysis of translocations in the mouse[END_REF]. This was of particular interest since multiple human diseases have been linked to chromosomal translocations. Cattanach, Beechey and Searle used these mice with chromosomal translocations to generate uniparental disomies (UPD),

where two copies of an entire or a part of a chromosome is derived from only one parent [START_REF] Cattanach | Differential activity of maternally and paternally derived chromosome regions in mice[END_REF][START_REF] Searle | Complementation studies with mouse translocations[END_REF]. They noted that in some cases of UPD, but not all, the resulting progeny exhibited abnormal phenotypesmostly related to growth and behavior-and/or lethality. This demonstrated that both maternal and paternal copies were required for only some specific genomic regions in mice [START_REF] Cattanach | Parental origin effects in mice[END_REF]. Most interestingly, mice carrying reciprocal UPDs displayed an opposite phenotype. Using this method, they could screen the whole mouse genome for regions which function differently depending on their parental dosage [START_REF] Cattanach | Interactions between imprinting effects: summary and review[END_REF]. This method provided the first genome-wide map of imprinted regions in the mouse (Figure 8). This led to the hypothesis that the imprinting phenomenon could lie in the genes contained in these regions and that potential "

imprinted" genes could harbor parent-of-origin expression that accounted for the nonreciprocal observed phenotypes.

1.3.d First imprinted genes in mice

The mapping of chromosomal regions functioning differently depending on their parental origin by Cattanach and colleagues allowed the identification of the chromosomal regions subject to genomic imprinting in the mouse genome. Although this implied that imprinting was likely a phenomenon restricted to specific genes, none of them had been identified. The year 1991 was marked by the identification of the first three endogenous imprinted genes in the mouse. The observation made by Johnson in 1974 [START_REF] Johnson | Hairpin-tail: a case of post-reductional gene action in the mouse egg[END_REF] at the Tme locus on chromosome 17 was previously not recognized as an imprinting phenomenon per se, but it happened to pave the path of the discovery of the first imprinted locus in mice. The Tme mutation was identified as having a maternal effect in mice and has been demonstrated to result from defective nuclear activity (McGrath and Solter, 1984b), but the causative gene was not mapped.

By analyzing the genes falling into the minimal deletion containing the Tme locus, Denise Barlow identified the gene encoding the insulin-like growth factor type-2 receptor (Igf2r) as the cause of the parental-origin effect observed by [START_REF] Johnson | Hairpin-tail: a case of post-reductional gene action in the mouse egg[END_REF][START_REF] Barlow | The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus[END_REF]. Igf2r was thus the first imprinted gene ever described in the mouse genome. The expression of Igf2r was proved to be restricted to the maternal allele only, the paternal allele being silenced despite being genetically intact (Figure 7A).

The same year, two studies identified Igf2 (insulin-like growth factor typer-2)the gene encoding for the ligand of IGF2R-as being mono-allelically expressed as well but from the opposite parental allele [START_REF] Dechiara | Parental imprinting of the mouse insulin-like growth factor II gene[END_REF][START_REF] Ferguson-Smith | Embryological and molecular investigations of parental imprinting on mouse chromosome 7[END_REF]. Igf2 had been previously reported as having a physiological function in embryonic growth [START_REF] Dechiara | A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting[END_REF]. The first study was based on the analysis of mice carrying a genetic deletion of Igf2, which showed severe growth deficiency when the mutation was inherited from the male germline, while no apparent phenotype was observed upon maternal transmission [START_REF] Dechiara | Parental imprinting of the mouse insulin-like growth factor II gene[END_REF]. The second study used embryos with a maternal UPD of the distal region of chromosome 7, which showed repression of Igf2 [START_REF] Ferguson-Smith | Embryological and molecular investigations of parental imprinting on mouse chromosome 7[END_REF]. In addition, this analysis also suggested that the region of UPD contained more than one imprinted gene. Indeed, while the maternal duplication of the distal part of chromosome 7 led to embryonic lethality, the only embryos deficient for Igf2 were viable. Together, these two independent results demonstrated that Igf2 was an imprinted gene expressed exclusively from the paternal allele. Finally, the third gene recognized as imprinted in 1991 was H19, a non-coding RNA localized on the same distal region of chromosome 7 as Igf2 [START_REF] De Chiara | Parental imprinting of the mouse insulin-like growth factor II gene[END_REF]. The H19 RNA is highly expressed in the embryo but repressed from somatic tissues after birth, except for the skeletal muscle [START_REF] Pachnis | Locus unlinked to alphafetoprotein under the control of the murine raf and Rif genes[END_REF]. Interestingly, ectopic expression of H19 in transgenic mice caused embryonic lethality, supporting the importance of this gene's dosage [START_REF] Brunkow | Ectopic expression of the H19 gene in mice causes prenatal lethality[END_REF]. By generating hybrid crosses from two different mouse strains to infer parental origin using transcribed nucleotide polymorphisms, the Tilghman laboratory could show that H19 was an imprinted gene with a maternal active copy and silenced expression from the paternal allele [START_REF] De Chiara | Parental imprinting of the mouse insulin-like growth factor II gene[END_REF]. Igf2 and H19 appeared therefore as two adjacent genes on chromosome 7 with opposite parental expression, from the paternal and maternal allele, respectively (Figure 7B). This observation was instrumental for prompting the hypothesis that imprinted genes may be localized in clusters. Interestingly, in 2011, a study suggested that the appropriate expression of H19 and Igf2 in parthenogenetic embryos was sufficient to restore viability [START_REF] Kono | Produced with a Trial Version of PDF Annotator -www.PDFAnnotator.com letters to nature Birth of parthenogenetic mice that can develop to adulthood Produced with a Trial Version of PDF Annotatorwww.PDFAnnotator.com letters to nature[END_REF].

1.3.e Nowadays

Following the discovery of the first three mouse imprinted genes, the identification and characterization of imprinted genes became an active area of research and more imprinted genes were subsequently identified [START_REF] Schulz | Wamidex: A web atlas of murine genomic imprinting and differential expression[END_REF]. The development of microarray approaches and then high throughput sequencing technologies were notably instrumental for identifying parental-specific imprinting at a genome-wide level, without relying on targeted functional genetics at specific loci (Proudhon et al., 2012;[START_REF] Xie | Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome[END_REF]. In total, around 140 imprinted genes have been identified among the 25,000 genes in the mouse genome (Figure 8 and https://www.mousebook.org).

Imprinting has also been described in the human genome, which contains about 100 imprinted genes, many of them being conserved with mice [START_REF] Morison | A census of mammalian imprinting[END_REF]. Most imprinted genes tend to be grouped in clusters, although there are also some singletons (Figure 8). At the end of the 20 th century, besides providing increasingly accurate census of the number of imprinted genes, many efforts were deployed In yellow are represented the regions tested by Cattanach and colleagues for uniparental disomies leading to abnormal embryonic and/or post-natal phenotypes.

Image from https://www.mousebook.org.

Epigenetic control of genomic imprinting in mammals

Knowing the existence of imprinted genes, the next question was to understand what regulates their parent-specific expression pattern. As early as 1991, Denise Barlow suggested that parent-specific epigenetic modifications likely controlled the monoallelic expression of imprinted genes [START_REF] Barlow | The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus[END_REF]. In 1942, the first use of the term "epigenetics" was attributed to Conrad Waddington who defined it as "the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being" [START_REF] Waddington | The Epigenotype[END_REF]. However, the definition of what are epigenetic processes in biology has evolved since that time [START_REF] Haig | The ( Dual ) Origin of Epigenetics[END_REF].

Today, even if the word "epigenetics" is not always used for its original meaning [START_REF] Greally | A user's guide to the ambiguous word "epigenetics[END_REF], I hope not to offend anyone in the field by defining epigenetics as a cellular memory process that is stable, heritable and reversible. Epigenetic mechanisms thus provide critical memories of earlier developmental decisions.

The differential expression of imprinted genes as well as the intergenerational inheritance of genomic imprinting imply that the imprint must be 1) influencing transcription, 2) established in the germline and 3) transmissible through cell divisions.

In this part, I will address the epigenetic mechanisms that direct the heritable allelespecific expression of imprinted genes, focusing mainly on DNA methylation as the chief epigenetic modification controlling genomic imprinting and the first one which has been attributed to it [START_REF] Barlow | Methylation and imprinting: From host defense to gene regulation?[END_REF]. I chose to describe the processes that underlie the genomic imprinting cycle in their temporal order during embryogenesis, from the mature gametes undergoing fertilization, followed by the epigenetic reprogramming of the embryo and to germline differentiation.

DNA methylation: general concepts

2.1.a Transcriptional silencing

DNA methylation is one of the best-studied epigenetic marks. This modification involves the addition of a methyl group (CH3) on the fifth carbon of the aromatic ring (5mC) by DNA methyltransferase enzymes (DNMTs) (Figure 9A). Although the bacterial type of methylation on adenines has been recently described in mouse embryonic stem cells (Wu et al., 2016b), the vast majority of DNA methylation occurs on cytosines and mostly within the context of a cytosine-guanine dinucleotide, connected by a phosphodiester bond (CpG) in mammalian cells. As a whole, about 4-6 % of all cytosines and 70 to 80% of CpG motifs are methylated in mammalian somatic cells. DNA methylation is involved in many important cellular processes in mammals including genomic imprinting, X chromosome inactivation, cellular differentiation and silencing of transposable elements [START_REF] Smith | DNA methylation: Roles in mammalian development[END_REF]. In 1975, two key papers proposed that DNA methylation could be involved in the transcriptional control of gene expression and in cellular memory processes in vertebrates [START_REF] Holliday | DNA modification mechanisms and gene activity during development[END_REF][START_REF] Riggs | X inactivation, differentiation, and DNA methylation[END_REF]. A few years later, a large number of studies, including several based on the X inactivation process, supported the role of DNA methylation in gene control [START_REF] Doerfler | DNA Methylation and Cene Activity[END_REF][START_REF] Gartler | Mammalian X-Chromosome Inactivation[END_REF][START_REF] Monk | Methylation and the X chromosome[END_REF]. Notably, a strong correlation was established between hypermethylation of promoters and gene silencing.

It is now widely accepted that DNA methylation on promoter regions is associated with gene repression, while gene body methylation is a secondary consequence of transcriptional activity [START_REF] Jones | Functions of DNA methylation: Islands, start sites, gene bodies and beyond[END_REF]. The link between DNA methylation and gene silencing appears to be indirect, through either the recruitment of silencing factor or by preventing the binding of transcriptional activators. DNA methylation is stable through multiple somatic divisions and transmissible between generations. It has been established that methylation is a default state and has to be removed at specific regions [START_REF] Lister | Human DNA methylomes at base resolution show widespread epigenomic differences[END_REF][START_REF] Stadler | DNA-binding factors shape the mouse methylome at distal regulatory regions[END_REF]. The high level of DNA methylation exhibited by mammalian genomes has an evolutionary cost: while deamination of unmethylated cytosines is recognized by the base-excision repair machinery, the loss of amine groups from a methylated cytosine results in a C to T transition, leading to a global depletion of CpGs compared to the expected frequency [START_REF] Bird | DNA methylation and the frequency of CpG in animal DNA[END_REF]. However, CpG dinucleotides can be found at a high frequency at some places of the genome: those are named "CpG islands" (CGIs), are globally unmethylated [START_REF] Bird | A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA[END_REF] and act as major regulatory regions of the mammalian genome. About 50% of CpG islands are within gene promoter regions, but another 25% lie in gene bodies and may serve as alternative, intragenic promoters (Figure 9B).

2.1.b Maintenance through cell division

The observed symmetry of methylated CpGs on both strands [START_REF] Bird | Use of restriction enzymes to study eukaryotic DNA methylation. II. The symmetry of methylated sites supports semi-conservative copying of the methylation pattern[END_REF] leads to the early hypothesis that a maintenance system must exist for DNA methylation to be somatically heritable. It was thus proposed that hemi-methylated cytosines generated upon DNA replication should be targeted by a DNA methylation maintenance enzyme to convert the newly synthesized strand [START_REF] Riggs | X inactivation, differentiation, and DNA methylation[END_REF]. This enzyme was identified and cloned in the 1980's [START_REF] Bestor | Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA[END_REF][START_REF] Bestor | Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells: the carboxyl-terminal domain of the mammalian enzymes is related to[END_REF]. As it was the first DNA methyltransferase (DNMT) enzyme ever described in mammals, it was named DNMT1. The overall structure of DNMT1 shows conservation with its homolog MET-1 in plants. In mitotic cells, DNMT1 is responsible for transferring methyl groups on hemi-

H NH2 O NH N C CH3 NH2 O NH N me C A B CpG island Repeated sequence Transcript Intragenic CpG Intergenic CpG
methylated CpGs, which leads to a full-methylation status in the daughter cells. During the S phase, DNMT1 is recruited to the replication fork and its direct interaction with URHF1 allows its tethering to the DNA [START_REF] Bostick | UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells[END_REF][START_REF] Sharif | The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA[END_REF]. Both

Dnmt1 and Urhf1 knock-out mice (KO) are embryonic lethal around mid-gestation (Figure 10A) [START_REF] Lei | De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells[END_REF]Li et al., 1992;[START_REF] Sharif | The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA[END_REF], showing the essential role of DNA methylation maintenance. Another DNMT-related enzyme, DNMT2, appears to be highly conserved from mammalian to plant genomes but despite its analogy with DNMT1, it was finally shown to have a little affinity with DNA but rather methylate tRNAs [START_REF] Goll | Eukaryotic Cytosine Methyltransferases[END_REF].

2.1.c De novo methylation

In Dnmt1 knock-out mouse embryonic stem cells (mESCs), DNA methylation is low but still detectable at a significant level and cells are able to methylate an exogenous DNA sequence [START_REF] Lei | De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells[END_REF]. This observation motivated the discovery of two de novo DNA methyltransferases, DNMT3A and DNMT3B [START_REF] Okano | Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases[END_REF]. These two proteins are able to methylate naïve, unmethylated DNA and are essential for de novo methylation in the early mouse embryo. A DNMT3-like (DNMT3L) protein was then described as the third essential component of the de novo DNA methylation machinery in mammalian genomes. However, although it shares strong homology with DNMT3A and DNMT3B, DNMT3L lacks the catalytic activity domain essential to the transmethylation reaction [START_REF] Aapola | Isolation and Initial Characterization of a Novel Zinc Finger Gene, DNMT3L, on 21q22.3, Related to the Cytosine-5-Methyltransferase 3 Gene Family[END_REF]. DNMT3L is thus not a DNMT enzyme per se but rather acts as a stimulatory factor of de novo DNA methyltransferases (Chen et al., 2005b;[START_REF] Suetake | DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction[END_REF]. The biological role of these three proteins has been extensively studied in constitutive and conditional KO mouse models. The strongest phenotype has been shown for Dnmt3b-KO embryos, which die at mid-gestation (Figure 10B and 10C) (Okano et al., 1999). Dnmt3a-deficient mice survive until birth but they then die within the first four weeks of life (Figure 10D). DNMT3B seems therefore to exert a more essential role than DNMT3A in methylating the embryonic genome, while DNMT3A is mostly required in adult stem cells. Accordingly, germ cell depletion of Dnmt3a leads to male and female sterility, while conditional KO of Dnmt3b

in the germline has no effect [START_REF] Kaneda | Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting[END_REF]. Interestingly, mouse ESCs can tolerate Dnmt1, Dnmt3a and Dnmt3b triple-KO and complete lack of DNA methylation in the undifferentiated state [START_REF] Tsumura | Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b[END_REF]. Finally, Dnmt3l-KO mice develop normally but adult males and females are sterile (Figure 10E) [START_REF] Bourc'his | Dnmt3L and the establishment of maternal genomic imprints[END_REF].

Recently, Dnmt3C -a 5 th member of the family-has been identified as a rodent-specific DNMT, active only in the male germline and which selectively methylates the promoter of evolutionarily young transposons [START_REF] Barau | The DNA methyltransferase DNMT3C protects male germ cells from transposon activity[END_REF]. Mutant mice for Dnmt3C show a male infertility phenotype.

2.1.d De-methylation mechanisms

While DNMTs catalyze the reaction from an unmethylated to a methylated cytosine, two mechanisms exist for the transition from a methylated to an unmethylated state. A passive demethylation process is directed by the exclusion of DNMT1 from the replication fork and dilution of the mark over cell divisions. An active demethylation process is also known to be mediated by the TET1/2/3 enzymes of the ten-eleven translocation (TET) family, which can successively oxidize methylated cytosines to hydroxyl-methylated cytosines (5hmC), 5-formylcytosines (5fC) and 5carboxylcytosines (5caC), followed by thymine DNA glycosylase (TDG)-mediated excision and repair to generate unmodified cytosines (Tahiliani et al., 2009;[START_REF] He | Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA[END_REF]. TET-mediated 5mC oxidation can also promote passive DNA demethylation, as DNMT1 appears to have very low affinity for hemi-hydroxymethylated cytosine substrates [START_REF] Frauer | Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain[END_REF][START_REF] Hashimoto | Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation[END_REF].

2.1.e Parental-specificity at imprinting control regions (ICRs)

Rapidly after the discovery of the first three imprinted genes in the mouse, DNA methylation has been reported as the mechanism underlying the parental-specific expression of these genes [START_REF] Bartolomei | Epigenetic mechanisms underlying the imprinting of the mouse H19 gene[END_REF][START_REF] Ferguson-Smith | Parental-origin-specific epigenetic modification of the mouse H19 gene[END_REF][START_REF] Stöger | Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal[END_REF]. This same year, the definitive recognition that DNA methylation was the essential regulator of the maintenance of imprinting came from the analysis of Dnmt1-KO mice [START_REF] Li | Role for DNA methylation in genomic imprinting[END_REF]. The hypomethylation status caused by Dnmt1 deletion lead to a loss of imprinting and abnormal expression -either lack or biallelicof imprinted genes. It has been now clearly established that the mono-allelic expression of imprinted genes is directed by differential parental DNA methylation at imprinting control region (ICRs). ICRs are essential cis-acting regulatory elements responsible for the imprinted pattern.

In the coming paragraph, I will be describing [START_REF] Borsos | Building up the nucleus: nuclear organization in the establishment of totipotency and pluripotency during mammalian development[END_REF] the process of maintenance of the methylation at ICRs in the embryos after fertilization, (2) the establishment of ICRs in the differentiated gametes and (3) the different types of ICRs.

Maintenance of ICRs in the embryos

2.2.a Epigenetic asymmetry in the gametes

The morphological appearance of sperm and oocyte is strikingly divergent (Figure 11).

But the two mature gametes exhibit extensive epigenetic differences far beyond their mere appearance. The sperm genome is highly methylated (about 90% of CpG methylation) and packed with protamines that replace the great majority of histones during the final steps of spermatogenesis [START_REF] Gaucher | From meiosis to postmeiotic events: The secrets of histone disappearance[END_REF]. Histone-borne information carried by the sperm is therefore limited. Protamines are small proteins that allow the ultra-condensed state of the sperm genome that is not compatible with transcriptional activity, but facilitates the motility of the sperm and the protection if its DNA. Sequences harboring DNA methylation in the sperm genome include repeated elements and intergenic regions [START_REF] Howlett | Methylation levels of maternal and paternal genomes during preimplantation development[END_REF][START_REF] Smith | A unique regulatory phase of DNA methylation in the early mammalian embryo[END_REF]) (Figure 11). In contrast, the oocyte genome is highly transcribed during its growing phase, to allow the production of a maternal storage of RNAs and proteins for the future embryo, and presents with an extensively open chromatin conformation (Wu et al., 2016a;[START_REF] Zheng | Resetting Epigenetic Memory by Reprogramming of Histone Modifications in Mammals[END_REF]. The maternal genome is characterized by a low methylation level (less than 40%), which preferentially localizes into the body of actively transcribed genes, including at intragenic CpG islands (Kobayashi et al., 2012a;[START_REF] Smallwood | Dynamic CpG island methylation landscape in oocytes and preimplantation embryos[END_REF][START_REF] Smith | A unique regulatory phase of DNA methylation in the early mammalian embryo[END_REF] (Figure 11). The sperm is globally highly methylated with CpG methylation (about 80%) mostly localized in intergenic regions and transposons sequences. The oocyte genome is less methylated (about 40%), highly transcribed and acquires DNA methylation on gene bodies, including intragenic CpG islands.

2.2.b Epigenetic reprogramming of the early embryo

Cell fate reprogramming is needed to produce a pluripotent embryo from the union of the two differentiated gametes through the process of fertilization. Notably, the oocyteto-zygote transition is characterized by dynamic epigenetic remodeling of gene transcription, histone modifications and DNA methylation in the early embryo [START_REF] Eckersley-Maslin | Dynamics of the epigenetic landscape during the maternal-to-zygotic transition[END_REF][START_REF] Seah | From Germline to Soma: Epigenetic Dynamics in the Mouse Preimplantation Embryo[END_REF]. Indeed, the gametic profiles need to be erased and replaced by a new epigenetic landscape that will reflect the embryonic identity. Embryonic epigenetic reprogramming includes a genome-wide erasure of DNA methylation marks followed by an extensive remethylation (Figure 12).

The reset of DNA methylation profiles occurs concomitantly to the restoration of After fertilization, in the 1 cell-stage embryo, the sperm-derived genome undergoes a rapid loss of DNA methylation and protamines are replaced by maternal histones. In theory, loss of DNA methylation can be mediated either by an active process, mediated by the TET oxidizing enzymes, or by passive dilution through lack of post-replicative maintenance of DNA methylation. The rapid transition from high to low methylation content suggests that erasure of DNA methylation from the male pronucleus is active rather than passive. Indeed, in the zygote, the male genome is subject to active demethylation through the conversion of 5mC in 5hmC by the maternal store of TET3 enzymes [START_REF] Gu | The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF].

Immunostaining experiments have shown that 5hmC is then oxidized and gradually eliminated during pre-implantation development (Inoue and Zhang, 2011;Inoue et al., 2011). In contrast, DNA methylation is erased by a slower process on the maternal genome, suggesting a passive loss over multiple cell divisions as a consequence of the lack of embryonic DNMT1/ URHF1 [START_REF] Howell | Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene[END_REF]. However, more recent studies suggested that such a simple model might not reflect the functions of TET enzymes and DNA replication in the demethylation process that the embryonic genome undergoes post-fertilization. TET1 was also proposed to play a role in this process, as suggested by the an abnormal embryonic phenotype of double Tet1;Tet3 mouse KO [START_REF] Kang | Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis[END_REF]. Importantly, it has been reported that TET3-mediated demethylation also affects the maternal genome, while the paternal genome is also subject to passive loss of DNA methylation throughout replication [START_REF] Shen | Tet3 and DNA replication mediate demethylation of both the maternal and paternal genomes in mouse zygotes[END_REF][START_REF] Wang | Programming and inheritance of parental DNA methylomes in mammals[END_REF]. Finally, TET3-mediated accumulation of 5hmC seems to depend on the activity of DNMT3A and DNMT1, suggesting a role for TET3 in compensating de novo methylation in the early embryo [START_REF] Amouroux | De novo DNA methylation drives 5hmC accumulation in mouse zygotes[END_REF]. In summary, both active and passive demethylation of the maternal and paternal genome is likely taking place in the early embryo and TET3 could play a role in protecting unmethylated regions from de novo methylation.

Subsequent to genome-wide DNA demethylation and concomitant to the acquisition of pluripotency, the genome reaches a really low level of DNA methylation (about 20% of CpG methylation) at the blastocyst stage. This low methylation state is only transient during mouse development, as the embryo is subject to genome-wide de novo methylation immediately at implantation, to establish the new epigenetic landscape characteristics of the loss of naïve pluripotency (Figure 12). At E6.5, CpG methylation levels are estimated at 60% and the gain of methylation is completed by stage E8.5, with a global level of 80% of CpG methylation. Embryonic establishment of DNA methylation appears to be mostly DNMT3B-dependent, as the absence of DNMT3A has only limited consequences on DNA methylation at this stage (Auclair et al., 2014;[START_REF] Borgel | Targets and dynamics of promoter DNA methylation during early mouse development[END_REF]. This process has also been shown to be mostly independent from DNMT3L [START_REF] Guenatri | Plasticity in Dnmt3L-dependent and -independent modes of de novo methylation in the developing mouse embryo[END_REF]. The developmental pluripotency associated 3 (Dppa3) gene, also known as Pgc7 or Stella, was initially described as restricted to the primordial germ cells (PGCs) [START_REF] Saitou | A molecular programme for the specification of germ cell fate in mice\rPrimordial germ cells in the mouse embryo during gastrulation[END_REF]. However, further analysis demonstrated the importance of STELLA during the early stages of mouse embryogenesis, whereas its role in germ cell development seems rather unclear [START_REF] Bortvin | Dppa3 / Pgc7 / stella is a maternal factor and is not required for germ cell specification in mice[END_REF][START_REF] Nakashima | Effects of Dppa3 on DNA Methylation Dynamics During Primordial Germ Cell Development in Mice1[END_REF][START_REF] Payer | stella Is a Maternal Effect Gene Required for Normal Early Development in Mice[END_REF]. In adult mice, Stella is not expressed in the male germline. In contrast, Stella transcripts are detected at high levels in the immature oocytes from young female ovaries and are maintained in fully grown oocytes. After fertilization, its expression persists in the newly formed pre-implantation embryo [START_REF] Sato | Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells[END_REF].

2.2.c Protection of genomic imprints during pre-implantation development

In Stella mutant mice, crosses between heterozygous animals lead to normal embryogenesis and viable progenies. However, the resulting homozygote females are sterile: the embryos lacking maternal contribution of Stella are compromised in preimplantation development and rarely reach the blastocyst stage [START_REF] Payer | stella Is a Maternal Effect Gene Required for Normal Early Development in Mice[END_REF].

STELLA was thus defined as a maternal factor with an essential function during embryogenesis, resembling other cases of maternal-specific effect previously described [START_REF] Christians | Embryonic development: maternal effect of hsf1 on reproductive success[END_REF][START_REF] Tong | Mater, a maternal effect gene required for early embryonic development in mice[END_REF][START_REF] Wu | Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyteto-embryo transition[END_REF]. In embryos derived from Stella-null oocytes, the assessment of DNA methylation shows a strong demethylation of the maternal genome [START_REF] Nakamura | PGC7/Stella protects against DNA demethylation in early embryogenesis[END_REF], while no change in DNA methylation was detected in Stella-null oocytes, before fertilization. Importantly, several imprinted loci (notably Peg1, Peg3, Peg10, H19 and Rasgrf1) showed significant less methylation in Stella-null zygotes compared to age-matched controls.

This suggested that Stella could function as a factor protecting some maternally and paternally imprinted regions from the DNA demethylation changes in the early preimplantation embryo. The current proposed mechanism predicts that STELLA protects DNA methylated sites from TET3-mediated active demethylation, through its binding to maternally H3K9me2 inherited marks [START_REF] Bian | PGC7 suppresses TET3 for protecting DNA methylation[END_REF][START_REF] Nakamura | PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos[END_REF].

Interestingly, a recent study reported that oocytes derived from females subject to a high-fat diet (HFD)-based model of obesity display a reduction of STELLA [START_REF] Han | Embryonic defects induced by maternal obesity in mice derive from Stella insufficiency in oocytes[END_REF]. The embryos resulting from the fertilization of these oocytes show accumulation of 5hmC and hypomethylation at specific regions, including transposons and some imprinted genes. The embryonic deficiency observed in embryos derived from maternal obesity might therefore be due to STELLA maternal insufficiency.

DNMT1 is the necessary and sufficient enzyme responsible for the faithful maintenance of methylation at imprinted loci during the reprogramming of the early embryo [START_REF] Branco | Safeguarding parental identity: Dnmt1 maintains imprints during epigenetic reprogramming in early embryogenesis[END_REF][START_REF] Hirasawa | Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development[END_REF]. Indeed, by analyzing the level of DNA methylation at ICRs in pre-implantation embryos deficient for both Dnmt3a/3b, the de novo DNMTs have been ruled out from general imprinting maintenance in the early embryo. In contrast, the lack of both maternal and zygotic Dnmt1 results in the complete hypo-methylation of imprinted loci in pre-implantation embryos [START_REF] Howell | Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene[END_REF]. However, because of its exclusion in the zygotic nucleus, the level of DNMT1 in the pre-implantation embryo is really low and the protein thus needs to be specifically targeted to imprinted loci. This non-canonical recruitment of DNMT1 to ICRs may be mediated through the KRAB zinc finger protein ZFP57. ZFP57 has been identified as being required for the maintenance of both maternal and paternal imprints [START_REF] Li | A Maternal-Zygotic Effect Gene, Zfp57, Maintains Both Maternal and Paternal Imprints[END_REF][START_REF] Takahashi | ZFP57 and the targeted maintenance of postfertilization genomic imprints[END_REF]. Embryos lacking maternal and zygotic contribution of Zfp57 are unable to maintain DNA methylation at ICRs and die around E14.5.

However, ZFP57 by itself is not able to recruit DNMT1 [START_REF] Zuo | Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain[END_REF]. KRAB zing finger proteins often act as epigenetic silencers by interacting with the heterochromatin factor KAP1/TRIM28 [START_REF] Schultz | SETDB1: A novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zincfinger proteins[END_REF]. KAP1 being part of a multifunctional repressor complex that includes DNMT1, it acts as a co-factor of ZFP57 to recruits DNMT1 at ICRs. Accordingly, maternal depletion of KAP1 leads to hypomethylation of ICRs, followed by embryonic lethality [START_REF] Alexander | TRIM28 Controls Genomic Imprinting through Distinct Mechanisms during and after Early Genome-wide Reprogramming[END_REF][START_REF] Messerschmidt | Trim28 is required for epigenetic stability during mouse oocyte to embryo transition[END_REF]. Further ChIP-sequencing analyses have revealed an hexanucleotide or heptanucleotide consensus motif (TGCCGC or TGCCGCN) recognized by ZFP57/KAP1 complex when the internal CpG is methylated [START_REF] Quenneville | In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions[END_REF][START_REF] Strogantsev | Allelespecific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression[END_REF]. Importantly, this motif is present in multiple instances at all ICRs, demonstrating the sequence-specific recruitment of ZFP57/KAP1 complex to confer DNMT1-mediated protection. Whereas STELLA has been proposed as protecting against active demethylation, the ZFP57/KAP1 complex would rather protect the methylated allele of ICRs from passive demethylation (Figure 13). Recently, maternal storage of URHF1 has been shown to be essential for pre-implantation development [START_REF] Maenohara | Role of UHRF1 in de novo DNA methylation in oocytes and maintenance methylation in preimplantation embryos[END_REF]. Urhf1 maternal-KO embryos have an hypomethylated genome and die at the blastocyst stage, suggesting a role of URHF1 in maintaining ICR methylation during pre-implantation embryos. During the phase of epigenetic reprogramming, the methylated allele of ICRs has been proposed to be protected both by STELLA from active demethylation and by ZFP57/KAP1 from e passive demethylation. After implantation, ICRs are probably protected from remethylation by the binding of specific transcription factors. This figure shows the example of a maternally DNA methylated region; the same kind of mechanism applies for regions that inherit DNA methylation from the paternal allele. The grey line represents the global changes in genomic DNA methylation levels.

Establishment of ICRs in the germline

2.3.a Epigenetic reprogramming in the germline

To fully accomplish embryo development and start a new life cycle, germline differentiation is required. In mammals, a small group of germ cell precursors -the primordial germ cells (PGCs)-originates from the embryonic endoderm at E6.5/E7.5 in response to inducing signals from the nearby extra-embryonic ectoderm and the visceral endoderm [START_REF] Saitou | Primordial germ cells in mice[END_REF]. These cells are thus derived from an embryonic lineage already primed for further somatic differentiation and with high genomic methylation. To establish a germline identity, PGCs need to undergo a second round of epigenetic reprogramming, to remove the somatic program and activate germline genes (Figure 14) [START_REF] Reik | Epigenetic Reprogramming in Mammalian Development[END_REF][START_REF] Seisenberger | Conceptual links between DNA methylation reprogramming in the early embryo and primordial germ cells[END_REF]. During this process, DNA methylation at ICRs is erased together with the rest of the genome, providing a blank state for the establishment of sex-specific patterns during male and female gametogenesis [START_REF] Tada | Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells[END_REF]. PGC demethylation is orchestrated in two phases of passive and active loss of DNA methylation. The passive phase is characterized by low levels of the de novo DNMT3A and DNMT3B enzymes and nuclear exclusion of URHF1, which are then completely repressed by E9.5 [START_REF] Kagiwada | Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice[END_REF][START_REF] Yabuta | Gene Expression Dynamics During Germline Specification in Mice Identified by Quantitative Single-Cell Gene Expression Profiling1[END_REF]. During this replication-dependent phase of demethylation when PGCs are actively dividing, imprinted regions, together with a subset of repetitive elements and CGIs on the active X chromosome in females, follow a slower demethylation kinetics than the rest of the genome [START_REF] Guibert | Global profiling of DNA methylation erasure in mouse primordial germ cells[END_REF][START_REF] Reik | F1000Prime recommendations of: The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells[END_REF]. A non-canonical targeting of DNMT1 -not URHF1-dependent-may maintain DNA methylation at some ICRs until approximately E10.5 [START_REF] Hargan-Calvopina | Stage-Specific Demethylation in Primordial Germ Cells Safeguards against Precocious Differentiation[END_REF].

After E11.5, an active, TET-dependent demethylation of specific loci is engaged [START_REF] Hackett | Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine[END_REF][START_REF] Lee | Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells[END_REF]. TET1 and TET2 enzymes are present in PGCs, and indeed, several studies have shown the requirement of TET1/2 as well as the accumulation of 5hmC concomitant with the loss of 5mC at some imprinted regions (including at H19, KvDMR, Peg10, Igf2r, and Peg3 loci) [START_REF] Piccolo | Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion[END_REF][START_REF] Yamaguchi | Role of Tet1 in erasure of genomic imprinting[END_REF]. However, while it seems clear that TET1/2 are playing a role in ICR demethylation through 5hmC conversion, combined inactivation of both genes is still compatible with normal embryo development, suggesting an additional mechanism involved [START_REF] Dawlaty | Loss of tet enzymes compromises proper differentiation of embryonic stem cells[END_REF]. Moreover, a recent study proposed a role for TET1 in the removal of de novo DNA methylation at specific loci rather than in the global demethylation process, reminiscent of the role of TET3 in protecting from de novo DNA methylation during epigenetic programming of the early embryo [START_REF] Hill | Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte[END_REF]. In summary, the epigenetic reprogramming of the PGC genome is subject to two waves of demethylation, the first one (from E6.5 to E10.5) corresponds to a global and passive decrease of DNA methylation, whereas the second one (from E11.5 to E13.5) appears to be an active, locus-specific demethylation that mediates the resetting of ICRs. From E6.5, the embryo begins PGC (in green) differentiation in a process called germline specification. PGCs will then migrate to the embryonic endoderm, to the hindgut and finally towards the genital ridges, while undergoing passive and active genome-wide demethylation. By E13.5, the level of PGC DNA methylation is around 10%.

2.3.b Germline specification

During epigenetic reprogramming, PGCs are proliferating and migrating to the genital ridges. By E13.5, PGCs contain the lowest CpG methylation level that a physiologically normal cell can attain, less than 10% of CpG methylation [START_REF] Popp | Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency[END_REF]. From that point, sex determination has been initiated and ICRs need to be established. The re-establishment of DNA methylation occurs in different times and environments between the two sexual germlines to ultimately lead to sperm or oocytespecific methylation patterns. In the oocyte, the gain of methylation initiates postnatally, during the growing phase, while arrested in prophase of meiosis I. In contrast, paternal remethylation initiates as early as E14.5, in mitotically arrested prospermatogonia.

Thus while female germ cell methylation is still really low at E16.5, half of paternal DNA methylation is set at that stage [START_REF] Kota | Epigenetic Transitions in Germ Cell Development and Meiosis[END_REF]. At birth, DNA methylation patterns are completed in the male germline and stay more or less unaltered through the subsequent stages of spermatogenesis, until the production of spermatozoa. Despite this different timing in DNA methylation establishment, the methylation enzymes directing ICR methylation are the same during male and female gametogenesis: DNMT3A, together with the DNMT3L co-factor are strictly required and their absence leads to sterility in both sexes [START_REF] Bestor | Transposon silencing and imprint establishment in mammalian germ cells[END_REF][START_REF] Bourc'his | Dnmt3L and the establishment of maternal genomic imprints[END_REF][START_REF] Kaneda | Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting[END_REF]. However, the details of the sterility phenotype are different in the two sexes. Dnmt3aor Dnmt3L mutations do not compromise oocyte production and fertilization, despite the lack of DNA methylation, but act as maternal effect mutations: the progeny die around E10.5 in the context of abnormal expression of maternally imprinted genes [START_REF] Bourc'his | Dnmt3L and the establishment of maternal genomic imprints[END_REF][START_REF] Kaneda | Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting[END_REF]. In males, loss Dnmt3L causes a meiotic catastrophe, as transposon methylation is also affected and this is incompatible with sperm production (Bourc'his and Bestor, 2004). As emphasized before, while oocyte DNA methylation is mostly found in the body of actively transcribed genes (Kobayashi et al., 2012a;[START_REF] Smallwood | Dynamic CpG island methylation landscape in oocytes and preimplantation embryos[END_REF][START_REF] Veselovska | Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape[END_REF], male germ cell methylation mostly occurs outside of genes.

Accordingly, maternal ICRs are all CpG islands that were located in transcription units, while all paternal ICRs have an intergenic location [START_REF] Duffié | Parental Epigenetic Asymmetry in Mammals[END_REF]. Genomic imprinting is established in the male and female gametes during pre-and post-natal life, respectively, through the action of DNMT3A and the DNMT3L co-factor. Following fertilization, the methylated allele of ICRs is protected from epigenetic reprogramming by STELLA and the ZFP57/KAP1 complex. When DNA methylation is re-established at implantation, transcription factor binding could protect the unmethylated allele of ICRs.

During germline differentiation, genomic imprinting is erased through active TET demethylation in both male and female germlines.

2.4.a Canonical imprinting

Imprinted loci typically contain parent-specific differentially methylated regions (DMR).

These DMRs can be classified into two categories: (1) the ones established during gametogenesis, when the parental genomes are physically separated, and called germline DMR (gDMRs) and ( 2) the ones acquiring differential DNA methylation after fertilization and which are referred to as secondary (or somatic) DMRs (sDMRs). To be classified as an ICR, a DMR has to show autonomous cis regulatory properties when genetically deleted, in the sense that genes in the vicinity will show loss of imprinted expression. This is why ICRs are usually gDMRs. In contrast, sDMRs can have regulatory properties, but they are not autonomous as their sDMR status always depends on the activity of a gDMR, usually through the control of a long noncoding RNA with long-range chromatin effects. At the end, ICRs are exceptional and only a small subset of sequences are capable of maintaining the memory of the methylation status they had in the parental germlines, throughout life and in all somatic tissues: only 20 of them have been characterized, the majority of which are located in the imprinting effect regions defined by Cattanach (Figure 8).

In agreement with the general pattern of sperm and oocyte methylation, maternal and paternal ICRs have sexually dimorphic genomic features: maternal ICRs correspond to CGIs that are located within oocyte-transcribed gene units, while paternal ICRs are found in CpG-poor, intergenic regions. Also, although the number of paternally and maternally expressed genes is roughly similar, the number of maternal ICRs exceeds the number of paternal ICRs: only three ICRs (H19, Rasgrf1 and Dlk1-Glt2) carry DNA methylation of paternal origin among the 20 currently described ICRs.

Because of their higher CpG enrichment, maternal ICRs are indeed more likely to harbor TGCCGC ZFP57-binding sites, and to maintain methylation post-fertilization.

Also, because of their association with CGIs that have intrinsic promoter properties, maternal gDMRs are more likely to impact on gene expression than intergenic, paternal gDMRs. As a consequence, there is a dominant role of the oocyte and more specifically of maternally inherited DNA methylation in genomic imprinting control.

The unique profile of DNA methylation established at ICRs is also associated with relevant chromatin histone modifications. Notably, tri-methylation of the lysine 4 on histone H3 (H3K4me2/3) is commonly found on the active allele whereas the inactive copy is decorated H3K9me2/3 and H4K20me3 [START_REF] Henckel | Transcription and histone methylation changes correlate with imprint acquisition in male germ cells[END_REF]. Overall, ICRs harbor an asymmetric pattern of DNA methylation, histone methylation and gene expression between the two parental alleles. While their expression can be tissuespecific, the dogma was that differential DNA methylation was kept unaltered in all tissues and all developmental stages. However, this canonical life-long and tissuewide form of imprinting is rare and DNA methylation imprints were more recently found to be sometimes tissue-specific or stage-specific (Proudhon et al., 2012).

2.4.b Tissue-specific imprinting

Contrary to what was thought during the first 15 years of the study of genomic imprinting, a number of ICRs do not maintain differential methylation in all somatic tissues. This realization came from a screen that was carried out in our laboratory to identify new maternal gDMRs/ICRs, by a MeDIP (Methylated DNA ImmunoPrecipitation)-sequencing analysis of E8.5 mouse embryos coming from either wild-type or Dnmt3L-/+ DNA methylation-free oocytes (Proudhon et al., 2012). This screen allowed the identification of maternal ICRs that lose imprinting in a tissuespecific manner, through the gain of DNA methylation on the previously unmethylated paternal allele. For example, the Cdh15 DMR on mouse chromosome 8 fulfills all the developmental criteria of a maternally inherited ICR, and harbors ZFP57 binding sites (Proudhon et al., 2012). However, in adult mice, although maternal-specific DNA methylation is maintained in a number of somatic tissues, the parental specificity is lost in a subset of brain tissues. In mouse adult cortex and cerebellum, the Cdh15 gDMR is bi-allelically methylated. The Cdh15 DMR corresponds to an intragenic promoter, that promotes paternal-specific expression of a shorter Cdh15 isoform in tissues where imprinting is kept. Importantly, the case of Cdh15 is not isolated and other ICRs have been shown to harbor tissue-specific patterns of DNA methylation imprints (Proudhon et al., 2012).

Imprinted genes can also show tissue or cell-specificity in allelic expression without an initial change in the DNA methylation status of their associated ICRs but rather through secondary epigenetic control. This characteristics seems to mostly relate to imprinted genes under the control of a paternal ICR, therefore with intergenic location. Dlk1, a member of the Dlk1-Dio3 imprinted cluster on mouse chromosome 12, is paternally expressed during embryogenesis and controlled by an intergenic paternally methylated ICR. The expression of Dlk1 becomes biallelic in the neurogenic niche after birth, without the paternal ICR being altered [START_REF] Ferrón | Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis[END_REF]. Similarly, biallelic expression of the paternally imprinted Igf2 gene is observed in two parts of the brain, the choroid plexus and leptomeninges, whereas it is imprinted elsewhere [START_REF] Dechiara | Parental imprinting of the mouse insulin-like growth factor II gene[END_REF]. Another example of tissue-specific expression of an imprinted gene is the unique case of Grb10: this transcript is maternally expressed in the embryo but becomes paternally expressed, from an alternative promoter, specifically in the post-natal brain [START_REF] Charalambous | Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism[END_REF]. Interestingly, this tissue-specific form of imprinting without alteration of the imprint often regulates the dosage of imprinted genes in the brain, suggesting that this organ is an important site for the dosage-control of imprinted gene functions. Overall, the identification of tissue-and cell-specific imprinted regions revisited the initial concept of life-long maintenance of genomic imprinting. It emphasizes how epigenetic control of mono-allelic expression at imprinted genes could be modulated in time and space, depending on the cellular requirement.

2.4.c DNA methylation-independent transient imprinting

A growing number of data support the evidence that the oocyte does not only provide DNA methylation to control expression of the embryonic genome but also histonebased information [START_REF] Dahl | Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition[END_REF][START_REF] Hanna | MLL2 conveys transcription-independent H3K4 trimethylation in oocytes[END_REF]Wu et al., 2016a;[START_REF] Zheng | Resetting epigenetic memory by reprogramming of histone modifications in mammals[END_REF]. Indeed, histone modifications associated with both active and repressive transcriptional states are detected in great amount in the mouse oocyte genome and their distribution pattern is different from somatic cells. A recent study emphasized the essential role of oocyte-derived H3K27me3 in the control of mono-parental gene expression at specific loci in the early embryo, defining a form of non-canonical genomic imprinting, independent of DNA methylation [START_REF] Hanna | Genomic imprinting beyond DNA methylation: A role for maternal histones[END_REF][START_REF] Inoue | Maternal H3K27me3 controls DNA methylation-independent imprinting[END_REF][START_REF] Pathak | Oocyte-derived histone H3 lysine 27 methylation controls gene expression in the early embryo[END_REF]. This chromatin modification, mediated through the activity of the polycomb group proteins, ensures a repressive state (Piunti and Shilatifard, 2016). Interestingly, oocyte-derived H3K27me3 was also reported to be heritable in flies [START_REF] Zenk | Germ line-inherited H3K27me3 restricts enhancer function during maternal-tozygotic transition[END_REF]. Drosophila embryos lacking maternal contribution of polycomb epigenetic memory are not viable and show spurious activation of enhancers. In mice, maternal H3K27me3 was shown as essential for mouse embryonic growth in a previous model of oocyte-specific deletion of the H3K27 methyltransferase EZH2 [START_REF] Erhardt | Consequences of the depletion of zygotic and embryonic enhancer of zeste 2 during preimplantation mouse development[END_REF].

This is by mapping DNaseI-hypersensitive sites that Inoue and colleagues identified in the early embryo paternal-specific open chromatin regions that were marked by oocyte-derived H3K27me3 [START_REF] Inoue | Maternal H3K27me3 controls DNA methylation-independent imprinting[END_REF]. Interestingly, despite

showing monoallelic, paternal-specific expression, these regions did not exhibit differential DNA methylation. Their results showed that maternally inherited

H3K27me3 was able to repress gene expression at a subset of existing and new imprinted loci in the pre-implantation embryo until the blastocyst stage. However, while DNA methylation-depending imprinting at ICRs can be maintained after implantation, maternally inherited H3K27me3 marks are rapidly lost at the time of implantation in the embryo and only persist at a small subset of loci in extra-embryonic tissues [START_REF] Inoue | Maternal H3K27me3 controls DNA methylation-independent imprinting[END_REF]. Altogether, this suggests that both DNA methylation and H3K27me3 inherited from the oocyte can control parent-specific expression. The existence of H3K27me3-related transient imprinting opens new questions in the field of imprinting and enlarges the number of regions that are controlled in a parent-specific manner in the mouse genome.

2.4.d DNA-methylation dependent transient imprinting

While the work of Inoue et al. provided evidence for H3K27me3-mediated transient imprinting [START_REF] Inoue | Maternal H3K27me3 controls DNA methylation-independent imprinting[END_REF], a form a DNA methylation-dependent transient imprinting had been previously uncovered in our laboratory, using a MeDIP-seq screen (Proudhon et al., 2012). These so-called transiently imprinted regions inherit DNA methylation from the oocyte, are protected from genome-wide demethylation during embryo reprogramming (they harbor ZFP57 binding motifs) but then lose their parental-specificity at the time of embryonic implantation. Indeed, by either gaining DNA methylation on the initially unmethylated paternal allele (Figure 16A) or losing DNA methylation on the oocyte-inherited allele (Figure 16B), transiently imprinted regions display symmetrical 5mC profiles in the post-implantation embryo and later on, in adult tissues. The differential DNA methylation at these regions is thus detectable only when looking at stages prior to implantation. Interestingly, transient ICRs could control the mono-allelic expression of genes at the beginning of development, but these genes will then be either completely shut down or bi-allelicaly expressed after implantation. Such genes will thus be imprinted for a really short window of time that corresponds to about four days in the mouse pre-implantation embryo. This observation is intriguing and whether the role of transiently imprinted genes is restricted to the early embryo or could rather have life-long consequences was unknown when I started my PhD.

2.4.e The Gpr1-Zdbf2 locus: a paradigm of transient imprinting

The Gpr1-Zdbf2 locus is the only transiently imprinted locus that has been extensively studied so far [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF]. It spans 137 kb and is localized on chromosome 1 in mice (1qC2). The overall structure of the locus is conserved in humans on chromosome 2 (2q33.3) (Figure 17). Within this locus and its vicinity, only Zdbf2 has been confirmed to have imprinted expression [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF], despite an original study that reported that Gpr1 may also be imprinted [START_REF] Hiura | A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip[END_REF]. Zdbf2 in mice and humans likely arose from a segmental duplication that included the Dbf4 gene (Figure 17). The DBF4 protein is an activator of the CDC7 kinase, which has a conserved role in DNA replication [START_REF] Hughes | Crystal structure of human CDC7 kinase in complex with its activator DBF4[END_REF]. In terms of function, Zdbf2 encodes the DBF-type zinc finger-containing protein 2, whose biological role is unknown and that contains as the only annotated protein domain a C2H2-type zinc finger [START_REF] Razin | Cys2His2 zinc finger protein family: Classification, functions, and major members[END_REF], homolog to the DBF4 yeast protein [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF].

GPR1 is a G-protein coupled receptor of Chemerin, an adipose-derived signaling protein that is involved in regulating adipocyte differentiation and function, immune function, metabolism, and glucose homeostasis [START_REF] Banas | The expression and regulation of chemerin in the epidermis[END_REF][START_REF] Rourke | Gpr1 is an active chemerin receptor influencing glucose homeostasis in obese mice[END_REF]. Although Gpr1 is lowly expressed in adult tissues and its function has been poorly described, it was shown in mammals to be expressed in adipocyte tissues and skeletal muscles. Gpr1-KO mice display no obvious phenotype but were reported to develop glucose intolerance when fed on a high-fat diet, without weight difference [START_REF] Rourke | Gpr1 is an active chemerin receptor influencing glucose homeostasis in obese mice[END_REF]. This locus was originally identified in the mouse by a microarray-based transcriptional screening comparing parthenogenetic and normal embryos [START_REF] Kobayashi | Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2[END_REF]. The Zdbf2 gene was reported as paternally expressed and paternally imprinted, exhibiting paternal DNA methylation at an intergenic differentially methylated region (DMR) in somatic tissues [START_REF] Hiura | A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip[END_REF][START_REF] Kobayashi | Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2[END_REF].

However, neither ZFP57 binding motifs nor ZFP57/KAP1 enrichment were found at this region, suggesting that it did not meet the criteria of a gDMR/ICR. This DMR was rather demonstrated to be a secondary DMR (sDMR) resulting from the activity of a maternal ICR [START_REF] Duffié | The Gpr1/Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF][START_REF] Kobayashi | Imprinted DNA methylation reprogramming during early mouse embryogenesis at the Gpr1-Zdbf2 locus is linked to long cis-intergenic transcription[END_REF]. A maternal germline DMR (gDMR) is indeed located at a CGI in the second intron of Gpr1 and located 65kb upstream of the paternal sDMR (Figure 18). This gDMR coincides with the transcription start site (TSS) of an alternative transcript of Zdbf2, called long isoform of Zdbf2 (Liz).

Liz and the canonical Zdbf2 transcript only differ by their 5'UTR but have the same coding potential [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF]. As the gDMR inherits DNA methylation from the oocyte, Liz is mono-allelically expressed in a paternal-specific manner in the pre-implantation embryo. However, at the time of the implantation, Liz expression is shut down as the gDMR gains DNA methylation on the previously unmethylated paternal allele. This defines the gDMR as a transiently imprinted region that controls the mono-allelic expression of Liz (Figure 18). Liz expression is limited to the pre-implantation embryo but its short-lived expression is associated with a cascade of events occurring in cis, on the same allele it was transcribed, suggesting a role as a regulatory RNA or through the mere act of its transcription: DNA methylation is gained on the sDMR that is located on its path and Zdbf2 becomes activated from the same paternal allele, both of these features being stably maintained through somatic life. Importantly, the complex imprinting scenario of this locus is conserved in humans [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF].

When I started my PhD, the role and regulation of Liz transcription in the preimplantation embryo as well as the biological function of Zdbf2 as an imprinted gene in adults were not resolved. My PhD work shed light onto these unresolved issues and this will be extensively described in the results section of this manuscript.

Physiological functions and evolution of imprinted genes

One fundamental question in the field of genomic imprinting is to understand why such a phenomenon -that could be deleterious to the organism by exposing to haploinsufficiency-has been selected throughout evolution? Indeed, the mono-allelic expression of imprinted genes ultimately infers that a deleterious mutation on the expressed allele cannot be compensated for via the second allele. When embryology experiments revealed that uniparental embryos could not develop, it was first suggested that imprinted genes evolved to prevent parthenogenesis [START_REF] Solter | Differential Imprinting and Expression of Maternal and Paternal Genomes[END_REF] and ovarian diseases resulting from the development of an unfertilized egg [START_REF] Varmuza | Genomic imprinting [mdash] defusing the ovarian time bomb[END_REF]. Accordingly, genomic imprinting has specifically occurred in viviparous mammals, especially eutherians but also marsupials to a lesser extent (less imprinted genes have been reported). Conversely, the oviparous mammals, the monotremes, do not seem to have genes expressed in parent-of-origin manner, although only a limited number of tissues have been investigated. This phylogenetic confinement suggests that genomic imprinting may be linked to the evolution of the placenta (Figure 19).

Because of the similarities in terms of mechanisms and functions during embryogenesis, it has been suggested that imprinting in mammals and flowering plants evolved in response to similar selective pressure [START_REF] Pires | Different yet similar: evolution of imprinting in flowering plants and mammals[END_REF].

Currently, there is not a single model to explain the evolution of imprinted genes in mammals but rather multiple theories that have been proposed during the past 30 years [START_REF] Patten | The evolution of genomic imprinting : theories , predictions and empirical tests[END_REF]. I will develop three of them, including the most discussed theory of parental conflict. The next fundamental question in the field was why tight gene dosage regulation needs to happen at imprinted genes? Ultimately, knowing the function of a gene can illuminate why it has been maintained in a certain environment through evolution. Moreover, the fact that imprinted genes have been selected for a complex mono-allelic regulation strongly suggests that they have essential physiological functions. Since the last 25 years, major progress has been made towards understanding imprinted gene functions in mammals, mostly through functional genetics in mice. Although protection of genomic imprinted regions is essential during pre-implantation development, imprinted genes appear to exert their function after the implantation of the embryo, during pre-and post-natal life [START_REF] Cleaton | Phenotypic Outcomes of Imprinted Gene Models in Mice: Elucidation of Pre-and Postnatal Functions of Imprinted Genes[END_REF]. I will notably address the described function of some imprinted genes in mouse embryonic and post-natal growth regulation as well as in adult behavior and their implication in human neuro-developmental syndromes.

Theories of genomic imprinting evolution

3.1.a The host defense hypothesis

Following the discovery of genomic imprinting, DNA methylation has been rapidly proposed as the mark that allows the cell to distinguish the two parental alleles. Three concomitant studies reported variable DNA methylation patterns of an exogenous transgene depending on the parental transmission [START_REF] Reik | Genomic imprinting determines methylation of parental alleles in transgenic mice[END_REF][START_REF] Sapienza | Degree of methylation of transgenes is dependent on gamete of origin[END_REF][START_REF] Swain | Parental legacy determines methylation and expression of an autosomal transgene: A molecular mechanism for parental imprinting[END_REF]. However, the methylation imprint was partly or wholly influenced by the strain background. The clear demonstration finally came from the insertion of the TG.A transgene on an inbred genetic background [START_REF] Chaillet | Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis[END_REF].

The observation that the exogenous TG.A was methylated only when maternally transmitted led to the hypothesis that imprinting had evolved as a mechanism to silence foreign DNA, especially in the female germline [START_REF] Barlow | Methylation and imprinting: From host defense to gene regulation?[END_REF]. This theory is based on the host defense function of DNA methylation [START_REF] Yoder | Cytosine methylation and the ecology of intragenomic parasites[END_REF] and proposed that imprinted regions are recognized as foreign DNA, as it is the case for the TG.A transgene. Genomic imprinting would thus have evolved from a protection mechanism against transposable elements and retrovirus invasion. The endogenous insertion of an IAP retrovirus element at the 5' of the Agouti locus also seems to function as an imprinting element that harbors gamete-of-origin methylation and expression [START_REF] Duhl | Neomorphic agouti mutations in obese yellow mice[END_REF][START_REF] Michaud | Differential expression of a new dominant agouti allele (A(iapy)) is correlated with methylation state and is influenced by parental lineage[END_REF]. Evolutionary evidence also supports the host-defense theory: expansion of retrotransposons seems to be concomitant to the occurrence of genomic imprinting in the genome of placental mammals (Figure 14). Moreover, Dnmt3L, the gene encoding the necessary co-factor for the establishment of DNA methylation at imprinted loci and transposable elements in male germ cells is also only present in therians but not in monotremes.

Interestingly, species-dependent imprinting could also be an argument for the host defense hypothesis. For example, the retinoblastoma gene Rb1 is not imprinted in rodents but has evolved as imprinted in humans after the retrotransposition of a pseudogene [START_REF] Kanber | The human retinoblastoma gene is imprinted[END_REF]. The inserted sequence has become a differentially methylated region that serves as an alternative promoter for Rb1 and skews its expression in favor of the maternal allele. The last example of an imprinted locus that supports the genome defense theory is the ICR of the paternally imprinted Rasgrf1 gene, which arose from an LTR-type transposable element and as such, requires PIWI-interacting RNAs (piRNAs) to acquire DNA methylation during spermatogenesis [START_REF] Watanabe | Role for piRNAs and moncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus[END_REF]. However, the previously described cases of methylationmediated silencing of foreign DNA were specific to the female germline. Rasgrf1 is the only imprinted locus that is treated as a transposable element and is therefore more the exception than the rule. In conclusion, although defense against transposons was certainly a driving force for the evolution of DNA methylation in mammals, DNA methylation targeting to imprinted genes in the mammalian germline cannot be assimilated to a host defense strategy.

3.1.b The co-adaptation theory

Two organs that are considered of major importance during pregnancy and the early post-natal period in mammals are the placenta and the hypothalamus. The placenta is the extra-embryonic tissue that allows the transfer of nutrients from the mother to the embryo during pregnancy. The hypothalamus is the part of the brain which controlsamong other important functions that I will detail later-maternal behavior after birth (nursing, inhibition of sexual behavior, maternal care, …). A more recent theory proposes that imprinted genes are positively selected when they favor the coadaptation of parent and offspring traits during mammalian development in utero [START_REF] Wolf | A maternal -Offspring coadaptation theory for the evolution of genomic imprinting[END_REF]. The genetic integration of parent-infant co-adapted traits is considered as an evolutionary force wthat increases the fitness of the offspring.

Several imprinted genes are expressed both in the placenta during development and in the mother's hypothalamus [START_REF] Keverne | Epigenetics, brain evolution and behaviour[END_REF]. Although Wolf and Hager's hypothesis predicted that these imprinted genes should be maternally expressed, the paternally expressed gene 3 (Peg3) is the perfect example of this mother-child coadaptation. Pups that inherit a Peg3 deletion from their father are born with growth retardation, remain smaller during adulthood and develop metabolic, behavior and reproductive phenotypes [START_REF] Curley | Coadaptation in mother and infant regulated by a paternally expressed imprinted gene[END_REF][START_REF] Curley | Increased body fat in mice with a targeted mutation of the paternally expressed imprinted gene Peg3[END_REF]. Peg3 mutation also severely affects maternal behavior in females, which in consequence raise growth-retarded pups [START_REF] Li | Regulation of Maternal Behavior and Offspring Growth by Paternally Expressed Peg3[END_REF]. Interestingly, while a maternally inherited deletion of Peg3 is silent in heterozygous (Peg3 being expressed from the paternal allele), they show a similar phenotype than the pups lacking Peg3 expression [START_REF] Curley | Coadaptation in mother and infant regulated by a paternally expressed imprinted gene[END_REF]. This example highlights the co-adapted function of an imprinted gene in the developing placenta, in the mother's hypothalamus before and after birth and in the pups' hypothalamus. Similar co-adaptation of embryonic placental and maternal behavior defects has been described with the paternally expressed Mest/Peg1 gene [START_REF] Lefebvre | Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest[END_REF].

This theory of the adaptation was further extended to the co-regulation of imprinted genes: the fitness of an individual could increase by selecting genes to become imprinted to match the imprinted expression pattern of their neighboring interacting partners [START_REF] Wolf | Evolution of genomic imprinting as a coordinator of coadapted gene expression[END_REF][START_REF] Wolf | Gene interactions in the evolution of genomic imprinting[END_REF]. This model is congruent with imprinted genes being in clusters and forming regulatory gene interaction networks [START_REF] Patten | Regulatory links between imprinted genes: evolutionary predictions and consequences[END_REF].

3.1.c The parental conflict theory

The two theories described above are part of the expanding list of the "non-conflict" theories [START_REF] Spencer | Non-conflict theories for the evolution of genomic imprinting[END_REF], as opposed to the kindship hypothesis proposed by David Haig (Moore and Haig, 1991). The theory was first suggested in an attempt to understand the unusual triploid composition of endosperm in flowering plants [START_REF] Haig | Parent-Specific Gene Expression and the Triploid Endosperm[END_REF]. Phenotypic similarities were reported in embryos with altered parental contribution between flowering plants and mammals, leading to the hypothesis that genomic imprinting has evolved in both species because the offspring is commonly nourished by maternal tissues during embryogenesis. In angiosperms, normal development requires a triploid endosperm formed with two maternal and one paternal gamete (2:1 ratio). When the endosperm receives a maternal excess (4:1) or a paternal excess (2:2), it displays reduced or enhanced mitotic activity, respectively, leading to impaired development [START_REF] Cooper | Caryopsis Development Following Matings between Diploid and Tetraploid Strains of Zea Mays[END_REF]. In mouse gynogenetic embryos (two maternal pronuclei), embryogenesis failure is due to poor extra-embryonic development, while the embryo proper is more or less normal. Conversely, androgenetic (2 paternal pronuclei) embryos do not grow but their extra-embryonic tissues are normally formed (Barton et al., 1984;[START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF]. Finally, it has been reported that human triploid fetuses formed with an extra-paternal genome display a large placenta, and in contrast, the ones derived from an extra-maternal genome develop a small placenta [START_REF] Hall | Genomic Imprinting : Review and Relevance to Human Diseases[END_REF]. In all these cases of unbalanced parental contribution, the unequal interest of the maternal and the paternal genome for the distribution of the resources in the embryo could be involved. On the one hand, acquiring more resources from the mother is beneficial for the size of the offspring and thus its chance of survival and reproduction. This is in the interest of the father, to have the most vigorous progeny. On the other hand, the more nutrients the mother gives to the embryos, the more it increases the cost to the reproduction for the next progenies.

The mother interest is thus to maintain a balance between embryo survival and her own survival, by reducing the transfer of nutrients during pregnancy.

Haig have thus proposed that mammalian imprinted genes have evolved from a parental "tug-of-war" [START_REF] Haig | The Kinship Theory of Genomic Imprinting[END_REF]Moore and Haig, 1991). The strength of this theory resides in the predictions arising from this model. Tom Moore and David Haig have predicted that imprinted genes in mammals must "influence placental growth, suckling, neonatal behaviour, appetite, nutrient metabolism and postnatal growth rate" and that "imprinting influences appetite control and hypothalamic function" (Moore and Haig, 1991). As it will be developed in the following part, most of the characterized imprinted genes in mice and humans have indeed brain-related function in growth and behavior. Notably, the classical example of an imprinted region that illustrates this theory is the Igf2/H19 locus. The Igf2 gene is paternally expressed and promotes placental growth as well as embryonic and post-natal growth in mice [START_REF] Dechiara | A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting[END_REF][START_REF] Dechiara | Parental imprinting of the mouse insulin-like growth factor II gene[END_REF]. H19 is a long-non-coding RNA maternally expressed that prevents Igf2 expression from the same allele. H19 abolition leads to over-weighted mice that overexpress Igf2 [START_REF] Leighton | Deletion of the H19 gene region in mice disrupts the imprinting of its neighbouring genes[END_REF]. Interestingly, Igf2r, which encodes a receptor of IGF2 that triggers its degradation, is also maternally expressed. Even if the Igf2 locus as well as a number of other imprinted genes fit the parental conflict theory, one question remains: why the genes controlling growth are not all subject to genomic imprinting? Moreover, it appears that a few imprinted genes are not fitting the theory and show the opposite parental-origin/phenotype link. Overall, it seems that one single evolutionary hypothesis cannot fully account for the diversity of imprinted loci and function. Genomic imprinting might rather result from different selective pressure acting at individual loci.

The large asymmetry of DNA methylation patterns in the oocyte and the sperm gives the potential for regulating gene dosage in an allelic manner in the progeny.

Functions of imprinted genes in mammals

3.2.a Placental and embryonic growth

Genomic imprinting is essential for proper embryogenesis in mammals (Barton et al., 1984;[START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF], highlighting a crucial role of imprinted genes during the in utero period. Accordingly, the first identified imprinted genes in mice were involved in fetal growth [START_REF] Barlow | The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus[END_REF][START_REF] Dechiara | A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting[END_REF][START_REF] Leighton | Deletion of the H19 gene region in mice disrupts the imprinting of its neighbouring genes[END_REF][START_REF] Ludwig | Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds[END_REF]. Since these first discoveries, a significant number of imprinted genes have been described as having major roles in placental function and embryonic growth.

As the kinship theory predicts, the products of paternally imprinted genes often enhance fetal growth, while maternally imprinted gene products act as growth repressors. Grb10, a maternally expressed gene, acts as crucial growth restrictor during fetal life: maternal deletion of Grb10 causes placental and embryonic hypertrophy, and biallelic expression leads to growth retardation during both fetal and neonatal development [START_REF] Charalambous | Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism[END_REF][START_REF] Charalambous | Maternally-inherited Grb10 reduces placental size and efficiency[END_REF][START_REF] Shiura | Paternal deletion of Meg1/Grb10 DMR causes maternalization of the Meg1/ Grb10 cluster in mouse proximal Chromosome 11 leading to severe pre-and postnatal growth retardation[END_REF]. Grb10, as well as the cluster of imprinted genes Igf2, Igf2r and Ins2 seem to regulate embryonic growth through the insulin pathway [START_REF] Holt | Grb10 and Grb14: enigmatic regulators of insulin action -and more?[END_REF]. Interestingly, their pattern of embryonic and placental expression resembles a lot what has been described for other imprinted genes, including Kcnq1, Peg3, Peg1, Dlk1/Pref1 and Gtl2. These genes have been shown to be part of an imprinted gene network (IGN), whose transcription is regulated by the zinc-finger type transcription factor ZAC1, as well as the trans action of the H19 RNA [START_REF] Gabory | H19 acts as a trans regulator of the imprinted gene network controlling growth in mice[END_REF][START_REF] Varrault | Zac1 Regulates an Imprinted Gene Network Critically Involved in the Control of Embryonic Growth[END_REF].

Knock-out of each of these co-regulated genes in mice results in severe feto-placental defects [START_REF] Fowden | Imprinted genes and the epigenetic regulation of placental phenotype[END_REF].

The placenta is a key organ during pregnancy, which acts as the interface between maternal and fetal environments. It has mainly two functions during embryogenesis: the transfer of nutrients from the mother to the embryo and an endocrine function. Placental hormones, cytokines and growth factors are key molecules that drive maternal behavior during pregnancy. Notably, placental lactogens stimulate maternal hypothalamic functions for increasing food intake, insulin resistance and pancreatic islet function [START_REF] Ladyman | Hormone interactions regulating energy balance during pregnancy[END_REF]. Impaired placental formation during embryogenesis often leads to embryonic lethality [START_REF] Rossant | Placental development: lessons from mouse mutants[END_REF]. It was recently reported that the importance of this organ in mouse mutant models resulting in embryonic lethality has often been under-estimated [START_REF] Perez-Garcia | Placentation defects are highly prevalent in embryonic lethal mouse mutants[END_REF]. Whereas some imprinted genes influence embryonic growth independently of their expression in the placenta-as it is the case for the paternally expressed Dlk1 gene (Da [START_REF] Da Rocha | Gene dosage effects of the imprinted delta-like homologue 1 (Dlk1/Pref1) in development: Implications for the evolution of imprinting[END_REF]-multiple cases of placental restricted effects have been reported. Notably, several imprinted genes regulate placental functions by modulating the size of the endocrine compartment. For instance, Mash2 (Ascl2) is a maternally expressed imprinted gene encoding a lineage-specific transcription factor that is required for the development of trophoblast progenitors cells [START_REF] Guillemot | Genomic imprinting of Mas2, a mouse gene required for trophoblast development[END_REF].

Mash-2-KO embryos die from placental failure around E10.5, as a consequence of the absence of the spongiotrophoblast, a major hormone-producing region of the placenta [START_REF] Guillemot | Essential role of Mash-2 in extraembryonic development[END_REF]. Two retrotransposon-derived paternally expressed genes, Peg10 and Rtl1, have also essential role in the developing placenta morphology.

Deletion of Peg10 results in the same mid-gestation lethality phenotype as in Mash2-KO embryos [START_REF] Ono | Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality[END_REF], while Rtl1 (also known as Peg11) is required for maintaining feto-maternal capillaries. Rtl1 mutation causes late embryonic or neo-natal lethality [START_REF] Sekita | Role of retrotransposon-derived imprinted gene, Rtl1, in the feto-maternal interface of mouse placenta[END_REF]. Conversely, the maternally expressed genes Cdkn1c and Phlda2, when deleted in mutant mice, cause expansion of spongiotrophoblast cells and increased placental size [START_REF] Fowden | Imprinted genes and the epigenetic regulation of placental phenotype[END_REF][START_REF] Zhang | Cooperation between the Cdk inhibitors p27(KIP1) and p57(KRP2) in the control of tissue growth and development[END_REF]. Recent work has highlighted the role of Phlda2 in suppressing the expression of placental hormones and glycoproteins, suggesting a role in the regulation of maternal resource allocation (Tunster et al., 2016a). It seems that a critical dosage of Mash2 and Phlda2 is essential to control the expansion of the spongiotrophoblast lineage (Tunster et al., 2016b).

Interestingly, elevated levels of human placental PHLDA2 is linked with increase placental lactogens in cases of human fetal growth restriction, consistent with the observations made in mouse models [START_REF] Janssen | Maternal prenatal depression is associated with decreased placental expression of the imprinted gene PEG3[END_REF]. Finally, fetal growth is also impaired by imprinted genes that control nutrient supply, as it is the case for the placenta-specific Igf2-P0 transcript [START_REF] Constância | Placental-specific IGF-II is a major modulator of placental and fetal growth[END_REF]. The placenta thus appears as a major site of imprinted gene expression and function to control proper embryonic growth.

3.2.b Post-natal growth and survival

Affecting fetal growth does not necessarily result in a lethality phenotype but can indirectly impact post-natal growth and survival. This is the case for a number of imprinted genes. For example, Igf2 mutant mice exhibit a 40% growth deficiency at birth but are otherwise morphologically normal and fertile [START_REF] Dechiara | A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting[END_REF].

Mouse mutants with a loss of imprint at the Dlk1-Dio3 locus, resulting in bi-allelic expression of Dlk1, die before birth from placental and embryo maturation defects [START_REF] Rocha | Genomic imprinting at the mammalian Dlk1-Dio3 domain[END_REF]. However, modest perturbation of gene dosage at this imprinted cluster on chromosome 12 leads to normal embryo development but has a critical impact on survivability and growth during the first weeks of life [START_REF] Charalambous | Imprinted gene dosage is critical for the transition to independent life[END_REF]. Overexpression of Dlk1 also alters fat storage and growth hormone feedback regulation in adult mice [START_REF] Charalambous | DLK1/PREF1 regulates nutrient metabolism and protects from steatosis[END_REF]. More recently, a study highlighted the role of DLK1 in the developing fetus as a source of expression in the maternal blood circulation to maintain fetal nutrition during periods of pregnancy [START_REF] Cleaton | Fetusderived DLK1 is required for maternal metabolic adaptations to pregnancy and is associated with fetal growth restriction[END_REF]. Dlk1 thus appears as an essential imprinted gene, both during fetal life and post-natal life, particularly during periods of nutrient partitioning [START_REF] Cassidy | Genomic imprinting, growth and maternal-fetal interactions[END_REF].

While genomic imprinting has been initially discovered as critical for mouse embryo development, an important number of imprinted genes does not influence in utero function but impacts mouse physiology after birth. The Rasgrf1 gene encodes a brain-specific factor expressed exclusively after birth [START_REF] Zippel | Ras-GRF, the activator of Ras, is expressed preferentially in mature neurons of the central nervous system[END_REF] and was the first imprinted gene that was reported to be a post-natal rather than an embryonic growth regulator [START_REF] Itier | Imprinted gene in postnatal growth role[END_REF]. Mutant pups for Rasgrf1 are born with no sign of physiological perturbation, implying that it is facultative during embryo development.

However, when paternally inherited, Rasgrf1 deletion leads to a growth deficiency visible from the first week of age and most prominent around weaning age (3-4 weeks of age), with a 25-30% decrease in weight [START_REF] Itier | Imprinted gene in postnatal growth role[END_REF]. The mice are nevertheless viable and a 15% growth reduction persists in adult mice. This phenotype has been shown to be due to the reduction of Rasgrf1 in both the hypothalamus-leading to a disruption of the growth hormone signaling-and the pancreatic islets, resulting in glucose intolerance [START_REF] Font De Mora | Ras-GRF1 signaling is required for normal -cell development and glucose homeostasis[END_REF].

Gnas, which encodes for a G-protein, and its longer isoform GnasXL provide another example of imprinting-linked post-natal growth effects. Gnas is maternally expressed in a tissue-specific manner in adipose tissues [START_REF] Yu | Variable and tissue-specific hormone resistance in heterotrimeric G s protein ␣-subunit (G s ␣) knockout mice is due to tissue-specific imprinting of the G s ␣ gene[END_REF]

, whereas

GnasXL is exclusively paternally expressed, preferentially in the brain neuro-endocrine tissues involved in feeding behavior [START_REF] Plagge | The imprinted signaling protein XLαs is required for postnatal adaptation to feeding[END_REF]. Fitting with the conflict of interest theory, Gnas and GnasXL have antagonistic effects on glucose metabolism (Chen et al., 2005a). Gnas loss-of-function results in high peri-natal lethality. The surviving mice are obese, develop hyperglycemia due to insulin resistance and increased adiposity (Chen et al., 2005a). Conversely, paternal deletion of GnasXL results in severe body weight reduction accompanied by high rate of lethality during the first days of life [START_REF] Plagge | The imprinted signaling protein XLαs is required for postnatal adaptation to feeding[END_REF]. The lack of GnasXL in neuro-endocrine tissues is associated with poor suckling, decreased level of blood glucose due to increased insulin sensitivity and low adiposity. These metabolic changes are due to stimulation of the central sympathetic nervous system rather than altered adipocyte tissues, suggesting a major role of GnasXL in feeding behavior after birth [START_REF] Xie | The alternative stimulatory G protein α-subunit XLαs is a critical regulator of energy and glucose metabolism and sympathetic nerve activity in adult mice[END_REF]. Another critical aspect of early life survival is the maintenance of neonatal body temperature by thermoregulation in the brown adipocyte tissues (BAT). Gnas, GnasXL, Dlk1 and also Ndn are expressed in BAT and their role in thermogenesis has been described during the pre-and post-weaning period (Peters, 2014).

3.2.c Behavior

As previously mentioned with the examples of GnasXL and Rasgrf1, the brain appears as a major site of imprinted gene expression. Further functional investigation highlighted the role of imprinted genes in brain development and neurological functions [START_REF] Wilkinson | Genomic imprinting effects on brain development and function[END_REF]. Imprinted genes have been shown to influence a wide-variety of behavioral phenotype such as olfaction [START_REF] Isles | Genetic imprinting: Urinary odour preferences in mice[END_REF][START_REF] Swaney | Genomic imprinting mediates sexual experience-dependent olfactory learning in male mice[END_REF], memory, maternal care or feeding behavior. Notably, suckling activity in newborns appears to be impaired in a number of imprinted gene mutant mouse models, including Gnas, GnasXL, Peg3, Magel2 and Dlk1 (Peters, 2014). Nesp is a maternally expressed transcript of the Gnas locus, which encodes a neuroendocrine secretory protein localized in the hypothalamus, the pons and midbrain regions [START_REF] Dent | Brain-expressed imprinted genes and adult behaviour: The example of Nesp and Grb10[END_REF]. Deletion of Nesp does not impact growth and survival after birth, but adult mice display an increased locomotor activity, independent of an anxiety behavior [START_REF] Plagge | Imprinted Nesp55 influences behavioral reactivity to novel environments[END_REF]. This comportment was rather due to an abnormal reactivity to a new environment, suggesting a role of Nesp in the novelty exploration behavior. More recent behavioral studies showed that Nesp-KO, conversely to wild-type mice, would chose an immediate small reward rather than a delayed larger reward [START_REF] Dent | Impulsive choices in mice lacking imprinted Nesp55[END_REF][START_REF] Dent | Impulsive Choice in Mice Lacking Paternal Expression of Grb10 Suggests Intragenomic Conflict in Behavior[END_REF]. This impulsive choice reaction was associated with impaired neurotransmitters involved in impulsive behavior. In the same brain area than Nesp, the paternal allele of Grb10 is expressed [START_REF] Dent | Brain-expressed imprinted genes and adult behaviour: The example of Nesp and Grb10[END_REF][START_REF] Garfield | Distinct physiological and behavioural functions for parental alleles of imprinted Grb10[END_REF] and its ablation leads to increased social dominance as demonstrated by several behavioral tests [START_REF] Garfield | Distinct physiological and behavioural functions for parental alleles of imprinted Grb10[END_REF]. As mentioned earlier, Grb10 can be also maternally expressed from a different promoter, and this parental expression is associated with growth regulation during fetal development [START_REF] Charalambous | Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism[END_REF].This finding highlighted for the first time the implication of an imprinted gene in two distinct phenotypes: fetal growth and social behavior. Recently, a similar bi-modal function was defined for the maternally expressed Cdkn1c gene, in both placental development and neurobiological function in the dopaminergic system [START_REF] Mcnamara | Dopaminergic and behavioural changes in a loss-of-imprinting model of Cdkn1c[END_REF].

Finally, recent investigations have further involved imprinted genes in circadian clock and sleep homeostasis [START_REF] Tucci | Genomic Imprinting: A New Epigenetic Perspective of Sleep Regulation[END_REF]. Notably, Gnas was reported to affect sleep in mice [START_REF] Lassi | Loss of Gnas imprinting differentially affects REM/NREM sleep and cognition in mice[END_REF] and this was further supported by a human GWAS of insomnia complaints, which identified GNAS as one of the strongest candidate gene [START_REF] Hammerschlag | Genome-wide association analysis of insomnia complaints identifies risk genes and genetic overlap with psychiatric and metabolic traits[END_REF]. These new roles of imprinted genes in a wide range of behaviors open the door for future investigation and shed the light on possible implications in psychological diseases. In conclusion, this non-exhaustive description of imprinted gene function identified via mouse models reveals the importance of their dosage regulation during the critical periods of both embryogenesis and early postnatal life.

Insight from human diseases

Uniparental disomy has long been recognized as a cause of spontaneous abortion in humans [START_REF] Engel | A new genetic concept: Uniparental disomy and its potential effect, isodisomy[END_REF]. Individually, imprinted genes play crucial role in human fetal growth and have been identified as misregulated in several neuro-developmental disorders. Both genetic disruption and epigenetic alteration can cause perturbed expression of imprinted genes and lead to disease phenotypes. I will briefly describe here four imprinted-related human syndromes that have been extensively characterized and are related to growth anomalies.

3.3.a Beckwith-Wiedemann and Silver Russel syndromes

The Beckwith-Wiedemann syndrome (BWS) was first described by Dr. J. Bruce Beckwith and Dr. Hans-Rudolf Wiedemann in the early 1960s. This disorder is characterized by in utero overgrowth, resulting in children born with an abnormally large size. The associated phenotypic traits are diverse, and include abdominal defects and large organs (visceromegaly), large tongue (macroglossia), low blood sugar (hypoglycemia), kidney abnormalities and predisposition to childhood tumors. The frequency of this disease is estimated to 1 in 13700 birth worldwide. In 1974, a parentof-origin effect was reported in a familial pedigree of BWS (Wu and Kushnick, 1974).

In the 1990's, BWS was linked to the 11p15.5 region and cases with uniparental paternal disomy of this region were reported [START_REF] Henry | Uniparental paternal disomy in a genetic cancer-predisposing syndrome[END_REF]. However, the causes of this disorder are complex and involve multiple epigenetic and genetic perturbation [START_REF] Cooper | Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome[END_REF]. The 11p15.5 region contains indeed two imprinted domains: [START_REF] Borsos | Building up the nucleus: nuclear organization in the establishment of totipotency and pluripotency during mammalian development[END_REF] the IGF2-H19 locus, regulated by the ICR1 and ( 2) the CDKN1C-KCNQ1 locus, regulated by the ICR2 (Figure 20). Half of the BWS cases are due to epigenetic causes, in which the DNA methylation pattern is altered at the ICR of one of the two imprinted domains [START_REF] Weksberg | Beckwith-Wiedemann syndrome[END_REF]. Notably, gain of DNA methylation at the ICR1 leads to silencing of the H19 RNA, and abnormal biallelic expression of IGF2. But the most common cause of BWS is characterized by the lack of DNA methylation at the ICR2 (also called KvDMR), which leads to biallelic maternal expression of the longnoncoding RNA KCNQ1OT and subsequent silencing of the cell cycle regulator CDKN1C [START_REF] Diaz-Meyer | Silencing of CDKN1C (p57KIP2) is associated with hypomethylation at KvDMR1 in Beckwith-Wiedemann syndrome[END_REF]. In about 20% of the cases, BWS is due to paternal uniparental disomy, with biallelic expression of either IGF2 or KCNQ1OT. Finally, genetic causes disrupting the CDKN1C gene are, to a lesser extent, found in cases of BWS [START_REF] Hatada | An imprinted gene p57 KIP2 is mutated in Beckwith-Wiedemann syndrome[END_REF].

The BWS is thus an overgrowth genomic imprinting disorder caused in most of the cases by loss of expression of the CDKN1C gene and in a few cases, to the gain of the paternally expressed IGF2 gene. Importantly, mouse models of gain and loss of imprinting for those genes recapitulate BWS phenotypic characteristics [START_REF] Tunster | Fetal overgrowth in the Cdkn1c mouse model of Beckwith-Wiedemann syndrome[END_REF]. Interestingly, Beckwith-Wiedemann patients show an increased rate of autism compared to the general population [START_REF] Kent | Beckwith Weidemann syndrome: A behavioral phenotype-genotype study[END_REF], which is reminiscent of the role of mouse Cdkn1c in social behavior. Another imprinted-related syndrome has been linked to the same chromosome 11 region but displays opposite phenotype: babies affected by the Silver Russel disorder are born smaller and exhibit dwarfism for the rest of their life. This phenotypic feature is, for the majority of reported cases, due to hypomethylation of the IGF2-H19 ICR1, and in turn biallelic H19 expression and IGF2 silencing (Peters, 2014). However, because maternal uniparental disomy at human chromosome 7 (MatUPD7) also causes Silver-Russel, GRB10 is another candidate gene for this pathology. Here are represented the two domains of the 11p15.5 region, containing two ICRs. Domain 1 contains the IGF2-H19 locus, while CDKN1C-KCNQ1 forms domain 2.

3.3.b Angelman and Prader-Willi syndromes

Angelman and Prader-Willi syndromes are two neuro-developmental disorders with distinct phenotypic outcomes but caused by a de novo deletion on the same genetic interval on human chromosome 15q11-q13 region, which contains a cluster of imprinted genes expressed in the brain [START_REF] Horsthemke | Mechanisms of imprinting of the Prader-Willi/Angelman region[END_REF]. When the region is deleted on the maternal allele, it causes Angelman syndrome whereas the paternal transmission of the deletion results in Prader-Willi disorder [START_REF] Knoll | Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental origin of the deletion[END_REF].

In 1965, the physician Harry Angelman described for the first time a curious set of clinical features he observed in three children: severe mental retardation, speech impairment, ataxia, seizure and microcephaly [START_REF] Angelman | Puppet Children A Report on Three Cases[END_REF]. These children also displayed a happy disposition characterized by social approach behaviors, smiling and eye contact, which owes them to be referred as "happy puppets". The Prader-Willi syndrome (PWS) is a form of obesity associated with mental retardation that was the first human disease reported as subject to genomic imprinting [START_REF] Nicholls | Genetic imprinting suggested by maternal heterodisomy in non-deletion Prader-Willi syndrome[END_REF].

Children born with PWS display neonatal hypotonia, early childhood obesity, and will develop later cognitive impairment, behavioral characteristics, mild mental retardation and hypogonadism.

The most common genetic cause of the Angelman disorder has been attributed to the loss of UBE3A expression from the maternal allele [START_REF] Horsthemke | Mechanisms of imprinting of the Prader-Willi/Angelman region[END_REF], which can be due to de novo deletion, paternal disomy or mutation of the ICR at the locus. UBE3A encodes a ubiquitin protein with tissue-specific imprinted expression in neurons, and plays a role in GABAergic and glutamatergic neurotransmission [START_REF] Albrecht | Imprinted expression of the murine angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons[END_REF][START_REF] Rougeulle | An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript[END_REF]. PWS results from the loss of expression of several paternally expressed genes in the 15q11-q13 region rather than a single causal gene. Notably, two genes in this genomic interval, MAGEL2

and NDN, are lost in PWS and are the focus of several studies because of their role in the hypothalamic regions that control feeding behavior [START_REF] Nicholls | Genome organization, function, and imprinting in Prader-Willi and Angelman syndromes[END_REF].

Magel2-null mice display growth retardation at birth but an excessive gain in weight after weaning, followed by increased adiposity and altered metabolism, and ultimately, obesity [START_REF] Bischof | Inactivation of the mouse Magel2 gene results in growth abnormalities similar to Prader-Willi syndrome[END_REF]. These characteristic features of PWS appear to be due to the hypothalamic regulation of feeding behaviors at two different levels. First, decreased oxytocin production leads to impaired suckling behavior [START_REF] Schaller | A single postnatal injection of oxytocin rescues the lethal feeding behaviour in mouse newborns deficient for the imprinted Magel2 gene[END_REF]. Second, loss of Magel2 also causes deficiency in leptin sensing, an hormone secreted by adipocyte tissues that regulate feeding behavior and energy expenses [START_REF] Mercer | Magel2 is required for leptin-mediated depolarization of POMC neurons in th[END_REF]. Ndn-deficient mice exhibit the same impaired suckling behavior due to low hypothalamic levels of oxytocin but also show specific memory behavior, reminiscent of human PWS [START_REF] Muscatelli | Disruption of the mouse Necdin gene results in hypothalamic and behavioral alterations reminiscent of the human Prader-Willi syndrome[END_REF]. Altogether, it appears that Ube3a, Ndn and Magel2 mutant mice provide important insight into understanding the molecular events leading to the Angelman and Prader-Willi human disorders, supporting the mouse as a good model to gain insight into imprinted-related diseases in humans.

The hypothalamo-pituitary axis

The hypothalamus is a central part of the brain involved in multiple physiological functions that are essential for health and survival. The hypothalamus notably influences hormonal balance through its interactions with the pituitary gland. As previously emphasized in this Introduction, imprinted genes have major roles in metabolism and growth regulation in mice and their mis-regulation in humans causes neuro-developmental disorders such as the Prader-Willi syndrome. The hypothalamus thus appears as a logical site for imprinted gene action (Charalambous et al., 2007;[START_REF] Ivanova | Imprinted genes and hypothalamic function[END_REF]. Moreover, during the path of my PhD, we identified Zdbf2 as highly expressed in the pituitary and the hypothalamus in mice. My results suggest that the role of Zdbf2 is intimately linked to a biological function controlled by the hypothalamo-pituitary axis. In this last section, I will briefly describe the function and structure of the hypothalamus and the pituitary gland and give an overview of the behavior of imprinted genes in these organs. I will finally address the developmental aspect of the hypothalamo-pituitary axis during embryogenesis and how it controls growth in mice.

The hypothalamus

4.1.a Structure and function of the hypothalamus

The hypothalamus is a major homeostatic regulator of metabolic, behavioral and endocrine processes. It is involved in survival -food intake, temperature regulation, circadian rhythms-and in major neuro-endocrine functions through communication with the pituitary gland [START_REF] Saper | The hypothalamus[END_REF]. Located in the ventral part of the brain, the hypothalamus is a complex brain structure with an enormous cell-diversity, containing both neuronal and non-neuronal cells [START_REF] Chen | Single-Cell RNA-Seq Reveals Hypothalamic Cell Diversity[END_REF]. It is symmetrical to the third ventricle (3V), forming a boundary between each part in the middle line (Figure 21). It is structured in hypothalamic nuclei that are formed during embryonic neurogenesis, from E10 to E16, under the control of specific transcription factors [START_REF] Shimogori | A genomic atlas of mouse hypothalamic development[END_REF]. The hypothalamus can be separated in three areas: the preoptic area in the anterior part, the tuberal area in the lateral part and the posterior area [START_REF] Saper | The hypothalamus[END_REF]. The hypothalamic nuclei of the anterior part are involved in thermoregulation, circadian rhythms, sleep control and sexual behavior, while the posterior part plays a crucial role in stress responses. The lateral hypothalamus contains key integrative signals for feeding behavior, sexual behavior and many endocrine functions. Notably, the ventral surface of the lateral hypothalamus contains the median eminence (Me), which establishes contacts with the pituitary stalk for the exchanges between these two organs (Figure 21).

Within the lateral hypothalamus, the arcuate nucleus (ARC) control endocrine input to the pituitary gland and food intake together with neural structures of the ventroand dorso-medial hypothalamic nuclei (VMH and DMH). The key player in food regulation signaling is the leptin, an hormone secreted by white adipocyte tissues when substrates are available (i.e. after food consumption for example) and one of the main causal gene in mouse and human obesity [START_REF] Zhang | Positional cloning of the mouse obese gene and its human homologue[END_REF]. Leptin acts in the hypothalamus, on specific reception of the ARC, VMH and DMH nuclei to secrete neuro-peptides that regulate the metabolic balance between food intake/energy expenditure [START_REF] Friedman | Leptin and the regulation of body weight in mammals[END_REF]. After feeding, increased leptin levels activate POMC and inhibit AgRP/NPY neurons, while in fasting condition, the low leptin levels induce the opposite. This activation of specific neuro-peptides by the hypothalamus is responsible of the satiety/hunger behavior. The paraventricular nucleus (PVN) -localized between the anterior and the lateral hypothalamic area-also controls food intake but mostly during pre-weaning period [START_REF] Pei | AVP neurons in the paraventricular nucleus of the hypothalamus regulate feeding[END_REF]. This endocrine control is mediated through the expression of oxytocin by the PVN, which is then secreted to the pituitary gland and provided to the entire body. The oxytocin is responsible of the suckling behavior of the newborn mice, being essential for survival during the critical early-life period. Importantly, most of the hypothalamic nuclei and their function are conserved between mice and humans.

4.1.b Imprinted genes in the hypothalamus

About a decade after the first demonstration of the requirement of both parental genomes in mouse embryonic development through generation of uniparental embryos (Barton et al., 1984;McGrath and Solter, 1984a;[START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF], elegant experiments on mouse chimeras demonstrated the importance of genomic imprinting for brain development [START_REF] Allen | Distribution of parthenogenetic cells in the mouse brain and their influence on brain development and behavior[END_REF][START_REF] Keverne | Genomic imprinting and the differential roles of parental genomes in brain development[END_REF]. Chimeric mice were generated with gynogenetic (two maternal contribution) or androgenic (two paternal contribution) cells transplanted in biparental embryos. Conversely to the pure gynogenetic (GG) or androgenic (AG) embryos, these chimeras survived during embryogenesis and developped to term. However, these mice displayed severe brain and body size phenotypes: GG chimeric embryos had large brain although their body was smaller than their wild-type littermates, while AG chimeric embryos showed oppositely a smaller brain with a general larger body size [START_REF] Allen | Distribution of parthenogenetic cells in the mouse brain and their influence on brain development and behavior[END_REF][START_REF] Keverne | Genomic imprinting and the differential roles of parental genomes in brain development[END_REF]. Using reporter genes specific to GG or AG cells, the authors were able to follow the localization of those cells in the developing brain. Interestingly, these cells display reciprocal distribution in the brain: AG cells localized preferentially in the hypothalamus and the pre-optic area, while GG cells colonized the cortex, striatum and hippocampus and were excluded from the AG-occupied regions. These analyses provided the first demonstration that the two parental genomes contribute differentially to brain development. This also highlighted the importance of imprinted genes in specific brain structures, a statement that has been further elaborated in genome-wide studies [START_REF] Gregg | High-Resolution Analysis of Parent-of-Origin Allelic Expression in the Mouse Brain[END_REF](Gregg et al., , 2010a;;[START_REF] Perez | New Perspectives on Genomic Imprinting, an Essential and Multifaceted Mode of Epigenetic Control in the Developing and Adult Brain[END_REF].

The reported role of imprinted genes in growth metabolism and behavior, the function of the hypothalamus, together with the restricted localization of AG cells in this organ, define the hypothalamus as an imprinting "hot spot" [START_REF] Ivanova | Imprinted genes and hypothalamic function[END_REF].

Widespread imprinting has been reported in the hypothalamus of both mice and humans [START_REF] Babak | Genetic conflict reflected in tissue-specific maps of genomic imprinting in human and mouse[END_REF]. Notably, Ndn and Magel2 are highly expressed in the hypothalamus and are involved in the phenotype of Prader-Willi patients [START_REF] Schaller | A single postnatal injection of oxytocin rescues the lethal feeding behaviour in mouse newborns deficient for the imprinted Magel2 gene[END_REF]. Additionally, the paternally expressed imprinted gene Nnat is an hypothalamic target of the leptin signal, involved in energy homeostasis [START_REF] Vrang | The Imprinted Gene Neuronatin Is Regulated by Metabolic Status and Associated With Obesity[END_REF]. Other examples of imprinted genes, including the already mentioned Peg3, Gnas, and Dlk1 genes, are highly expressed and imprinted in hypothalamic nuclei where they exert their function.

The pituitary gland

4.2.a Structure and function of the mature pituitary gland

The pituitary, as small as it is, is a major endocrine gland controlling a large range of vital processes, such as puberty, reproduction, growth, lactation, metabolism and stress response. It is localized in the skull basis and is physically linked to the hypothalamus through the pituitary stalk and the median eminence. This connection between the pituitary and the hypothalamus is crucial to control the hormonal secretion of the gland. Notably, two hypothalamic nuclei, the arcuate hypothalamic nucleus (ARC) and the paraventricular nucleus (PVN), are keys controllers of pituitary gland secretion. The mature adult pituitary gland is composed of three lobes -anterior, intermediate and posterior-which are morphologically and functionally distinct (Figure 22). The posterior lobe originates from the neurohypophysis, whereas the intermediate

4.2.b Formation of the pituitary gland during development

In the mouse, the pituitary gland emanates from an ectodermal structure, the hypophyseal placode, at E7.5. This placode is in contact with the oral ectoderm and the neuro-epithelium that corresponds to the primitive hypothalamus (Figure 23). At E9.5, the hypophyseal placode forms a surface ectoderm structure, the Rathke's pouch (RP), which will give rise to the anterior and intermediate lobe of the future pituitary [START_REF] Mollard | A tridimensional view of pituitary development and function[END_REF]. The posterior pituitary originates from a neural ectodermal structure, the infundibulum, which evaginates towards RP at E12.5. This will give rise not only to the posterior lobe of the pituitary but also to the median eminence and the pituitary stalk (Figure 23). Pituitary morphogenesis and differentiation requires the expression of a cascade of transcription factors and signaling molecules [START_REF] Gaston-Massuet | Genetic interaction between the homeobox transcription factors HESX1 and SIX3 is required for normal pituitary development[END_REF][START_REF] Kelberman | Genetic Regulation of Pituitary Gland Development in Human and Mouse[END_REF].

Abnormalities in these signaling pathways result in a spectrum of disorders known as hypopituitarism.

Around E10. for the morphogenesis and the future endocrine function of the pituitary gland.

Meanwhile, the LIM-homeodomain Lhx3/4 transcription factors are required for the development of both the pituitary gland and the central nervous system. Lhx4

expression is restricted to the early development of the anterior lobe, while Lhx3 is maintained throughout development and in the adult gland to define cell lineages and activate pituitary hormones [START_REF] Mullen | Roles of the LHX3 and LHX4 LIM-homeodomain factors in pituitary development[END_REF]. Finally, another homeodomain transcription factor, Prop-1, which peaks at E12.5, is necessary for pituitary cell lineage differentiation. Prop-1 is activating Pit-1, the transcription factor that is expressed from E13.5 onwards and is essential for the terminal differentiation of the somatotrophs (GH), lactotrophs (PRL) and thyrotrophs (TSH) lineages, as well as their post-natal expansion [START_REF] Mollard | A tridimensional view of pituitary development and function[END_REF][START_REF] Rizzoti | Genetic regulation of murine pituitary development[END_REF]. Pit-1 thus appears a a major factor of cellular identity in the posterior pituitary.

The differentiation of the various cell linages of the pituitary gland is not a synchronous process. TSH cells are the first ones to emerge at E11.

Infundibulum

Future intermediate lobe

Median eminance

Posterior lobe

Intermediate lobe

Anterior lobe

Hormone-producing cells (ACTH, GH, LH, FSH, TSH, PRL)

Growth control in mice

4.3.a General principles of mouse growth control

Growth defines the process of increasing in size. In biology, growth can be defined at the level of a single cell, multiple cells or ultimately, a whole organism. Body size growth influences a number of biological processes: the bigger you are, the more energy you will need and the bigger your organs will be, thus requiring an increased physiological demand. Growth is the basis of the formation of an entire organism:

development starts with a cell of few nanograms to finally reach a multicellular weight of approximately 30 grams in adult mice. However, during the first days of embryonic life, from the fertilized egg to the blastocyst stage, the embryo is not subject to any increase in growth. While cells are dividing, the mass of the embryo is maintained.

During this period of pre-implantation development, the embryo transitions from one cell to about 128 cells at the blastocyst stage [START_REF] Streffer | Kinetics of Cell Proliferation in the Pre-Implanted Mouse Embryo in Vivo and in Vitro[END_REF]. Subsequently to the implantation in the uterus, the embryo slows down its growth rate until gastrulation when the growth rate will immediately increase, concomitantly with organogenesis. At birth, the mouse pups weight 1.5g in average, which corresponds to a 6.5 x 10 6 increase in size compare to the 230ng zygote [START_REF] Efstratiadis | Genetics of mouse growth[END_REF].

During post-natal life, the growth rate is characterized by three phases. From the first days up to about two weeks of age, the growth rate continues at a similar speed as before birth. Then, between 2 to 3 weeks of age, there is an important transient change: the growth rate declines compared to what it was before. Finally, the initial growth rate is restored after weaning, until about 8 weeks, when the mouse has reached adult size. As it is such a precise mechanism, growth needs to be tightly regulated. Growth control genes are defined as genes that lead to altered growth, both upon knock-out and overexpression. A growth-promoting gene is associated with growth retardation in a mouse KO, while its overexpression should increase global growth; the inverse applies for a growth-inhibiting gene [START_REF] Efstratiadis | Genetics of mouse growth[END_REF].

4.3.b Hypothalamo-pituitary axis-dependent control of growth

The growth hormone (GH) pathway is the major regulator of growth during the mouse post-natal period. The GH is secreted by the somatotroph cells from the anterior pituitary gland, which are activated during development by Prop-1 and Pit-1 [START_REF] Ryan | POU domain family values: Flexibility, partnerships, and developmental codes[END_REF]. These cells produce GH in response to the hypothalamic growthhormone releasing hormone (GHRH) that binds to the GHRH-receptor in the pituitary.

Circulating GH then binds to GH-R receptors, which leads to transduction signals of growth and proliferation in multiple tissues. Accordingly, mutations in Gh, Ghrh, Ghrhr, Gh-r, Prop-1 or Pit-1 genes all result in severe dwarfism in mice [START_REF] Snell | Dwarf, a New Mendelian Recessive Character of the House Mouse[END_REF][START_REF] Sornson | Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism[END_REF][START_REF] Voss | Anterior pituitary development: Short tales from dwarf mice[END_REF]. The most well-characterized Gh-deficient mice with dwarfism are the Snell (dw), the Ames (df) and the little (lit) mutants. They all grow normally during embryogenesis and are born with a similar weight as their wild-type littermates. However, severe growth retardation starts at around 2 weeks of age, resulting in a 25-60% of normal growth weight [START_REF] Efstratiadis | Genetics of mouse growth[END_REF]. These observations demonstrated that the growth hormone gene has no impact on embryonic growth but is rather essential for growth from 2 weeks of age, at the onset of puberty, under the activation of hypothalamic GhRH [START_REF] Ebling | The neuroendocrine timing of puberty[END_REF].

Rather than being dependent upon the growth hormone, embryonic growth is regulated by the IGF signaling pathway. The IGF1 and IGF2 ligands are produced by many somatic tissues, act in an autocrine/paracrine manner and circulate as plasmatic hormones. Their action depends upon the IGF1R and IGF2R receptors: IGF1 binds to IGF1R and IGF2 binds to both receptors [START_REF] Fernandez | The many faces of insulin-like peptide signalling in the brain[END_REF]. As describe earlier in this thesis, Igf2 and Igf2r show opposite parental pattern of imprinted expression and their products have antagonistic roles on embryonic growth. While IGF2 role is restricted to the pre-natal life, IGF1 has continuous function during development and after birth. Igf1-deficient mice are born with a 60% normal size, as it is the case for Igf2 mutants, and the post-natal growth rate is lower than wild-type mice.

The surviving pups become 30% to normal weight at adult age [START_REF] Liu | Mice carrying null mutations of the genes encoding insulinlike growth factor I (lgt-1) and type 1 IGF receptor (lgf1r)[END_REF]. The post-natal production of IGF1 from the liver is controlled by the release of GH from the anterior pituitary gland. At 1-2 weeks, GH levels increase and this stimulates the production of circulating IGF1 by the liver which then mediates hormonal effects to increase general body growth [START_REF] Lupu | Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth[END_REF]. Importantly, IGF1 signaling exerts a negative feedback on the hypothalamo-pituitary axis to regulate GH secretion [START_REF] Murray | The hypothalamo-GH axis: The past 60 years[END_REF]. Importantly, the TSH has also growth-mediated effect in post-natal mice:

mutants lacking Thra, the gene encoding the main thyroid hormone (T3), exhibit normal

RESULTS

Context and summary of my PhD work.

Genomic imprinting is a paradigm of epigenetic regulation that allows the control of expression of a few mammalian genes in a mono-allelic and parent-specific manner.

A few years ago, our laboratory has made an important contribution to the field through the identification of tissue and stage-specific imprinting (Proudhon et al., 2012). The not the case and we were able to show for the first time the importance of an early embryonic and transient transcript in controlling adult physiological processes in mice (Greenberg et al., 2017). We demonstrated that the early transcription of Liz is responsible for programming adult growth later on, through the activation of the canonical promoter of Zdbf2 in the brain. As a large impact in the field of developmental biology, this discovery highlighted the physiological importance of an early embryonic event and shed the light on how critical the early embryonic period is for long-term biological processes. Theoretically, early programming of gene expression requires an epigenetic switch: a regulatory element acquires a chromatin state in the early embryo, but this state is read out only later, after being stably maintained throughout development. Cytosine DNA methylation represents a possible mediator of early programming of adult phenotypes, as this mark stably propagates across cell divisions 2 . In mammals, DNA methylation patterns are acquired across the genome as the pluripotent embryo implants into the uterus and starts forming the different tissue precursors; these patterns are then globally maintained throughout somatic life 3,4 .

What is the function of

The distribution of DNA methylation is linked to underlying histone modifications. In particular, through their PWWP domains, the de novo DNA methyltransferases (DNMTs) DNMT3A and DNMT3B bind trimethylated lysine 36 of histone H3 (H3K36me3), a mark that is linked to transcriptional elongation 5,6 . In contrast, they are antagonized by the presence of methylation at H3K4 residues 7,8 . An anticorrelative relationship also exists between DNA methylation and H3K27 trimethylation, which is catalyzed by Polycomb repressive complex 2 (PRC2). Although both marks associate with transcriptional repression, H3K27me3 and DNA methylation seem to be mutually exclusive in CpG-rich regions, such as CpG island (CGI) promoters 9-12 . The interplay between H3K36, H3K4 and H3K27 methylation can explain the majority of the specificities of mammalian genomic methylation patterns: exclusion from most CGI promoters and preferential localization to the bodies of actively transcribed genes.

Despite increasing knowledge on how and where DNA methylation patterns are established during embryogenesis, an understanding of the impact of these patterns on adult physiology has remained elusive, in large part because mutations in the DNMT family severely affect early development 13,14 . Similarly, mutants for core components of PRC2 fail to undergo gastrulation 15-17 . As a result, although their existence has been postulated, events of early epigenetic programming of adult phenotypes have thus far failed to be clearly demonstrated.

To discern the long-lasting effects of an early epigenetic switch, we took advantage of the Zdbf2 locus (encoding zinc finger, DBFtype containing 2), which is under the control of transient genomic imprinting 18 . Imprinting control regions (ICRs) acquire different DNA methylation patterns during male and female gametogenesis and maintain these parent-of-origin differences from fertilization throughout life 19 . In the case of transient imprinting, maternal-specific methylation exists only from fertilization to implantation, which corresponds to 4 d in mice and 7 d in humans 20 . The biological relevance of transient imprinting is currently unknown, and it is unclear whether transient imprints impact early development or entail lifelong epigenetic consequences.

At the Gpr1/Zdbf2 locus, the transcription start site (TSS) of an alternative transcript of Zdbf2 -Liz (long isoform of Zdbf2 )-coincides Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth Maxim V C Greenberg 1,5 , Juliane Glaser 1,5 , Máté Borsos The potential for early embryonic events to program epigenetic states that influence adult physiology remains an important question in health and development. Using the imprinted Zdbf2 locus as a paradigm for the early programming of phenotypes, we demonstrate here that chromatin changes that occur in the pluripotent embryo can be dispensable for embryogenesis but instead signal essential regulatory information in the adult. The Liz (long isoform of Zdbf2) transcript is transiently expressed in early embryos and embryonic stem cells (ESCs). This transcription locally promotes de novo DNA methylation upstream of the Zdbf2 promoter, which antagonizes Polycomb-mediated repression of Zdbf2. Strikingly, mouse embryos deficient for Liz develop normally but fail to activate Zdbf2 in the postnatal brain and show indelible growth reduction, implying a crucial role for a Lizdependent epigenetic switch. This work provides evidence that transcription during an early embryonic timeframe can program a stable epigenetic state with later physiological consequences.

with a maternal germline differentially methylated region (gDMR) 18,21,22 (Fig. 1a). Liz is expressed from the paternal allele in the pluripotent blastocyst, before ceasing expression at implantation for the rest of somatic life, in parallel with de novo DNA methylation of the gDMR (Supplementary Fig. 1a). Following Liz expression, DNA methylation is acquired in a paternal-specific manner upstream of the Zdbf2 promoter, generating a somatic DMR (sDMR), and the canonical Zdbf2 promoter becomes active from the same allele. This setting, established at implantation, exhibits lifelong stability. The Zdbf2 locus therefore represents an attractive model to study the potential for early embryonic events that may confer lifelong epigenetic effects.

By using in cellula and in vivo genetic approaches in the mouse, we report here that Liz transcription is required for the disruption of a repressive block of H3K27me3 modifications via the deposition of antagonistic DNA methylation marks at the sDMR and that this disruption is required for Zdbf2 activation. Notably, failure to express Liz in the early embryo is not detrimental for the embryo itself but has a lifelong impact on growth, as Zdbf2 fails to activate in the hypothalamus-pituitary axis postnatally. Our study provides one of the very few demonstrations of the potential for early and transient transcriptional events being epigenetically transmitted and relevant for adult physiology.

RESULTS

EpiLC differentiation recapitulates the embryonic dynamics of the paternal Liz/Zdbf2 allele

To gain mechanistic insight into the epigenetic switch occurring at the Zdbf2 locus, we used a culture-based system of ESC differentiation, where we could recapitulate de novo DNA methylation (Fig. 1b). As a starting point, we relied on serum-free culture medium supplemented with vitamin C and inhibitors of MEK and GSK3 (2i+vitC) to obtain a naive, DNA-methylation-free sDMR (Fig. 1c,d and Supplementary Fig. 1b), as assessed by bisulfite pyrosequencing (BS-pyro) and bisulfite cloning sequencing (Supplementary Fig. 1b). As parental methylation imprints are not maintained in 2i+vitC medium 23,24 (Fig. 1c,d and Supplementary Fig. 1b), this system converted the two alleles into a paternal-like state, as observed in vivo before implantation, in the pluripotent blastocyst (Supplementary Fig. 1a).

To induce de novo DNA methylation, we performed epiblast-like cell (EpiLC)-directed differentiation, which induces a transition from naive to primed pluripotency 25 (Fig. 1b). Within 4 d, CpG methylation increased across the genome from less than 10% to 75%, in line with that of the late epiblast in vivo (Fig. 1b-d and Supplementary Fig. 1b) 4 . This level was then globally maintained until the end of differentiation at day 7. At the Zdbf2 locus, the sDMR acquired methylation at a similar rate as the genome, while the gain observed at the gDMR was delayed, as observed in vivo (Fig. 1c,d and Supplementary Fig. 1b) 18 . The sDMR exhibited full allelic methylation in day 7 EpiLCs: both alleles resembled the paternal allele, as observed in vivo from the epiblast stage onward.

With regard to transcriptional regulation, Liz expression spiked immediately at day 1 of EpiLC differentiation, slowly decreased from day 2 onward, and was undetectable by day 7 (Fig. 1e). Liz upregulation therefore coincides with the exit from naive pluripotency in culture, with a dynamics and expression levels similar to what is observed in the blastocyst (Supplementary Fig. 1c) 18 . In contrast, Zdbf2 expression remained low until day 3, after which transcript levels rose until day 7 (Fig. 1e), concordant with its post-implantation induction in vivo. The mirror-image pattern of Liz and Zdbf2 kinetics indicated a possible switch between their respective promoters during differentiation, which we investigated by single-cell RT-qPCR (Fig. 1f). Liz downregulation preceded the methylation of its promoter, the gDMR (Fig. 1c-e and Supplementary Fig. 1b): methylation acquisition at this region seems to be a consequence rather than a cause of Liz transcriptional shutdown. In contrast, Zdbf2 levels increased only after the sDMR was fully methylated, past day 4. As in vivo, this suggests a causative, positive role of sDMR methylation on the activity of the Zdbf2 promoter located some 10 kb downstream.

As a whole, the differentiation of 2i+vitC-grown ESCs toward an EpiLC path recapitulates the overall dynamics of de novo DNA methylation and transcriptional regulation that occur in vivo on the paternal allele of the Zdbf2 locus, from which the Liz RNA is originally produced. Therefore, this cellular system provides a suitable tool to probe the underpinnings of the regulatory effects of Liz. 112

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Liz transcription is involved in sDMR DNA methylation and Zdbf2 activation Across the genome, DNA methylation is preferentially targeted to the body of actively transcribed genes 26 . To test whether the gDMR and the sDMR acquire DNA methylation upon transcriptional elongation of the genes wherein they lie-Gpr1 and Liz (Fig. 1a)-we performed CRISPR/Cas9-or TALEN-driven editing. DNA methylation of the gDMR was unaffected upon homozygous deletion of the Gpr1 TSS (Supplementary Fig. 2a-c). However, a loss-of-function Liz mutation-obtained through a 1.7-kb deletion of the gDMR, which spans the TSS and first exon of Liz (Fig. 2a-c and Supplementary Fig. 3a)-severely impaired sDMR methylation (Fig. 2d).

Concomitantly, activation of Zdbf2 transcription was drastically compromised in Liz EpiLCs (Fig. 2e). As Liz cells showed proper differentiation kinetics and normal regulation of the DNA methylation machinery (Supplementary Fig. 3b,c), the observed defects are likely to reflect a genuine impact of Liz on local chromatin features. Using a construct that interrupts Liz transcription (Liz::2×STOP) (Fig. 2a-c and Supplementary Fig. 3d,e), we were able to conclude that Liz itself, and likely the process of Liz transcriptional elongation, was involved in the epigenetic setting of the locus. Although the Liz::2×STOP mutation behaved as a hypomorph in comparison to Liz mutants, a significant effect was observed on sDMR methylation (Fig. 2d) and Zdbf2 activation (Fig. 2e).

Available chromatin immunoprecipitation (ChIP) data sets show an enrichment of H3K36me3 along the path of the Liz transcriptincluding at the sDMR-in ESCs 18,27 . By ChIP-qPCR, we found that this H3K36me3 block was lost in Liz ESCs (Supplementary Fig. 4a). Given the tight correlation between H3K36me3 and DNA methylation across the genome 6 , we hypothesized that the effect of Liz transcription on sDMR methylation could rely on intermediate H3K36me3 marks. However, while H3K36me3 was severely depleted throughout the path of Liz upon deletion of Setd2 -the sole mammalian H3K36 trimethyltransferase-encoding gene (Supplementary Fig. 4b-e) 6 , the sDMR DNA methylation was normal in Setd2 -/- EpiLCs (Supplementary Fig. 4f). These results exclude a major role of H3K36me3 in recruiting de novo DNMTs at the sDMR. Preceding work suggested that the link between H3K36me3 and DNA methylation is specifically mediated by DNMT3B in mouse ESCs, whereas DNMT3A methylates its targets in a SETD2-independent manner 6 . We found that DNA methylation levels of the sDMR were normal in single Dnmt3a -/-or Dnmt3b -/-mutant embryos at embryonic day (E) 11.5 (Supplementary Fig. 4g). In sum, the lack of an sDMR methylation phenotype in Setd2 -/-cells may reflect the redundant activity of DNMT3B and DNMT3A at this locus. As a result, we conclude that the sDMR gains full methylation in a Liz-transcription-dependent but H3K36me3-independent manner.

Zdbf2 activation requires H3K27me3 eviction

We previously reported the presence of a block of repressive H3K27me3 marks extending over the sDMR in a tissue-wide manner 18,27,28 , which we also observed in 2i+vitC-cultured ESCs (Fig. 3a,b and Supplementary Fig. 5a,b). This domain spans ~25 kb and is bordered by two DNA-methylation-free CGIs 18 : an intergenic CGI and the Zdbf2 CGI promoter. Interestingly, the Zdbf2 locus showed dynamic H3K27me3 patterns upon EpiLC differentiation: the block decreased in intensity from day 0 to day 7 over the sDMR and the Zdbf2 promoter (Fig. 3a,b and Supplementary Fig. 5b), in correlation with the timing of Zdbf2 activation (Fig. 1e).

To genetically ascertain whether H3K27me3 prevents Zdbf2 expression, we used ESCs deficient for embryonic ectoderm development (EED), a core component of PRC2 (ref. 29). While EED is dispensable in ESCs cultured in serum-based or in 2i-only medium 30 , we previously noticed a proportion of Eed -/-ESC lethality during 2i+vitC conversion 24 . The surviving cell population recovered and continued to proliferate, but DNA methylation levels never reached the same nadir as in wild-type cells (Supplementary Fig. 5c,d). Nevertheless, we observed an alleviation of Zdfb2 repression in undifferentiated 2i+vitC-grown Eed -/-ESCs (Fig. 3c); levels were similar to wildtype expression after 4 d of EpiLC differentiation, in agreement with H3K27me3 preventing Zdbf2 expression before differentiation. The effect of the Eed mutation on Zdbf2 was no longer significant upon differentiation, consistent with the normal depletion of H3K27me3 in EpiLCs at this locus. The Eed mutation resulted in cell death after day 4, which limited our analysis to this last time point. Finally, we assessed whether Liz transcription could be responsible for clearing the Zdbf2 locus of H3K27me3 marks. The H3K27me3 block was already stronger in Liz relative to wild-type ESCs at day 0 (Fig. 3a,b and Supplementary Fig. 5a,b). At day 4 and day 7 of differentiation, instead of being depleted, H3K27me3 accumulated in Liz EpiLCs, with a five-to tenfold enrichment at the sDMR and Zdbf2 CGI promoter in comparison to wild-type cells (Fig. 3a,b and Supplementary Fig. 5b). Notably, introduction of the Eed mutation onto the Liz background (Supplementary Fig. 5e) suppressed the Liz phenotype: in Liz; Eed -/-cells, Zdbf2 transcription was restored and even higher than wild-type levels (Fig. 3d and Supplementary Fig. 5d).

In sum, analyses of Liz and EED mutants demonstrate that Liz transcription antagonizes H3K27me3 upstream of and at the Zdbf2 promoter, and that this is required for Zdbf2 expression upon differentiation. Interestingly, despite a major reduction in Zdbf2 expression, H3K4me3 enrichment was largely unaffected at the Zdbf2 promoter in Liz-depleted cells (Fig. 3e and Supplementary Fig. 5b): this suggests that Liz-mediated effect on Zdbf2 activity is derepression, as opposed to activation.

DNA methylation antagonizes

A R T I C L E S

for DNA methylation 31 . Upon EpiLC-directed differentiation, DNMT triple-knockout cells exhibited the same trend of pluripotency factor expression (Supplementary Fig. 6a) and displayed similar morphological traits (Supplementary Fig. 6b) as wild-type cells. This finding underscores the ability of DNA-methylation-free DNMT tripleknockout ESCs to adopt a fate akin to EpiLCs, while they cannot undergo more advanced differentiation states 32,33 .

As expected, the gDMR and sDMR did not gain DNA methylation in DNMT triple-knockout EpiLCs (Supplementary Fig. 6c). While Liz displayed proper expression levels and dynamics (Fig. 4a), Zdbf2 completely failed to activate in DNMT triple-knockout EpiLCs at day 4 or day 7, showing an even stronger phenotype than the Liz mutation (Fig. 4b). This was not due to a delayed activation: Zdbf2 was still silent in DNMT triple-knockout EpiLCs at day 10 (data not shown).

The lack of Zdbf2 activation during DNMT triple-knockout EpiLC differentiation could reflect the misregulation of essential transcription factors in this background. Alternatively, the continued transcriptional repression of Zdbf2 could be due to an inability to displace PRC2 in the absence of DNA methylation. Indeed, instead of being depleted, H3K27me3 remained strongly enriched at the sDMR and at the Zdbf2 promoter upon differentiation of DNMT triple-knockout EpiLCs (Fig. 4c), while H3K27me3 levels at control loci were similar between wild-type and mutant cells (Supplementary Fig. 6d). Taken together, our results suggest that it is primarily DNA methylation at the sDMR that antagonizes H3K27me3-mediated silencing of Zdbf2 , and not the act of Liz transcription itself.

The Liz knockout regulatory phenotype is recapitulated in embryos

In differentiating ESCs, the Liz transcript seems to orchestrate the removal of H3K27me3 upstream of the Zdbf2 gene through the deposition of antagonistic DNA methylation marks. Liz expression is turned off at the exit of naive pluripotency, but the epigenetic switch promoted by Liz ensures permissive Zdbf2 expression later on. To decipher the biological relevance of such complex Liz-based regulation, we generated Liz knockout mice (Supplementary Fig. 7a). We followed two lines, derived from two independent founders carrying the expected 1.7-kb deletion.

As Liz is only expressed in preimplantation embryos, the effect of the gDMR deletion on Liz transcription was investigated specifically at that stage. Moreover, as Liz is paternally expressed, only paternally inherited deletions should produce loss-of-function alleles, while maternally inherited deletions should behave as silent mutations. Liz transcript levels were analyzed by RT-qPCR in individual blastocysts upon paternal or maternal transmission of the gDMR deletion, generated by crosses between wild-type C57Bl6/J mice and either Liz heterozygote male or female mice, respectively (Fig. 5a). While blastocysts from Liz +/-mothers homogeneously accumulated wild-type levels of Liz, blastocysts from Liz +/-fathers fell into two populations: one that exhibited wild-type levels (Liz WT) and the other with undetectable Liz (paternal knockout). Deletion of the gDMR therefore abolishes Liz transcription in preimplantation embryos when transmitted paternally.

We then assayed the effect of Liz deficiency on sDMR DNA methylation and Zdbf2 expression in E9.5 embryos, a stage following completion of de novo methylation of the embryonic genome. Maternally inherited Liz deletions were silent; maternal knockout embryos displayed a wild-type average of 50% of DNA methylation at the sDMRas expected from its imprinted status (Fig. 5b and Supplementary Fig. 7b). The Liz paternal-knockout embryos, conversely, exhibited much reduced DNA methylation, indicating an in vivo requirement of Liz for proper DNA methylation deposition; while the pyrosequencing assay gave an estimate of 15% of methylation, allelic analysis after cloning sequencing showed virtually no methylation at the sDMR (Supplementary Fig. 7c). Notably, prevention of Liz expression during preimplantation development resulted in complete elimination of Zdbf2 expression in E9.5 Liz paternal-knockout embryos (Fig. 5c and Supplementary Fig. 7d). The in cellula and in vivo data are therefore highly congruent for the Liz mutation, and the associated effects are even more pronounced within the in vivo context.

Liz paternal-knockout mice exhibit lifelong epigenetic consequences

Having established that Liz is required for sDMR DNA methylation in embryos, we next asked whether this phenotype was epigenetically maintained throughout adulthood. At 8 weeks, the sDMR remained constitutively hypomethylated in tissues representative of the three germ layers, in Liz paternal-knockout as well as in Liz homozygote mice (Fig. 5d). Analysis of crosses between a Mus musculus molossinus (JF1) dam and a Mus musculus musculus Liz +/-sire confirmed the lack of DNA methylation at paternally inherited sDMR alleles (Fig. 5e). The abnormal epigenetic setting was irreversibly maintained upon aging, as observed at 8 months (Supplementary Fig. 7e).

In wild-type adult tissues, RT-qPCR showed Zdbf2 expression primarily in brain derivatives and most markedly in mid-brain, hypothalamic and striatal regions (Supplementary Fig. 7f). Analysis of a mouse line with a Zdbf2 gene-trap allele showed strong LacZ staining in the hypothalamus and the pituitary gland (Fig. 5f). Modest Zdbf2 expression was observed in the labyrinth layer of the placenta (Supplementary Fig. 7f,g).

As was the case in E9.5 embryos, Zdbf2 expression was completely abolished in the hypothalamus and pituitary gland of both Liz paternal-knockout and Liz homozygote adult mice (Fig. 5g and Supplementary Fig. 7h). In Liz paternal-knockout mice, biallelic repression of Zdbf2 correlated with biallelic H3K27me3 enrichment upstream and at the Zdbf2 promoter, while this mark was only enriched on the maternal allele in wild-type mice (Fig. 5h,i). These results demonstrate that the transient expression of Liz in the early mouse embryo signals for permissive expression of the Zdbf2 gene by counteracting H3K27me3 deposition through DNA methylation; this epigenetic programming is then maintained throughout life.

Mice lacking Liz display a growth defect

Finally, to study the physiological impact of Liz-dependent events, we phenotyped cohorts of Liz knockout mice. Liz maternal-and paternal-knockout mice were born at expected Mendelian and sex ratios (Supplementary Fig. 8a,b), their fertility was normal and we did not observe obvious behavioral anomalies. However, Liz paternal-knockout mice exhibited a consistent growth deficiency phenotype. An average 7% reduction in body weight was measurable from the first day post-partum (d.p.p.), when normalized to the average weight of wild-type siblings from the same litter (Fig. 6a). The difference became more pronounced from the first to the fourth week of life, as a result of an attenuated rate of weight increase (Fig. 6a-c). At 2 weeks of postnatal age, male and female paternal-knockout mice were visibly smaller, with a 20% weight reduction (Fig. 6a,b,d,e) that affected all organs uniformly (Supplementary Fig. 8c). The unimodal distribution of the body weight data into 10% bin groups was consistent with high penetrance and low variance of the phenotype (Fig. 6f), but with a stronger effect in females (Fig. 6a-d). The undergrowth phenotype tended to minimize with age, but it was nevertheless persistent, as measured at 30 weeks (Fig. 6c). Comparatively, the growth defect was not seen upon maternal transmission of the Liz deletion in maternal-knockout mice (Supplementary Fig. 8d,e). Finally, prenatal growth was relatively normal in Liz paternal-knockout conceptuses, as assessed from embryo and placenta weights at E18.5 (Supplementary Fig. 8f,g). Collectively, our results provide evidence that the lack of paternal Liz expression in the early embryo results in diminished postnatal growth with a lifelong persistence.

DISCUSSION

In this study, we suggest that a dynamic competition between DNA methylation and H3K27me3 epigenetically programs the Zdbf2 locus during early development. Using ESCs, we demonstrated that Liz transcription releases PRC2-dependent repression of Zdbf2 by promoting the deposition of antagonistic DNA methylation marks. In vivo, Liz is a paternally expressed gene and its brief transcription in the pluripotent embryo is required for signaling information to later stages of development: the epigenetic signature conferred by Liz seems to prime Zdbf2 expression in postnatal tissues, ensuring full growth potential (Fig. 7). The synthesis of our findings leads to new insights into chromatin biology and early epigenetic programming of adult physiology.

PRC2 occupancy represents a default state at the Zdbf2 locus. In ESCs and in the inner cell mass of blastocysts 34,35 , a block of H3K27me3 occludes the Zdbf2 CGI promoter and Liz transcription is necessary to release Zdbf2 from PRC2-mediated repression at the exit from naive pluripotency. However, the act of transcriptional elongation itself does not seem sufficient but rather relies on the deposition of DNA methylation marks at the nearby sDMR: DNA-methylation-deficient DNMT triple-knockout cells are unable to displace H3K27me3 and derepress Zdbf2 , despite showing proper Liz expression levels and dynamics. How does Liz transcription promote de novo DNA methylation of the sDMR? Nascent Liz transcripts may act as a scaffold for DNMT recruitment. However, although de novo DNMTs can bind to RNAs in vitro 36 , there is limited in cellula evidence for such a mechanistic link. Alternatively, transcriptional elongation of Liz could confer favorable changes to the chromatin. PRC2-mediated repression is associated with chromatin compaction 37 , which may constitutively hinder the access of DNMTs to the sDMR. By remodeling the structure and composition of nucleosomes 38 , passage of the RNA polymerase II may promote de novo DNA methylation. Interestingly, the molecular impact of Liz deficiency is stronger in vivo than in cellula: DNA methylation of the sDMR is almost completely impaired in Liz paternal-knockout mice, whereas it is only partially impaired in Liz EpiLCs. Artifacts of the culture system, such as availability of the DNMT enzymes and/or cell division rate, may explain this discrepancy.

We showed that DNA methylation at the sDMR locally antagonizes PRC2 activity. This is not unusual, as DNA methylation and H3K27me3 are recognized as mutually exclusive marks, specifically at CGIs 9-12 . Underlying mechanisms of this antagonism are known: in their unmethylated form, CGIs are targeted by proteins with a CXXC domain, which protect them against de novo DNA methylation while promoting PRC2 recruitment 39-42 . However, the sDMR is not a CGI, but it displays genome-average CpG density and is located 10 kb upstream of the Zdbf2 CGI promoter. Moreover, the DNA methylation there has a positive, non-canonical role in stimulating Zdbf2 transcription. Although atypical, this situation may not be exceptional: DNA methylation promotes neurogenic gene transcription in neural stem cells, by antagonizing Polycomb-group proteins in non-promoter regions 43 . Future work should be dedicated to the identification of the mechanisms that mediate DNA methylation antagonism over H3K27me3 maintenance in non-CGI regions.

In vivo, Liz expression is subject to transient imprinting: as the maternal copy of the gDMR/Liz TSS is methylated, Liz is exclusively expressed from the paternal allele in the pluripotent embryo, before being turned off for the rest of somatic life at implantation 18 . We found that abolition of paternal Liz expression in embryos results in severe sDMR hypomethylation and Zdbf2 repression from the same paternal allele, which is maintained in all somatic cells of Liz paternalknockout mice. In agreement with Liz coordinating an H3K27me3 to DNA methylation switch, persistence of the H3K27me3 block was observed over the silent paternal Zdbf2 allele in Liz paternalknockout tissues. Genetically, our results indicate that the gDMR acts as a bona fide ICR, with a functional impact on regulation of the locus. It is worth emphasizing that the genetic information of the Zdbf2 gene is intact in Liz paternal-knockout mice-only the DNA methylation of the sDMR is altered. The Liz genetic deletion, therefore, produces a secondary 'epimutation' , with lifelong persistence.

Notably, we demonstrate that transiently imprinted genes can program adult phenotypes, despite being only briefly expressed at the beginning of development. Liz transcription does not have an immediate role in the early embryo but indelibly affects postnatal body weight: as they fail to derepress Zdbf2 , Liz paternal-knockout mice exhibit lifelong growth retardation. Liz/Zdbf2 therefore appears to be a new growth-controlling imprinted locus, and its parental direction of expression is concordant with the 'genetic conflict' hypothesis, according to which paternal expression tends to favor growth 44 . This raises the question of the function of the ZDBF2 protein, of which very little is known other than the presence of a DBF4 zinc-finger domain 18 . The major sites of Zdbf2 expression-the hypothalamus and the pituitary gland-control the regulation of several endocrine hormones, including the growth hormone. However, the hypothalamus coordinates other facets of physiology that are relevant to growth regulation, such as appetite and energy homeostasis 45 . Meanwhile, Zdbf2 is also expressed in the striatum, which modulates food consumption 46 . We are currently exploring the molecular and cellular functions of ZDBF2.

Although present in hundreds of locations in the mouse genome 20,47-49 , transient imprints remain poorly studied. In the case of the Zdbf2 locus, the synteny and regulation seem to be conserved from mouse to human 18 , which further underscores the evolutionary importance for an underappreciated component of mammalian biology. Moreover, given that Liz functions as an epigenetic programmer within the first days of development, genetic and environmental perturbations of Liz expression in gametes and early embryos might predispose to variable growth potential throughout life. Finally, our work provides one of the very few mechanistic and functional demonstrations of the potential for an early chromatin event-independently of its imprinted or nonimprinted status-to influence later adult phenotypes.

METHODS

Methods and any associated references are available in the online version of the paper.

Accession codes. ChIP-seq data have been deposited to the Gene Expression Omnibus (GEO) under accession GSE79549.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACKNOWLEDGMENTS

We would like to thank members of the Bourc'his laboratory for insightful experimental and conceptual input; E. Heard, R. Margueron, J. Barau

ONLINE METHODS

ESC lines. E14 was the primary ESC line used in this study and was the background for all in-house genome-edited lines. DNMT triple-knockout cells were a gift from M. Okano (RIKEN) 31 and the Eed -/-cell line was a gift from A. Wutz (ETH Zurich) 29 ; for both, the original J1 cell line was used as a wildtype control.

ESC cell culture and differentiation. Feeder-free ESCs were grown on gelatin-coated flasks. Serum culture conditions were as follows: Glasgow medium (Sigma) supplemented with 2 mM l-glutamine (Gibco), 0.1 mM MEM nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 15% FBS, 0.1 mM -mercaptoethanol and 1,000 U/ml leukemia inhibitory factor (LIF; Chemicon). Cells were passaged with trypsin replacement enzyme (Gibco) every 2 d. 2i culture conditions were as follows: N2B27 medium (50% Neurobasal medium (Gibco) and 50% DMEM/F12 (Gibco), supplemented with 2 mM l-glutamine (Gibco), 0.1 mM -mercaptoethanol, NDiff Neuro2 supplement (Millipore) and B27 serum-free supplement (Gibco)) with 1,000 U/ml LIF and 2i (3 M Gsk3 inhibitor CT-99021 and 1 M MEK inhibitor PD0325901). Cells were passaged every 2-4 d with Accutase (Gibco). Vitamin C (Sigma) was added at a final concentration of 100 g/ml. To induce EpiLC differentiation, cells were plated at a density of 1 × 10 4 cells/cm 2 on plates coated with fibronectin (10 g/ml) in 2i+vitC medium. After 1 d, cells were gently washed with 1× PBS and cultured in N2B27 medium supplemented with 12 ng/ml Fgf2 (R&D) and 20 ng/ml activin A (R&D). EpiLCs were passaged with Accutase at day 4 of differentiation. The VenorGeM Classic mycoplasma detection kit (Minerva Biolabs) was used to assess mycoplasma-free status.

Generation of edited ESCs. The Liz cell line was generated by deletion of a 1.7-kb region encompassing the gDMR using two TAL effector nuclease (TALEN)-generated double-strand breaks (DSBs). TALEN plasmids were cloned as previously described 50 . The TALENs were targeted to two sequences, one upstream (5 -TCATAAAATGTAACATCTGTgagaatgtgttataaACATGACA GTGCCCAAAACA-3 ) and one downstream (5 -TCTTTGTATATCCTGCTG ATgagctataaaccacctgGCTTGGGAACTAAAGAATTA-3 ) of the gDMR, where the lowercase sequences are the spacer regions between the TALEN-binding sites. All other deletions (Gpr1, Setd2 and Eed) were generated with CRISPR single guide RNAs (sgRNAs) specific to the target sequences followed by Cas9 nuclease activity. sgRNAs were designed using the online CRISPR Design Tool (http://crispr.mit.edu/) 51 from the laboratory of F. Zhang and cloned into the X330 plasmid harboring the Cas9 gene 52 . All sgRNA sequences are listed in Supplementary Table 1. Around 5 million E14 serum-grown ESCs were transfected with 1-3 g of plasmid(s) using the Amaxa 4D Nucleofector (Lonza) and plated at a low density. Ninety-six individual clones were picked and screened by PCR. Mutated alleles were confirmed by Sanger sequencing of cloned PCR amplicons. Homology-dependent insertion of the Liz::2×STOP construct was performed by cotransfection with the donor plasmid containing 700-bp homology arms and a TALEN plasmid generating a single DSB downstream of the first exon of Liz. The 2×STOP cassette used is a modified version of the construct used in Luikenhuis et al. 53 . Cells were plated at high density, and 125 ng/ml Geneticin (Life Technologies) was added after 24 h. Resistant clones were picked and genotyped by PCR and were confirmed by sequencing. Homozygous clones were then transiently transfected with a plasmid expressing Cre, cultured without antibiotics and rescreened by PCR for proper Cre-loxP recombination.

Mice. Mice were hosted on a 12-h light/12-h dark cycle with free access to food and water in the pathogen-free Animal Care Facility of the Institut Curie (agreement C 75-05-18). All experimentation was approved by the Institut Curie Animal Care and Use Committee and adhered to European and national regulation for the protection of vertebrate animals used for experimental and other scientific purposes (directives 86/609 and 2010/63). For tissue and embryo collection, mice were euthanized by cervical dislocation. The Liz-mutant mouse line was derived by CRISPR/Cas9 engineering of a 1.7-kb deletion spanning the gDMR (Supplementary Fig. 7a) in embryos at the one-cell stage, according to published protocols 54 . Briefly, the Cas9 DNA sequence was amplified by PCR from the pX330 plasmid and cloned into the T7-promoter-containing pCR2.1-XL plasmid. After plasmid linearization and phenol-chloroform purification, in vitro transcription was performed using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). The Cas9 mRNA was then purified with the RNeasy Mini kit (Qiagen). The two deletion-promoting sgRNAs (Supplementary Table 1) were amplified by PCR with primers containing a 5 T7 promoter sequence. After gel purification, the T7-sgRNA PCR products were used as the template for in vitro transcription with the MEGAshortscript T7 kit (Life Technologies) and the products were purified using the MEGAclear kit (Life Technologies). Cas9 mRNA and the sgRNAs were eluted in DEPC-treated RNase-free water, and their quality was assessed by electrophoresis on a denaturing agarose gel. Zygote injection with the CRISPR/Cas9 system was performed by the Transgenesis Platform of the Institut Curie. Eight-week-old superovulated B6D2F1 (C57BL/6J × DBA2) females were mated to stud males of the same background. Cytoplasmic injection with Cas9 mRNA and sgRNAs (at 100 ng/ l and 50 ng/ l, respectively) was performed in zygotes collected in M2 medium (Sigma) at E0.5, with wellrecognized pronuclei. Injected embryos were cultured in M16 medium (Sigma) at 37 °C under 5% CO 2 , until transfer at the one-cell stage the same day or at the two-cell stage the following day to the infudibulum of the oviduct of a pseudogestant CD1 female at E0.5. Twenty-five to 30 embryos were transferred per female. Of the 44 pups generated, 20 carried at least one modified allele. Two founders (N 0 ) carrying the expected 1.7-kb deletion were selected (female 393 and male 403) (Supplementary Fig. 7a). After the absence of in silico-predicted off-target mutations was verified by Sanger sequencing (Supplementary Table 2), these two founders were bred with wild-type C57Bl6/J mice. Two additional backcrosses were performed to segregate out undesired genetic events, following a systematic breeding scheme of crossing Liz heterozygous females with wild-type C557Bl6/J males to promote maternal (silent) transmission of the deletion. Cohorts of female and male N 3 mice were then mated with wild-type C57Bl6/J mice to study maternal-and paternalknockout progeny, respectively.

The Zdbf2 LacZ reporter line was derived from ESCs obtained from the European Conditional Mouse Mutagenesis Program (EUCOMM Project Zdbf2_82543). Proper insertion of the lacZ construct was determined by long-range PCR. Chimeric mice were generated through blastocyst injection by the Institut Curie Transgenesis Platform. DNA from the limb buds of Dnmt3a -/-and Dnmt3b -/-embryos at E11.5 was a gift from M. Weber (University of Strasbourg) 55 .

DNA methylation analyses. Genomic DNA was isolated from cells using the GenElute Mammalian Genomic DNA MiniPrep kit (Sigma), with RNase treatment. Genomic DNA from embryos and adult tissues was obtained following overnight lysis at 50 °C (100 mM Tris, pH 8.0, 5 mM EDTA, 200 mM NaCl, 0.2% SDS and proteinase K). DNA was recovered by standard phenol/ chloroform/isoamyl alcohol extraction and resuspended in water.

Global CpG methylation was assessed using LUMA (luminometric methylation assay) as described previously 56 . Genomic DNA (500 g) was digested with MspI-EcoRI and HpaII-EcoRI (New England BioLabs) in parallel reactions. HpaII is a methylation-sensitive restriction enzyme, and MspI is its methylationinsensitive isoschizomer. EcoRI was included for an internal reference. The extent of the enzymatic digestions was quantified by pyrosequencing (PyroMark Q24, Qiagen; dispensation order 5 -GTGTGTCACACAGTGTGT-3 ), and global CpG methylation levels were then calculated from the HpaII/MspI ratio.

Bisulfite conversion was performed on 500-1,000 ng of DNA using the EpiTect Bisulfite kit (Qiagen). Bisulfite-treated DNA was PCR amplified and either cloned and sequenced or analyzed by pyrosequencing. For the former, 20-30 clones were Sanger sequenced and analyzed with BiQ Analyzer software 57 . Pyrosequencing was performed on the PyroMark Q24 according to the manufacturer's instructions, and results were analyzed with the associated software. All bisulfite primers are listed in Supplementary Table 1. Statistical analyses were performed with two-tailed unpaired t tests using GraphPad Prism 6 software.

Chromatin immunoprecipitation. ChIP-qPCR experiments in cells were performed exactly as described in Walter et al. 24 . Chromatin from mouse hypothalamus was prepared as follows. The whole hypothalamus was finely chopped and cross-linked in PBS containing 1% formaldehyde for 15 min. After quenching, cross-linked tissue was homogenized using a Tissue Master 125 (OMNI). Tissue was centrifuged and then simultaneously dounced and lysed in swelling buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP-40 and proteinase inhibitors). Chromatin was pelleted and resuspended in TSE 150 (20 mM Tris, pH 8.0, 2 mM EDTA, 0.1% SDS, 1% Triton, 150 mM NaCl and proteinase inhibitors) and then sonicated. For ChIP-PCR pyrosequencing, input and ChIP DNA from hybrid mice was analyzed with the PyroMark Q24 instrument and its associated software. The following antibodies were used: H3K4me3 (Millipore, 07-473), H3K27me3 (Millipore, 07-449 and Cell Signaling Technology, C3B11) and H3K36me3 (Abcam, ab9050). All ChIP primers are listed in Supplementary Table 1.

ChIP-seq was performed using 10 ng of ChIP or input DNA. Remaining large DNA fragments were first eliminated using SPRIselect beads (Beckman Coulter), and libraries were prepared using the TruSeq ChIP Sample Prep kit (Illumina). 100-bp single-end reads were generated using the Illumina HiSeq 2500 platform. Reads were mapped to the mouse reference genome (mm10) using Bowtie2 v2.1.0, allowing one mismatch in a 22-bp seed. Tracks were created using HOMER software v4. 7 (ref. 58).

RNA expression analyses.

Total RNA was extracted using TRIzol (Life Technologies). To generate cDNA, TRIzol-extracted total RNA was treated with DNase (Ambion) and then reverse transcribed with Superscript III reverse transcriptase (Life Technologies) primed with random hexamers. RT-qPCR was performed using SYBR Green Mastermix on the Viia7 thermal cycling system (Applied Biosystems). Relative expression levels were normalized to the geometric mean of the C t values for the Rrm2 and Rplp0 housekeeping genes, unless otherwise indicated, using the C t method. Primers are listed in Supplementary Table 1. Statistical analyses were performed with two-tailed unpaired t tests using GraphPad Prism 6 software.

For NanoString RNA quantification, 100 ng of total RNA for each sample was analyzed using the NanoString nCounter Analysis System with a custom expression codeset. Actb, Ppia, Gapdh and Rplp0 were used for normalization. Probe sequences are available upon request.

Single-cell RT-qPCR was performed as follows. EpiLCs were dissociated well at day 4 of differentiation, and 200,000 cells were pipetted into the Integrated Fluidic Circuit (IFC) from Fluidigm. The IFC was loaded into the C1 AutoPrep Single-Cell System for sorting. The IFC was then imaged with a microscope to ensure proper sorting of single cells into each well. Reverse transcription, lysis and preamplification mixes were prepared according to the manufacturer's instructions and pipetted into the IFC. Collected cDNA samples were diluted ~10×, and 1 l of each was used for qPCR. qPCR was performed and results were analyzed as described above, but using three normalizers (Rplp0 , Rrm2 and Actb). Data from cells with expression values less than five times the maximum value for both Liz and Zdbf2 were discarded.

For expression analysis in single blastocysts, RNA was extracted from embryos that were collected 92 h after human chorionic gonadotrophin (hCG) injection in a minimal amount of PBS and snap frozen in liquid nitrogen. Samples were lysed in water containing RNasin (Promega) by two cycles of heating from 4 °C to 90 °C. Reverse transcription reactions using target-specific primers for normalizers and Liz were carried out for 2 h at 50 °C with Superscript III reverse transcriptase supplemented with RNasin. A 15-cycle preamplification step was included for Liz with nested primers. Free primers were removed with ExoSAP (Affymetrix), and qPCR was performed and results were analyzed as described above. Primers are listed in Supplementary Table 1.

RNA FISH. RNA FISH against Liz was performed with intronic Stellaris probes according to the manufacturer's instructions (Biosearch Technologies). Briefly, slide-grown cells at day 2 of EpiLC differentiation were fixed in 4% formaldehyde, permeabilized in 70% ethanol, washed (10% formamide in 2× SSC) and then incubated at 37 °C overnight in hybridization buffer (125 nM probe, 100 mg/ml dextran sulfate and 10% formamide in 2× SSC). The next day, slides were rinsed in wash buffer at 37 °C and then mounted using DAPI-containing mounting medium (Vectashield). Imaging was performed on a Zeiss LSM710 confocal microscope. Probe sequences are listed in Supplementary Table 1.

Immunoblotting. Immunoblots were visualized using the ImageQuant Las-4000 Mini-Biomolecular Imager (GE Healthcare). The following antibodies were used: lamin B1 (Abcam, ab16048), histone H3 (Abcam, ab1791), H3K36me3 (Abcam, ab9050) and H3K27me3 (Cell Signaling Technology, C3B11). Antibody against EED was a gift from R. Margueron (Institut Curie). Incubations were performed with antibodies at 1:5,000 dilutions.

LacZ staining. Whole brains and pituitary glands were fixed in 4% paraformaldehyde in PBS (pH 7.2) overnight and for 30 min, respectively, washed in PBS and in wash solution (2 mM MgCl 2 , 0.02% NP-40 and 0.01% C 24 H 39 NaO 4 in PBS) and finally incubated at room temperature overnight in X-gal solution (5 mM K 4 Fe(CN) 6 3H 2 0, 5 mM K 3 Fe(CN) 6 and 25 mg/ml X-gal in wash solution). After several PBS washes, samples were kept at 4 °C in PBS with a few drops of 4% paraformaldehyde. For placental sections, E18.5 placentae were fixed overnight, incubated in sucrose gradients and embedded in OCT before cryosectioning and LacZ staining.

Phenotypic analysis of weight. Postnatal weight measurements were performed every day from 1 d.p.p. to weaning (21 d). Mice were then separated according to genotype, housed in equal numbers per cage (n = 5 to 6) and weighed once a week. As body weight is a continuous variable, we used a formula derived from the formula for the t test to compute the minimum sample size for each genotype: n = 1 + C(s/d) 2 , where C is dependent on the values chosen for the significance level ( ) and power (1 -), s is the s.d. and d is the expected difference in means. Using = 5%, = 90% and an expected difference in means of 1 g at 2 weeks of age and 2 g later on, we obtained a minimum n of between 10 and 20 mice and thus decided to double this number, using between 20 and 40 mice per genotype, depending on the age and sex of the mice. No mice were excluded from the analysis. Mice were weighed with blinding to genotype until genotyping was performed at weaning (3 weeks). E18.5 embryos and placentae were collected and individually weighed on a 0.001-g scale. All data were generated using four separate mating pairs. Statistical analysis. The significance of the data obtained was determined by performing two-tailed unpaired t tests. P values were considered significant when 0.05: ****P 0.0001, ***P 0.001, **P 0.01, *P 0.05. Nature Genetics: doi:10.1038/ng.3718

Liz RT R CTCTTCAACAGATCCTGAATCCTCGG Hprt1 RT R CCCTGAAGTACTCATTATAGTCAAGGGCAT F GTGCTGGTTTCCCTATCACTCCCTG R GTTCCTCTCTGCACCGCTATCTGC F CGGTGAACATCCCATGCCTT R TGACGCCACAACTTGACTGT F GAGAAAAGCCTCCTGGTTCC R TGAGCAGAAAAGAGCAAGCA F CAGGCCCTGGAATGAGTAAA R AGCCACAAGCAGCCATAGT F TTCAAACACCACGACAGCTT R TCCTTTGAGACTTCCATGATG F CCGGAAGGAGAGCAGGAG R GAGCAGAAAAGAGCAAGCA F TCCAGAGGCACCATTGAAATT R TCGCTGGCTCCCACCTT F CCGAGCTGGAAAGTAAAGCG R ATGGGAAAGACAACGAAGCG F AAGTGACGTTGACATCCG R GATCCACATCTGCTGGAAGG F GCTTGCTGGTGAAAAGGACCTCTCGAAG R CCCTGAAGTACTCATTATAGTCAAGGGCAT F TTGGGGATCGGTGTTTAGAG R CGTCTTCGCTCTTCCGATAG F TGCTTGCTGGCTCAGTGTAA R ATCTTGCTTCAGCTGGGGAC F ACTGGTGGGGTTTTGGAGAA R GCCACCACAGGACAAACGTA F AGTGCACATGCTAGGACACA R ATGAGTGTGCCACTGAACGT F CTTTAAGAGCGGGAGGAAGG R AACACTGGGCCCCTTCTG F GGCTCTCCAGAGGATGGCTGAG R TCGGATGCCCCATCGCA F GCAGGCCTAGTAACCGAGACA R AGTTTTGGCGATGGGTGCT F ATGGGAGTTTGTTTTCCTGTGT R AGTGATGTTTGGCCTAATCCTG F GCTCACTCTCACTGCTGCTG R TCCCATCTCAGGGGTTACTG F AACTTTAGAGCCCCAGAAGG R CCATCCGACAATTCAAAATC F GGCCTTGTGCATGTGAATCA R AGGTTCTGGTAGTGGTGTCATTCAA F AGTCCGCCTAGAAGCACTTG R GTCCACCTTCCAGCAGATGT F AGAAGGGACAACATGCTGCC R TTGTGCTTGGTGGCGTGT F CCTTCAGAGTTAACATGGAGGACG R TCCAAACAGGCAGGGAGATT F [Biotin]-GGCACATTTGCATTTGCATCCT R ACCACTTCATGAGTGTGCCACTG Seq GTGTGCCACTGAACG F [Biotin]-GAACGCCATTCTCCTTGCC R AAAATGGCTCGCGAAGCAAC Seq GCTCGCGAAGCAACA
F AGGGTTATATTGAGAGAAATATTGTG R [Biotin]-ATATTAAATTAAACCCTAAATTCCATTTCT Seq ATTTAAATTATTGATGTTTAAG F AGATGGAAGGAAAGAATTGAATTTTATA R [Biotin]-TACCAACCCACTATATACATTCTTCATTA Seq TGAATTTTATATATTATTTTTTGAT Outer F TGAGTATATTGTGGTTTATGTTATG Outer R ACCTAACTCAATTCTAATAAAATAAC Inner F AAGATTAAATTAAATTTAAATTATTGATG Inner R CAAAAATAACCAATATTTCCAATAAAT Outer F ATTTTATGTATTTATTAGTGAGTTG Outer R CTAATCACAACTCCCAATCAC Inner F GAATTGAATTTTATATATTATTTTTTG Inner R CCTATATACAACTTACCTTACTT F TCAGGTTGTGAGCGAAGCTGACT R AGCCCTGTCTGGACAACAAAACAA F TTTTTCTTTTGCCTCCAAGAA R CCTGGACAGCATTTCTTTCTC R TGGCTTTAAAACAGGGGATG F CAGGCCAGCCTGGAATCCCC R TCAGGACCTTCAGAAGAGCAGTCA R (Insert) CCTCAACCGCGAGCTGTGGAAA F TTTCCACAGCTCGCGGTTGAGG R TGGGTTCCAGCCCTGTCTGGA F AGGGTGGTGGCTAGAAGACA R GCTAAACTAGCCCCACCAGA F AGAGTCGAGTCGAGGATGACA R AGCTGAGCCTTGGCTAACTG F GGGGAAATCATCAAGATCGAA

Supplementary Table 2. Sequences of potential exonic off-target cut sites for sgRNAs used to generate Liz KO mice.

The sites chosen have ≤4 mismatches outside of the five base-pair seed immediately adjacent to the protospacer adjacent motif (PAM). The sequences were identified using the online software available at crispr.mit.edu. PCR followed by sequencing confirmed that founder mice "393" and "403" used in this study did not harbor any mutations (not shown).

Nature Genetics: doi:10.1038/ng.3718

INTRODUCTION

Genomic imprinting is an epigenetic-based regulatory process by which one copy of a gene is silenced in a manner determined by its parental origin. Typically, the imprinted status of a gene is controlled by differential DNA methylation patterns between alleles of so-called "imprint control regions" (ICRs). The sperm and the oocyte not only differ morphologically, but they also exhibit highly dissimilar DNA methylation profiles. The sperm genome is compacted and highly methylated, particularly at repeated sequences, whereas the oocyte DNA displays a lower level of DNA methylation that is primarily localized in gene bodies and intragenic CpG islands (CGIs), as a consequence of transcriptional read-through (Kobayashi et al., 2012a;[START_REF] Smallwood | Dynamic CpG island methylation landscape in oocytes and preimplantation embryos[END_REF][START_REF] Smith | A unique regulatory phase of DNA methylation in the early mammalian embryo[END_REF]. These asymmetries of DNA methylation profiles are established during germline specification and are characteristic of the mature gametes.

However, the vast majority will be erased following fertilization during the genome-wide loss of DNA methylation concomitant with the acquisition of embryonic pluripotency [START_REF] Seah | From Germline to Soma: Epigenetic Dynamics in the Mouse Preimplantation Embryo[END_REF]. Only a few genomic sequences, including ICRs, are able to maintain gametic patterns of DNA methylation. ICRs are protected from the epigenetic reprogramming occurring after fertilization through the binding of the KRAB zinc finger protein ZFP57 on the methylated allele, which in turn recruits the KRABassociated protein 1 (KAP1) heterochromatin associated complex [START_REF] Li | A Maternal-Zygotic Effect Gene, Zfp57, Maintains Both Maternal and Paternal Imprints[END_REF][START_REF] Messerschmidt | Trim28 is required for epigenetic stability during mouse oocyte to embryo transition[END_REF][START_REF] Quenneville | In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions[END_REF][START_REF] Schultz | SETDB1: A novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zincfinger proteins[END_REF]. The ZFP57/KAP1 complex likely protects DNA methylation at both maternally and paternally inherited ICRs through the recruitment of the DNA methylation maintenance enzyme DNMT1 [START_REF] Howell | Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene[END_REF]. To date, roughly 20 ICRs have been identified in the mouse and human genomes, and they control the mono-allelic and parent-oforigin expression of approximately 140 imprinted genes.

At the time of its discovery, genomic imprinting was shown to be required for the proper development of mouse embryos. Embryo manipulation generating androgenetic (two paternal genomes) and gynogenetic (two maternal genomes) zygotes, which fail to develop further than embryonic day 9.5 (E9.5), reveal that the two parental genomes have reciprocal contributions to embryogenesis (Barton et al., 1984;McGrath and Solter, 1984a;[START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF]. However, while a lot of imprinted genes are required to regulate embryonic growth in placental mammals, not all are essential for embryogenesis. A number of imprinted genes have been shown to exert their function only after birth on a broad range of physiological processes [START_REF] Cleaton | Phenotypic Outcomes of Imprinted Gene Models in Mice: Elucidation of Pre-and Postnatal Functions of Imprinted Genes[END_REF][START_REF] Plasschaert | Genomic imprinting in development, growth, behavior and stem cells[END_REF]. Both loss of expression and overexpression of imprinted genes in mouse models have been shown to affect notably post-natal growth, feeding behavior, maternal care as well as neurogenesis [START_REF] Charalambous | DLK1/PREF1 regulates nutrient metabolism and protects from steatosis[END_REF][START_REF] Ferrón | Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis[END_REF][START_REF] Leighton | Deletion of the H19 gene region in mice disrupts the imprinting of its neighbouring genes[END_REF][START_REF] Li | Regulation of Maternal Behavior and Offspring Growth by Paternally Expressed Peg3[END_REF][START_REF] Plagge | The imprinted signaling protein XLαs is required for postnatal adaptation to feeding[END_REF]. In humans, imprinted gene misregulation is linked with several neuro-developmental metabolic syndromes (Peters, 2014). Imprinting thus reflects the profound importance of gene dosage in development and adult physiology.

Imprinted genes have two defining characteristics: they are expressed at one dose, in a mono-allelic manner, and they have an epigenetic memory from the gametes. However, the question of whether the dose or the parental origin matter most for function has been scarcely addressed in the field. Here we report the very sensitive dose regulation of post-natal growth through the imprinted Zdbf2 (DBF-type zinc fingercontaining protein 2) gene in a parent-of-origin-independent manner. Zdbf2 is a paternally expressed gene on mouse chromosome 1 and human chromosome 2 [START_REF] Kobayashi | Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2[END_REF]. The ICR at the Zdbf2 locus is a germline differentially methylated region (gDMR) of maternal origin [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF][START_REF] Kobayashi | Imprinted DNA methylation reprogramming during early mouse embryogenesis at the Gpr1-Zdbf2 locus is linked to long cis-intergenic transcription[END_REF]. However, this gDMR has the particularity of being only transient during mouse embryo development, disappearing by gain of DNA methylation at the time of embryonic implantation [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF]Proudhon et al., 2012). The paternal expression of Zdbf2 in adult tissues is controlled by a somatic DMR (sDMR), localized ~10kb upstream of its promoter and paternally methylated. Our previous work showed that the transient expression of the Long isoform of Zdbf2 (Liz), initiating from the paternal, unmethylated gDMR allele, is responsible for the programming of Zdbf2 expression in the adult brain (Greenberg et al., 2017). In the pre-implantation embryo, Liz acts as a cis-regulatory RNA that is required for a chromatin-based switch from polycomb to DNA methylation occupancy at the sDMR. This epigenetic setting established by Liz in the early embryo mediates the de-repression of the paternal Zdbf2 promoter, which is then primarily expressed in the hypothalamo-pituitary axis. We have reported that Liz-deficiency does not affect embryogenesis itself, but rather leads to a strong down-regulation of Zdbf2 in the hypothalamus and the pituitary gland, resulting in a post-natal growth reduction (Greenberg et al., 2017). However, how the dose of

RESULTS

Generation of a Zdbf2-KO genetic mutant.

We previously showed that the transcription of Liz in the early embryo is required to program adult growth later on. Liz-LOF mutant embryos give rise to adult mice that fail to activate Zdbf2 in the brain -despite an intact genetic sequence-and display a postnatal growth defect (Greenberg et al., 2017). To rule out the Liz RNA per se as a direct regulator of post-natal growth control, we generated a ZDBF2 loss-of function mutant by engineering a 700bp deletion of the entirety of exon 6 of Zdbf2 (Figure 1A), which is included in all detectable isoforms of the gene product. The Zdbf2-∆exon6 deletion leads to a frame-shift, generating a severely truncated protein. Heterozygote mutant mice were born at the expected sex and Mendelian ratio upon both maternal and paternal transmission of the deletion (Figure S1A andS1B). Because of the imprinted status of Zdbf2 in the brain, we would expect the maternally inherited deletion to be silent, whereas it should result in a loss-of-function protein when inherited from the father. The mutant generated upon paternal transmission of the Zdbf2-∆exon6 deletion will thus be termed as Zdbf2-KO. It should be noted that the transcription of Zdbf2 as well as the establishment of DNA methylation at the sDMR were not affected in this mutant (Figure S1C andS1D). We have therefore generated specific Zdbf2 mutant mice as a model to interrogate the biological function of the imprinted Zdbf2 gene in mammals.

The product of Zdbf2 controls post-natal growth and pre-weaning survival.

To investigate the effect of the deletion on growth physiology, cohorts of Zdbf2-KO mice were examined for general morphology and body growth. We identified a partial post-natal lethality phenotype in the Zdbf2-KO mice (Figure 1B). During pre-weaning period (from birth to 3 weeks of age), mutant males and females were more prone to die than their wildtype (WT) littermates. In particular, about 40% of Zdbf2-KO pups die within the first week of life. At 3 weeks, ~50% of the mutant pups had died (Figure 1B).

The reproducibility of this phenotype was confirmed with the analysis of the Liz-LOF pups, which displayed a similar decrease in the survivability rate (Figure S1E). In contrast, both in the case of the Liz-∆gDMR and the Zdbf2-∆exon6 mutation, the survivability rate was similar to WT when the deletions were maternally inherited (Figure S1F andS1G). (A) Graphical model of the deletion of Zdbf2 generated using two sgRNAs upstream and downstream of exon 6. This deletion generates a frame-shift leading to a loss-of-function protein. (B) Survivability curves from the first day of life up to 12 weeks of age comparing the rate of survival between WT and Zdbf2-KO males and females. The partial post-natal lethality phenotype appears to be specific to the pre-weaning period, from the first day to 3 weeks of age. (C) Normalized body growth of Zdbf2-KO mice to their WT littermates (100%) followed at different ages in males and females. The weight reduction is seen as early as 1 d.p.p. and amplifies within the first weeks of life. Data are shown as means ± s.e.m. from individuals from n= 27 litters. (D, E) Growth curves of female and male mice, comparing the body weights of the WT and Zdbf2-KO, through the first 3 weeks of life (D) and through 6 months (E). (F) Representative photography of a smaller Zdbf2-KO male as compared to a WT littermate at 2 weeks of age. (G) Half dot-half violin plots showing the weight distribution at 2 weeks of age in males (left) and females (right) between WT and Liz gain-of-function. **** p ≤ 0.0001, *** p ≤ 0.001. (H) Frequency of weight distribution for normalized weights of 2-week-old paternal-knockout mice relative to WT mice (100%) grouped into 10% bins. The weight reduction phenotype appears to be highly penetrant.

Among the surviving pups, we found that the Zdbf2-KO mice displayed virtually the exact same growth defect as the previous reported Liz-LOF phenotype (Figure 1C-H) (Greenberg et al., 2017). The growth reduction was not present pre-natally (Figure S1H) but started at the first day of post-natal life, for both males and females (Figure 1C), and persisted through adulthood (Figure 1D and1E). The growth reduction was the most drastic during puberty: at 2 weeks, Zdbf2-KO mice exhibit a fully penetrant 20% decrease in weight compared to their WT littermates (Figure 1C and1F-H). As predicted, we confirmed that when present exclusively on the maternal allele, Zdbf2-∆exon 6 had no discernable growth effect (Figure S1I andS1J).

The growth phenotype appeared to be systemic, as it affected both the length and the weight of the animal and all organs uniformly (Figure S2A-C). To establish a more detailed characterization of the body weight defect, adult WT and Zdbf2-KO males at 7 weeks were analyzed by dual-energy X-ray absorptiometry (DEXA) scan. This method allows for accurate in vivo measurement of the volume fraction of the three dominant contributors to total body composition in mammals: adipose, lean, and skeletal tissues [START_REF] Chen | Abdominal fat analyzed by DEXA scan reflects visceral body fat and improves the phenotype description and the assessment of metabolic risk in mice[END_REF][START_REF] Nagy | Precision and accuracy of dual-energy X-ray absorptiometry for determining in vivo body composition of mice[END_REF]. We found that the lean mass, but neither the fat nor the skeletal composition, was affected (Figure S2D-H).

These results suggest a developmental rather than a metabolic origin of the growth phenotype. Finally, we examined the body weight of aged mice at 2 years. We observed a reduced-but not significant-general body weight, confirming the pubertyspecific timing of this growth defect (Figure S2I). Collectively, the above analyses demonstrated that the post-natal growth defect observed upon the downregulation of Zdbf2 expression in the Liz-LOF mice (Greenberg et al., 2017) is due to the lack of the protein, confirming the role of ZDBF2 as a growth-controlling factor.

Zdbf2 is expressed at puberty in the hypothalamo-pituitary axis.

To gain insight into the function of Zdbf2 in the post-natal hypothalamus and pituitary gland, we examined its developmental expression dynamics and localization. Using RT-qPCR, we monitored Zdbf2 steady-state transcript levels from 4 to 70 day postpartum in the pituitary and hypothalamus (Figure 2A and2B). The expression of Zdbf2 during the first 10 weeks of age followed the same trend between males and females (Figure 2A and 2B), with slightly higher expression in the female pituitary gland.

136

In the pituitary gland and the hypothalamus, the expression of Zdbf2 stayed stable within the first 7 and 15 days of life. Interestingly, the transcript level reached a 5-fold increase around puberty time: at 2 weeks in the pituitary (Figure 2A) and 3 weeks in the hypothalamus (Figure 2B). In both tissues, the expression of Zdbf2 did not drop to pre-puberty levels at later time points.

We have previously generated a LacZ reporter Zdbf2 gene-trap mouse line, to monitor Zdbf2 expression in the pituitary and the hypothalamus (Greenberg et al., 2017). Of note, the insertion of the LacZ cassette downstream of Zdbf2 exon 6 likely did no fully abrogate ZDBF2 function, as we were able to detect proper exon 6-7 spliced products, albeit at reduced levels of only a 0.5 fold decrease compared to WT (Figure S3A and S3B). To elucidate if an incomplete reduction of Zdbf2 dose would impact post-natal body growth, we assessed the weight of males and females from the LacZ reporter line (Figure S3C). Interestingly, we found that mice harboring a knockin on the paternal allele displayed a significant weight reduction compared to their WT littermates, but less severe than Zdbf2-KO mice (Figure 1C). On average, we observed a 5 to 10% growth reduction from 7 to 21 days after birth. This suggests that the control of post-natal growth is highly sensitive to Zdbf2 dosage.

The mature mouse pituitary gland is divided into the adenohypophysis, which contains the anterior and the intermediate lobes, and the neurohypophysis consisting of the posterior lobe [START_REF] Mollard | A tridimensional view of pituitary development and function[END_REF]. Only the adenohypophysis is responsible for hormone production. The anterior lobe secretes hormones from five different specialized hormone-producing cells, whereas the intermediate lobe contains melanotrope cells [START_REF] Kelberman | Genetic Regulation of Pituitary Gland Development in Human and Mouse[END_REF]. Hormonal production from the anterior lobe is dependent on hypothalamic inputs. Analysis of the reporter line show X-gal staining in the anterior and intermediate lobes of the gland and almost undetectable signal in the posterior lobe, suggesting a role of Zdbf2 in the hormone secreting cell types of the pituitary (Figure 2C andS3D). In the brain, Zdbf2 expression was highly specific to the hypothalamic area (Figure 2D and2E). Several hypothalamic nuclei were positive for Zdbf2 expression, including the peri-and para-ventricular nuclei in the anterior area (Figure 2D) and the arcuate, the dorso-medial and the ventro-medial in the lateral hypothalamic area (Figure 2E). Notably, these hypothalamic nuclei from the lateral area are all involved in neuro-endocrine functions through their interaction with the pituitary gland, mediating hormone production [START_REF] Saper | The hypothalamus[END_REF]. To corroborate these results, we analyzed publically available clustering data from hypothalamic single-cell RNA-sequencing [START_REF] Chen | Single-Cell RNA-Seq Reveals Hypothalamic Cell Diversity[END_REF]. We identified Zdbf2 expression as being specific to neuronal cells and absent from non-neuronal cells (Figure S4A andS4B). In addition, the two clusters where Zdbf2 was the most highly expressed, Glu13 and Glu15, represent neuroendocrine cells from the arcuate and the periventricular hypothalamic neurons, respectively, confirming our own data (Figure S4A). Altogether, these results suggest that Zdbf2 may contribute to important neuroendocrine functions of the hypothalamo-pituitary axis during mouse puberty.

Regarding the specific expression pattern of Zdbf2 in the neuroendocrine cells of the hypothalamo-pituitary axis, we hypothesized that the origin of the growth reduction lay in a defect in the production of pituitary hormones, which might be due to impaired hypothalamic signal. Although we did not detect any embryonic phenotype (Figure S1F), the observed post-natal growth reduction could be due to an impaired developmental regulation of the pituitary gland in the embryo, as it is the case for the well described Ames dwarf mice [START_REF] Sornson | Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism[END_REF]. From E11.5 to E13.5, endocrine cells of the anterior lobe of the pituitary start differentiating [START_REF] Rizzoti | Genetic regulation of murine pituitary development[END_REF]. Thus, we first assessed the morphology and the cell lineage specification of the developing pituitary gland at both E13.5 and E15.5 but did not detect any morphological defect (Figure S5A). The major transcriptional regulator of cells from the anterior lobe Pit1, Lhx3, Pomc, and the common subunit α-GSU were normally expressed, suggesting a proper cell lineage differentiation in the Zdbf2-KO developing pituitary (Figure S5B) [START_REF] Kelberman | Genetic Regulation of Pituitary Gland Development in Human and Mouse[END_REF][START_REF] Raetzman | Lhx4 and Prop1 are required for cell survival and expansion of the pituitary primordia[END_REF][START_REF] Rizzoti | Genetic regulation of murine pituitary development[END_REF]. At E18.5, all the endocrine cell types were present and almost fully mature. We performed morphological and hormone quantification assessment at this stage and could not detect any abnormality in the Zdbf2-KO mutants (Figure S5C and S5D). To further investigate the possible role of Zdbf2 in regulating hypothalamic input, we analyzed the expression of three main markers of hypothalamic function [START_REF] Biran | Role of developmental factors in hypothalamic function[END_REF]. The RNA level of the arginine vasopressin (AVP) peptide, the oxytocin neuropeptide and the growth hormone-releasing hormone (Ghrh) appear unchanged in Zdbf2-KO hypothalamus compared to WT (Figure S5E). Finally, we investigated the expression of various pituitary hormones at the pituitary site (GH, ACTH, TSH and LH) both at the RNA and protein level but did not observe any obvious change (Figure S5F). Although we cannot exclude the occurrence of subtle changes, Zdbf2 deficiency did not seem to induce growth reduction through a developmental defect of the hypothalamo-pituitary axis, neither in the determination of neuroendocrine cells, 140 including growth-hormone producing cells.

Generation of a Liz-gain-of-function mouse line.

We reported the CRISPR/Cas9 mediated generation of Liz-LOF mutant mice carrying a deletion of the entire gDMR (Greenberg et al., 2017). From these founder mice, some of them carried a smaller deletion than expected, generated through serendipitous NHEJ repair. Two of these lines, the CRISPR-Liz_379 and 397 (Figure 3A andS6A) exhibited an unexpected pattern of DNA methylation at the locus: only upon maternal transmission, heterozygote embryos at E9.5 post-implantation showed a significant gain of sDMR methylation compared to their WT littermates (Figure S6B). We then assessed DNA methylation at the sDMR in adult mice, focusing on one of the two lines, the CRISPR-Liz_379 (Figure 3A). After birth, the sDMR remained constitutively hypermethylated in adult tissues representatives of the three germ layers (Figure 3B).

Increased sDMR DNA methylation levels was due to an ectopic gain of DNA methylation on the maternal allele, as demonstrated by allelic analysis after bisulfite cloning and sequencing from hybrid mice between C57Bl6/J and JF1 strains (Figure 3C and S6C). Interestingly, maternal DNA methylation gain was mosaic, occurring in roughly 70% of the cells. However, despite being providing gain-of-methylation on the maternal allele, the paternal transmission of the deletion behaved as a silent mutation, with no effect on the level of sDMR methylation, which stayed at about 50% as in the WT (Figure 3B).

We previously showed that in Dnmt3L -/+ embryos that lack maternal DNA methylation imprints, the biallelic expression of Liz results in both copies of the sDMR becoming methylated in post-implantation stage [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF]. We thus speculated that the small deletion in our two CRISPR-Liz_379 and 397 founder lines, when maternally transmitted, induced a Liz gain-of-function (Liz-GOF) mutation. Unlike Liz-LOF deletions, these two deletions only include the first 210bp of the gDMR.

Consequently, although the annotated transcription start site (TSS) of Liz is deleted, some promiscuous transcription is likely allowed from alternative unannotated transcription start sites (TSS). This transcript may initiate from the region directly downstream of the annotated TSS, which is marked by chromatin regulators characteristic of active promoters (Figure S6D) [START_REF] Rada-Iglesias | A unique chromatin signature uncovers early developmental enhancers in humans[END_REF]. Moreover, the gain of Liz expression appeared to be independent from the gDMR methylation as the maternal allele was fully methylated at the blastocyst stage (Figure S6E). At this 7.6% 87.2%

x x x x x x x x x x x x x x x x x x x x x x x x x x x x
93.8%

72.4%

Liz-GOF BxJ WT BxJ sDMR, 6 weeks Allelic ratio (%)

W T ( n = 2 ) L i z -G O F ( n = 4 ) W T ( n = 3 ) L i z -G O F ( n = 3 )
Zdbf2, 3 weeks pre-implantation stage, the sDMR is devoid of DNA methylation (Figure S6F).

Liz transcription in the early mouse embryo is able to program the expression of Zdbf2 in the adult brain (Greenberg et al., 2017). To decipher if a maternal ectopic transcript of Liz would be able to program Zdbf2 expression from the maternal allele, we assessed the level of Zdbf2 in the hypothalamus and the pituitary gland. Consistent with our previous finding, the Liz-GOF male and female mice displayed bi-allelic expression of Zdbf2 in the adult hypothalamus and pituitary (Figure 3D and3E).

Consistent with the incomplete gain-of-methylation in the those mutant mice, Zdbf2 transcripts originate at 60% from the paternal allele and 40% from the maternal allele (Figure 3E) and the global level of expression consequently showed a 1.6 fold increase (Figure 3D). These results are highly congruent with an incomplete gain of Liz expression from the maternal allele, leading to bi-allelic sDMR methylation and Zdbf2 expression, likely in a subset of cells. It is worthwhile to note that when paternally transmitted, this deletion has no significant impact on Zdbf2 expression albeit we detected a small decrease compare to the WT level (Figure 3D).

We previously reported that the Liz-dependent deposition of DNA methylation at the sDMR signals for Zdbf2 expression by counteracting the deposition of the H3K27me3 polycomb mark. In this direction, we observed a decrease in the level of H3K27me3 occupancy at the sDMR and Zdbf2 CGI in the Liz-GOF mutant, in association with increased Zdbf2 expression (Figure S6G). In sum, these results demonstrate that the transcription of Liz in the early mouse embryos is not only required but is necessary to program an epigenetic switch which senses Zdbf2 expression. The Liz-GOF mouse line consists of a loss-of-imprinting at the sDMR and thus leads to an abnormal increased dose of Zdbf2 expression.

Zdbf2 regulates post-natal growth in a dose-dependent manner.

Having established that the expression of Zdbf2 is increased in the hypothalamus and the pituitary gland of the Liz-GOF mice, we asked whether this impacted post-natal growth. The deletion had no effect on the sex and Mendelian distributions upon both parental transmission (Figure S7A et S7B) and the mice survived at the same rate as their WT littermates (Figure S7C). Moreover, we did not detect any fertility or behavioral phenotype. However, we did detect an abnormal post-natal growth phenotype: the Liz-GOF mutants showed consistent male-specific overgrowth (Figure 4A-C). As was the case for the growth retardation observed in the Liz-LOF mutant mice, increased body weight was more pronounced at puberty (Figure 4A) and affected all organs uniformly (Figure S7D). From 10 to 21 days of age, Liz-GOF males were 10% heavier when normalized to the average WT siblings from the same litter. At 2 weeks of post-natal age, males were visibly bigger than their WT littermates (Figure 4D-F) whereas the females displayed a standard normal distribution (Figure 4F). However, this overgrowth phenotype tended to disappear with age and was not detectable in aging mice after 2 years (Figure S7E). Comparatively, progenies from a paternal transmission of the deletion showed a growth progression similar to their WT littermates (Figure S7F and S7G). These data showed the importance of the dosage of Zdbf2 in the hypothalamus and the pituitary for the regulation of proper post-natal growth at puberty. The Zdbf2 imprinted gene thus appears to be a dose-dependent regulator of post-natal growth in mice.

Zdbf2 regulates post-natal growth in a parent-of-origin independent manner.

In WT context, Liz and Zdbf2 display paternal mono-allelic expression, in the early embryo and the adult brain, respectively. We generated two complementary mutant lines with a complete loss (0 dose) and a gain (increased dose) of Liz expression in the embryo, which are associated with reduction and increase of Zdbf2 in the brain, respectively (Greenberg et al., 2017 and Figure 3). Importantly, the dose of Zdbf2 in the hypothalamus and the pituitary correlates with body size at puberty.

To elucidate if the parental origin of Zdbf2 matters in the control of post-natal growth, we intercrossed the Liz-GOF and Liz-LOF lines to generate a mutant with an inversion of the imprinting at the locus. By crossing a Liz-GOF female with a Liz-LOF male (Figure 5A, left panel), we would expect to obtain four possible genotypes, as shown in Figure 5A. One quarter of these embryos would express Liz in a mono-allelic manner but with an inversion of the parental origin, from the maternal allele instead of the paternal one (Figure 5A, right panel). This mutant will receive a 0.6 dose of Liz from the GOF maternal allele but no dose from the LOF paternal allele. We thus named this line as "Liz-GOF/LOF". We assessed the level of sDMR methylation and Zdbf2 expression in adult tissues from the progenies of Liz-GOF females crossed with Liz-LOF males. The DNA methylation level at the sDMR was comparable to the WT level, with an average of 43.5% CpG methylation (Figure 5B and5C). The expression of Zdbf2 in the hypothalamus and pituitary gland of the Liz-GOF/LOF mutant reached an intermediate level between the WT and the Liz-LOF mutant (Figure 5D). On average, the dose of Zdbf2 in these compound mutants was about 0.6 fold compared to WT.

Because Liz is maternally expressed in the Liz-GOF/LOF embryos, the Zdbf2 transcript is in all likelihood exclusively coming from the maternal allele. Unfortunately the uniform background of the Liz LOF and GOF alleles did not allow doing hybrid crosses to experimentally prove it.

We finally asked how the body weight was affected in the mutant mice expressing a maternal Zdbf2 at a dose comparable to the WT level. In accordance with the expression data, the Liz-GOF/LOF mutant displayed an almost complete rescue of the growth phenotype (Figure 5E et 5F). At 2 weeks of age, these mutants exhibited only an average 5% decrease in weight compared to their WT littermates (Figure 5E). Altogether, these results crystallize the importance of the dosage of Zdbf2 in the post-natal brain to regulate proper body growth. They provide the crucial demonstration that the dose rather than the parental origin matters for Zdbf2 function.

Importantly, this is a rare demonstration of the inversion of parental-origin of an imprinted gene [START_REF] Leighton | Deletion of the H19 gene region in mice disrupts the imprinting of its neighbouring genes[END_REF].

DISCUSSION

Our study provides new insights into the biological function of ZDBF2 in mice. We reported growth retardation in mutants lacking the product of ZDBF2 and inversely, a body size increase upon overexpression (Figure 6). These are the two criteria essential to define Zdbf2 as a genuine growth-promoting gene [START_REF] Efstratiadis | Genetics of mouse growth[END_REF]. The molecular function of the ZDBF2 protein has not being characterized yet and the only annotated domain is a DBF4-like zinc finger, homolog to the DBF4 protein that is conserved from yeast to human and has a role in cell-cycle regulation [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF][START_REF] Hughes | Crystal structure of human CDC7 kinase in complex with its activator DBF4[END_REF]. The synteny of the locus supports that Zdbf2 in mice and humans arose from an ancestral duplication of the Dbf4 locus. However, there is no evidence that those two genes are related and their functions have more likely diverged.

Although our data support that Zdbf2 acts as a neuroendocrine-mediated regulator of growth through the hypothalamo-pituitary axis, the precise origin of the growth phenotypes is unclear. The expression of Zdbf2 in the anterior lobe of the pituitary gland at puberty suggests it could be involved in hormonal signaling pathways.

The anterior lobe contains five types of hormone-producing cells, including the somatotrophs that produce the Growth Hormone (GH). GH-deficient mice display postnatal dwarfism [START_REF] Snell | Dwarf, a New Mendelian Recessive Character of the House Mouse[END_REF][START_REF] Sornson | Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism[END_REF][START_REF] Wilson | Growth hormone and prolactin immunoreactivity in the pituitary gland of postnatal little (lit) mice[END_REF]. At birth, these mice have no size defect, grow normally for the first 12 days of life but start exhibiting a very clear general growth retardation from the 13 th day and then maintain consistent growth reduction during adult life [START_REF] Voss | Anterior pituitary development: Short tales from dwarf mice[END_REF]. The growth rate in Zdbf2-LOF mice is very similar, suggesting that these mutants might be GHdeficient. Sensitive assays are now needed to assess for potential subtle changes in hormonal production, such as cell-counting experiments and ELISA assays for hormonal blood secretion. Although both male and female Liz-GOF mutants show biallelic Zdbf2 expression in the hypothalamo-pituitary axis, only males exhibit an increased body weight, which affects general growth progression rather than adipose tissues specifically. This sex-specificity could reflect the higher basal growth rate of males compared to females, which implies that subtle increase of growth might be more visible in males than females. Alternatively, increased GH level could play a role, as the GH has been reported to have sex-specific effects. Indeed, the GH plasma concentration is naturally higher in males than in females [START_REF] Schindler | Growth Hormone Secretion and Control in the Mouse[END_REF] and GH interacts with male hormones [START_REF] Hull | Growth Hormone and Reproduction: A Review of Endocrine and Autocrine/Paracrine Interactions[END_REF][START_REF] Wanke | Overgrowth of skin in growth hormone transgenic mice depends on the presence of male gonads[END_REF].

While some aspects of the Zdbf2-associated growth phenotypes are consistent with altered GH signaling, some others may indicate that GH misregulation may not be the only driver. Longevity in mice is known to be enhanced upon decreased levels of GH [START_REF] Bartke | Somatotropic Signaling: Trade-Offs Between Growth, Reproductive Development, and Longevity[END_REF][START_REF] Brown-Borg | Growth hormone signaling is necessary for lifespan extension by dietary methionine[END_REF][START_REF] Sun | Longevity is impacted by growth hormone action during early postnatal period[END_REF]. However, we did not observe any change in lifespan in an ageing cohort of Zdbf2-LOF males (data not shown). Moreover, both the growth reduction and the overgrowth phenotypes were detected as early as 3 days of post-partum, while GH-specific phenotypes usually have a later onset [START_REF] Voss | Anterior pituitary development: Short tales from dwarf mice[END_REF]. Altogether these observations suggest a GH-independent component of the phenotype, which might be also responsible the partially penetrant lethality phenotype in Zdbf2-LOF mice. Alternatively, neo-natal growth retardation phenotypes are often associated with decreased survivability during the pre-weaning period [START_REF] Appelbe | Conditional deletions refine the embryonic requirement for Dlk1[END_REF][START_REF] Hsu | DAZAP1, an hnRNP protein, is required for normal growth and spermatogenesis in mice[END_REF][START_REF] Yamaguchi | Role of Tet1 in erasure of genomic imprinting[END_REF], suggesting that the Zdbf2-LOF early lethality could also be a secondary effect. Finally, our work provided one of the few demonstrations that the dose-but not the parental origin-of an imprinted gene is relevant for its function [START_REF] Leighton | Deletion of the H19 gene region in mice disrupts the imprinting of its neighbouring genes[END_REF]. In humans, the imprinted status of ZDBF2 is conserved and it is highly expressed in brain structures. Height determinism in humans is characterized by a large number of genetic and epigenetic components [START_REF] Benonisdottir | Epigenetic and genetic components of height regulation[END_REF][START_REF] Wood | Defining the role of common variation in the genomic and biological architecture of adult human height[END_REF]. Investigating genetic and epigenetic variations of ZDBF2 in human populations could impact the understanding of this complex trait. (chr1:63,246,196-63,246,948) 768 bp CRISPR-Liz_397

G A B A 1 G A B A 6 G A B A 5 G A B A 4 G A B A 3 G A B A 2 G A B A 1 8 G A B A 1 7 G A B A 1 6 G A B A 1 5 G A B A 1 4 G A B A 1 3 G A B A 1 2 G A B A G A B A 1 0 G A B A 8 G A B A 7 G A B A 9 G l u 1 G l u 3 G l u 2 G l u 1 5 G l u 1 4 G l u 1 3 G l u 1 2 G l u 1 1 G l u 1 0 H i s t a G l u 9 G l u 8 G l u 7 G l u 6 G l u 5 G l u 4

DNA methylation analyses

Genomic DNA from cells was isolated using the GenElute Mammalian Genomic DNA Bisulfite conversion was performed on 500-1000ng of DNA using the EpiTect® Bisulfite Kit (Qiagen). Bisulfite-treated DNA was PCR amplified and either cloned and sequenced, or analyzed by pyrosequencing. For the former, 20-30 clones were Sanger sequenced and analyzed with BiQ Analyzer software [START_REF] Bock | BiQ Analyzer: Visualization and quality control for DNA methylation data from bisulfite sequencing[END_REF].

Pyrosequencing was performed on the PyroMark Q24 (Qiagen) according to the manufacturer's instructions, and results were analyzed with the associated software.

Statistical analyses were performed by a two-tailed unpaired t-test using GraphPad Prism6 software.

Chromatin immunoprecipitation

Chromatin from mouse hypothalamus was prepared as followed: whole hypothalamus were finely chopped and crosslinked in PBS containing 1% formaldehyde for 15 minutes. After quenching, crosslinked tissue was homogenized using a Tissue Master 125 (OMNI). Tissue was centrifuged, and then simultaneously dounced and lysed in swelling buffer (5mM PIPES pH8, 85mM KCl, 0.5% NP-40, proteinase inhibitors).

Chromatin was pelleted and resuspended in TSE 150 (20mM TRIS pH 8, 2mM EDTA, 0.1% SDS, 1% Triton, 150mM NaCl, proteinase inhibitors) and sonicated.

RNA expression analyses

Phenotypic analyses of weight

Postnatal weight measurements were performed every day from 1dpp to weaning age (21 days). Then, mice were separated according to their genotype, hosted in equal number per cage (n=5 to 6) and weighted once per week. As body weight is a continuous variable, we used a formula derived from the formula for the t-test to compute the minimum sample size per genotype: n=1+C(s/d) 2 , where C is dependent on values chosen for significance level (α) and power (1-β), s is the standard deviation and d the expected difference in means. Using α =5%, β=90% and an expected difference in means of 1g at 2 weeks and 2g later on, we predicted a minimum n of between 10 and 20 and thus decided to increase this number using n size between 20 and 30 per genotype, depending of the age and the sex of the mice. No animals were excluded from the analysis. Animals were blindly weighed until genotyping at weaning age (three weeks). E18.5 embryos were collected and individually weighted on a 0.001g scale. All the data were generated using four separate mating pairs.

For aged mice, males and females were separated at weaning and then kept in cages of 3 to 6 mice for aging. These mice were thus never crossed and were not used for anything else than ageing and weight assessment.

DEXA scan analyses

Mutant and WT males at 2 weeks were sent from the animal facility of Institut Curie to the Mouse Clinic (Ilkirch, France) where they were hosted in a new animal house facility. The quarantine period lasted for 5 weeks before proceeding to the phenotypic analysis at 7 weeks of age. Mice were anesthetized prior analysis using an Ultrafocus DXA digital radiography system and finally scarified directly after the measurement without a waking up phase. The DEXA analysis allowed the assessment of the fat and lean mass, the bone area, the bone mineral content and the bone mineral density.

Phenotypic analyses of lethality

Material for genotyping was taken the first day of birth and then number of pups for a given litter was assessed every week. To avoid bias, we excluded from the analysis the litters where all the pups died due to neglecting mothers, not taking care of their pups.

Investigating the sDMR-dependent regulation of Zdbf2 expression

INTRODUCTION

Imprinting control regions (ICRs) are cis-regulatory sequences harboring parent-oforigin differential DNA methylation and which direct the mono-allelic expression of imprinted genes. To define a genomic region as an ICR, it needs to fulfill several criteria during development. ICRs have been shown to acquire their differential DNA methylation profiles during gametogenesis [START_REF] Christians | Embryonic development: maternal effect of hsf1 on reproductive success[END_REF][START_REF] Kono | Epigenetic modifications during oocyte growth correlates with extended parthenogenetic development in the mouse[END_REF][START_REF] Stöger | Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal[END_REF][START_REF] Surani | Imprinting and the initiation of gene silencing in the germ line[END_REF]. Subsequently to fertilization, ICRs are protected from the epigenetic reprogramming in the newly formed embryo. This is linked to the fact that ICRs harbor sequence motifs characteristic of the binding of specific proteins required to maintain differential DNA methylation patterns throughout development [START_REF] Messerschmidt | DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos[END_REF]. Notably, the binding of ZFP57, a zinc finger protein recruiting the KRAB-associated protein 1 (KAP1) complex confers the ability of ICRs to faithfully maintain DNA methylation on one parental allele during the genome-wide demethylation wave in the pre-implantation embryo [START_REF] Li | A Maternal-Zygotic Effect Gene, Zfp57, Maintains Both Maternal and Paternal Imprints[END_REF][START_REF] Messerschmidt | DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos[END_REF][START_REF] Quenneville | In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions[END_REF][START_REF] Schultz | SETDB1: A novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zincfinger proteins[END_REF][START_REF] Zuo | Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain[END_REF]. Moreover, what ultimately defines an ICR is the genetic demonstration that its deletion alters the expression of the surrounding imprinted gene(s). To date, about 20 ICRs have been described in the mouse and human genomes.

At the imprinted Liz/Zdbf2 locus, the ICR is a germline differentially methylated region (gDMR) of maternal origin, which controls the paternal expression of the long isoform of Zdbf2 (Liz) [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF]Kobayashi et al., 2009b). However, this DMR is only transiently imprinted and Liz is consequently expressed during a short period of time in the pre-implantation mouse embryo. Our previous results demonstrated the requirement of the Liz transcript to program Zdbf2 expression in the post-natal brain (Greenberg et al., 2017). Liz likely acts through its transcription to allow the deposition of DNA methylation at an intergenic somatic DMR (sDMR), initially occupied by a block of repressive H3K27me3 marks. Although being localized about 10kb away from the canonical promoter of Zdbf2, the epigenetic switch from polycomb to DNA methylation occupancy at the sDMR mediates the de-repression of Zdbf2 on the paternal allele in the post-implantation embryo (Greenberg et al., 2017). In somatic tissues, the sDMR is systematically paternally methylated and Zdbf2 is paternally expressed, mostly in brain tissues. It is worthwhile to note that the sDMR was originally defined as three discrete regions (sDMR 1, 2 and 3) [START_REF] Hiura | A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip[END_REF], but more recent whole genome bisulfite sequencing indicates a continuum of differential DNA methylation over the 8kb sDMR region. Importantly, we have demonstrated that the product of Zdbf2 in the hypothalamo-pituitary axis exerts a dose-dependent control of general post-natal growth in mice. While male and female mice lacking the expression of Zdbf2 in the hypothalamus and the pituitary gland are smaller, the biallelic expression of Zdbf2 in those two tissues results in a male specific overgrowth phenotype.

Between the two DMRs present at the locus, our data from the ∆gDMR in vivo deletion support that the gDMR acts as a bona fide ICR in the early pre-implantation embryo (Greenberg et al., 2017). The sDMR, on the other hand, does not fulfill any developmental criteria of an ICR but rather appears to be a major regulator of Zdbf2 in the post-implantation embryo. To get new insights into the sDMR-mediated regulation of the locus, I attempted to dissect the functional role of this regulatory region. To do so, I generated a ∆sDMR mouse line using CRISPR/Cas9 engineering and analyzed the effect on Zdbf2 expression upon maternal, paternal and homozygous transmission of the deletion. My data suggest that the sDMR negatively regulates Zdbf2 expression on the maternal allele in the hypothalamus but has no effect in the pituitary gland. This supports a two component regulatory mechanism at the locus, which involves a polycomb-dependent and a polycomb-independent repression of Zdbf2. Most importantly, the lack of a growth phenotype associated with the deletion of the sDMR allowed me to reveal the tissue specificity of the Zdbf2-mediated growth phenotype:

although Zdbf2 is highly expressed throughout the hypothalamo-pituitary axis, its physiological effect seems to depend upon its expression in the pituitary gland but not in the hypothalamus.

166

RESULTS

Hypothalamus-specific gain of Zdbf2 expression in ∆sDMR mutant mice.

To gain insight into the interplay between the sDMR and Zdbf2 expression, I generated a mutant mouse line carrying a 8.3kb deletion spanning the entire sDMR (Figure 1A).

The analysis of Zdbf2 expression in the hypothalamus and the pituitary gland in progenies from the three different types of transmission (i.e. maternal, paternal and homozygote) showed a tissue-specific effect of the deletion (Figure 1B). In the pituitary, the deletion of the sDMR did not impact Zdbf2 transcript levels, in all types of parental transmission. In the hypothalamus, we observed no effect when the deletion was paternally transmitted (sDMR-PKO). However, maternal knock-out (sDMR-MKO) and homozygous (sDMR-KO/KO) mice both exhibited increased Zdbf2 expression (Figure 1B), although it did not appear to be significant due to large variability among biological replicates (Figure 1B). We therefore compared the relative levels of hypothalamic Zdbf2 transcripts among an important number of mice and detected high interindividual variability in the sDMR-MKO and sDMR-KO/KO (Figure 1C). This variability was less important in the WT and sDMR-PKO hypothalamus, as well as in pituitary tissues (Figure 1C and1D). The molecular phenotype caused by the maternal deletion of the sDMR thus appears as partially penetrant. We then assessed the allelic contribution of Zdbf2 expression in both the hypothalamus and the pituitary gland of sDMR-MKO mice compared to WT, using pyrosequencing of material from hybrid crosses between C57Bl6 and JF1 strains. While Zdbf2 was exclusively paternally expressed in the pituitary gland of both mutant and WT mice, both paternal and maternal Zdbf2 expression was detected in the hypothalamus of sDMR-MKO mice only (Figure 1E). Importantly, analysis of both relative expression and allelic ratio demonstrated that sDMR-MKO individuals with a WT level of Zdbf2 showed only paternal expression, while increased Zdbf2 expression always correlated with biallelic expression (Figure 1F and1G).

Altogether, these data reveal that sDMR-MKO mice exhibit a partially penetrant tissue-specific Zdbf2-gain-of-function, in which the initially silenced maternal allele is reactivated only in the hypothalamus but not in the pituitary gland. Surprisingly, this supports the existence of differential Zdbf2 regulatory mechanisms between the hypothalamus and the pituitary gland. In the hypothalamus, the activation of Zdbf2 appears to be mediated through the sDMR, suggesting a polycomb-only-dependent repression. On the maternal allele, Zdbf2 is silenced by a block of polycomb whose binding is likely affected in the sDMR-MKO mice.

Conversely, our data points to a polycomb-independent regulation of Zdbf2 in the pituitary gland. To identify potential differential regulatory chromatin landscapes of the hypothalamus and the pituitary gland, I performed ATAC-sequencing on these tissues from of 3-weeks old WT mice (Figure S1). However, the ATAC-sequencing was not conclusive in identifying differential open chromatin states between these tissues and should be repeated (Figure S1).

Increased Zdbf2 expression in the hypothalamus does not impact post-natal growth.

I previously showed that the dose of Zdbf2 in the hypothalamo-pituitary axis is crucial for regulating post-natal growth regulation in mice (Glaser et al., in preparation). In Zdbf2-GOF mutants, the expression of Zdbf2 is biallelic, in both the hypothalamus and the pituitary gland; consequently, mice exhibit an overgrowth phenotype, specifically in males. In contrast, the sDMR-MKO mice display hypothalamus-specific biallelic Zdbf2 expression. To decipher if this tissue-specific double dose of Zdbf2 affects growth the same way as the complete gain-of-function does, I recorded body weights at 7, 14 and 21-post-partum days (Figure 2). Unexpectedly, I did not detect significant differences in weight from 1 to 3 weeks of age in both the sDMR-MKO (Figure 2A-C) and the sDMR-KO/KO mutants compared to sDMR-PKO and WT littermates (Figure 2D-I). Particularly, I did not observe a stronger variability in body weight in sDMR-MKO and sDMR-KO/KO that could reflect an inter-individual heterogeneity in Zdbf2 expression (Figure 2A-F). This showed that the gain of Zdbf2 expression in the hypothalamus has no impact on growth. Overall, when comparing the phenotypic outcome of the complete Zdbf2-GOF and the sDMR-MKO, it appears that only the enhanced expression of pituitary Zdbf2 causes overgrowth in males. These data may suggest that the role of Zdbf2 in growth regulation is restricted to the pituitary gland.

However, to attribute with certainty the post-natal growth phenotype to the expression of Zdbf2 in the pituitary gland, a pituitary-specific loss-of-function mutation is needed. Pituitary-specific Zdbf2 loss-of-function is sufficient to induce growth deficiency.

To decipher whether pituitary-specific Zdbf2 downregulation could induce growth reduction in mice in a context of normal hypothalamic expression, I used the previously generated Liz-LOF mice (Greenberg et al., 2017). In these mutants, both the hypothalamus and the pituitary tissues completely lack Zdbf2 expression. By crossing a female sDMR-KO with a male Liz-LOF, I generated sDMR-MKO/Liz-LOF double mutants (Figure 3A). Those mutants are of particular interest: we expect them to display one dose of (maternal) Zdbf2 in the hypothalamus and 0 dose in the pituitary gland (Figure 3A). Importantly, our previous results demonstrated that the effect of Zdbf2 on growth regulation is independent of its parental origin (Glaser et al., in preparation). In the sDMR-MKO/Liz-LOF double mutants, Zdbf2 contribution in the hypothalamus must be similar to what is normally found in WT hypothalamus and these mice should therefore exhibit a pituitary-specific Zdbf2 loss-of-function.

Preliminary measurement of body weight at 2 and 3 weeks of age showed that the sDMR-MKO/Liz-LOF double mutants have the exact same growth deficiency phenotype as their Liz-LOF littermates (Figure 3B and 3C). Accordingly to our previous analysis, the average body weight of sDMR-MKO mice was similar to the WT.

Importantly, the inter-individual variability was the same for all four different genotypes (Figure 3C). The precise cause of the growth phenotypes at the Zdbf2 locus is unknown. These data strongly support that the post-natal growth reduction culminating at puberty is exclusively due to the lack of Zdbf2 expression in the pituitary gland, while the expression of Zdbf2 in the hypothalamus has no apparent impact on growth regulation.

sDMR- expression induces growth retardation. Our investigation of the origin of the growth phenotype observed upon Zdbf2 dose changes should thus be focused on pituitaryrelated signaling. Although our preliminary results were not conclusive, the growth hormone pathway still appears as the best candidate and certainly requires further analyses. However, our results consistently show that the level of Zdbf2 in the hypothalamus is higher than in the pituitary (Figure 1A; Greenberg et al., 2017;Glaser et al., in preparation). This raises the question of the role of Zdbf2 in the hypothalamus. [START_REF] Boulant | Role of the Preoptic-Anterior Hypothalamus in Thermoregulation and Fever[END_REF][START_REF] Cannin | Brown Adipose Tissue: Function and Physiological Significance[END_REF].

Immediate future work will include assessment of the survivability in newborn double-sDMR-MKO/Liz-LOF mutants to decipher if the lethality phenotype is dependent on Zdbf2 hypothalamic input.

Overall, we propose that the mono-allelic expression of Zdbf2 is required in the pituitary gland to control proper post-natal growth whereas, in the hypothalamus, the level of Zdbf2 has no impact on growth. Accordingly, the regulation of Zdbf2 in those two tissues seems to involve different mechanisms.

sDMR-Figure S1 CpG Islands DMRs chr1: 63,197,198-63,310,005 (112,808 bp) 50 kb

Gpr1

Liz Zdbf2 For each tissues, we performed two replicates (Rep1 & 2). The regions deleted in the sDMR-KO mice is highlighted in light grey.

1. General conclusion and personal feelings.

Liz transient expression.

The first part of my PhD was centered around the question of the role of transient imprinting. The transiently imprinted status of the Liz/Zdbf2 locus was uncovered by two former PhD students in our laboratory [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF]Proudhon et al., 2012).

I teamed up with a post-doc fellow, Max Greenberg, to study the embryonic regulation of Zdbf2 through its cis-acting long isoform, Liz, using two complementary models of embryonic stem cells (ESCs) and in vivo genetic mouse mutants. We formally demonstrated that the transient transcription of Liz during early mouse development programs Zdbf2 expression and this in turns control post-natal growth. This was the first experimental proof of an early transient epigenetic event programming adult physiology.

Epigenetic switch

Although very brief, the paternal transcription of Liz in the early embryo operates in cis an epigenetic switch at the sDMR located on its transcription path, and this is essential for releasing Zdbf2 transcription from the same allele. Upon Liz transcription and concomitantly to the wave of de novo DNA methylation that the embryonic genome undergoes upon differentiation, the paternal sDMR transitions from H3K27me3 to DNA methylation occupancy.

Two aspects of the polycomb to DNA methylation switch at the sDMR are still unresolved. First, we could not identify how Liz brings DNA methylation at the sDMR.

Transcriptional elongation is associated with the deposition of the H3K36me3 marks, which then recruit the de novo DNMT3A and 3B through their PWWP domain [START_REF] Baubec | Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation[END_REF][START_REF] Dhayalan | The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation[END_REF]. However, our attempt to show a link between H3K36me3 deposition and gain of DNA methylation at the sDMR using mutant ES cells for the sole mammalian H3K36 trimethylase SETD2 indicated that sDMR de novo DNA methylation was, surprisingly, H3K36me3-independent (Greenberg et al., 2017).

Nevertheless, by interrupting the transcription of Liz without disturbing its TSS in ES cells (using an Liz::2×STOP cassette), Max demonstrated that the sDMR acquires methylation in a Liz transcription-dependent manner. Second, we initially did not understand how the sDMR methylation is associated with activation of Zdbf2 transcription. Indeed, although the sDMR is located about 10kb upstream from the canonical promoter of Zdbf2, the deposition of DNA methylation at this region seems to mediate an activating signal. DNA methylation and H3K27me3 are two repressive marks that have been shown to be antagonistic (Brinkman et al., 2012;[START_REF] Statham | Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIPseq) directly informs methylation status of histone-modified DNA[END_REF]. The sDMR at the Liz/Zdbf2 locus is the first example of a region switching from one repressive mark to another but with a de-repressing effect on transcription. The underlying mechanism may be related to the role of DNA methylation in the release of PCR2 binding. Future work screening for similar regions should shed the light onto the mechanisms of polycomb to DNA methylation switch.

Liz in the male germline

The gDMR/ICR of the Zdbf2 locus coincides with the Liz TSS and is regulated by maternal imprinting. Therefore, male germ cells have an unmethylated gDMR/Liz TSS and former work from our lab demonstrated that Liz is expressed during spermatogenesis [START_REF] Duffie | The Gpr1/ Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF]. Liz being transcribed in the male germline, the ∆gDMR in vivo deletion may impact sDMR methylation as well as Zdbf2 activation in this context -as it does in the early embryo-and this might have reproductive effects.

However, I found that both heterozygous and homozygous Liz-KO male mice were fully fertile. To gain insight into the regulation of Liz in the male germline, I assessed the sDMR methylation in sperm and Zdbf2 expression in adult testis. Surprisingly, both features were unaltered in the ∆gDMR mouse mutants (Figure 26). First, CpG methylation levels, assessed by bisulfite cloning sequencing from sperm DNA at the sDMR, were globally unchanged between WT, heterozygotes and homozygotes, despite a small decrease in the latter two (Figure 26B). Second, using RT-qPCR, I could not detect any differences in Zdbf2 expression levels between mutant and WT testes (Figure 26C). These results indicate that Liz transcription may still occur in the germline of males carrying a ∆gDMR deletion, suggesting promiscuous transcription from alternative promoters. Altogether, it seems that the Liz transcription may not be regulated in the same way in the embryo and the male germline. The characterization of Liz transcripts in the male germline and the generation of a conditional mutant provide a new area of research at this locus, which, unfortunately, I could not carry on due to lack of time and necessity to prioritize my efforts.

Liz/Zdbf2: an isolated case or the tip of the iceberg?

Our work provided the first demonstration that a transiently imprinted locus has functional relevance in programming a life-long phenotype. The Liz/Zdbf2 locus isstill-the only transiently imprinted region extensively characterized in the mouse genome. However, in silico analysis reveals that transient imprints are present at hundred locations in the mouse genome (Proudhon et al., 2012;[START_REF] Smallwood | Dynamic CpG island methylation landscape in oocytes and preimplantation embryos[END_REF][START_REF] Smith | A unique regulatory phase of DNA methylation in the early mammalian embryo[END_REF]. The case of the Liz/Zdbf2 locus might thus not be isolated.

Regarding the conservation of this locus in humans and the major physiological role we reported in mice, the identification and characterization of other transient imprinted regions appears as a primary concern in the field of mammalian biology. Alternatively, the functional relevance of the Liz/Zdbf2 locus could be an isolated case, which has been maintained throughout evolution for its important physiological role on post-natal growth regulation and survivability. Recent work reported the transient binding of DNMT3A to specific enhancers during early neurogenesis, which mediates a longlasting epigenetic neuronal effect on brain functions [START_REF] States | Early-Life Gene Expression in Neurons Modulates Article Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States[END_REF]. This further adds to our findings of the long-lasting epigenetic effects of early transient events.

Finally, what characterizes transiently imprinted regions is their inability to maintain parental DNA methylation differences after implantation of the embryo. 3. Liz, Zdbf2: when dose matters.

Generation of Liz-GOF mutant mice

When I started my PhD, the CRISPR/Cas9 system had been recently described as a tool to efficiently and easily engineer genomic deletions in vivo [START_REF] Wang | One-step generation of mice carrying mutations in multiple genes by CRISPR/cas-mediated genome engineering[END_REF]. To gain insight into the function of the Liz transcript, we decided to take advantage of this emerging tool and generated a 1.7 kb deletion spanning the entire gDMR (Greenberg et al., 2017). At that time, we had no idea that these mutants would reveal so much Overall, the Liz-GOF mutants, although unexpected, happened to be extremely useful to dissect the growth-control function of Zdbf2 in mice. However, as explained above, the mechanism underlying the gain-of-function is difficult to ascertain.

Therefore, I started entertaining the idea that Liz-GOF mutants could be generated in a "cleaner" way, by abrogating the transcription of Gpr1 in the oocyte [START_REF] Veselovska | Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape[END_REF]. Number of high-throughput sequencing studies have shown a role for transcription in the acquisition of DNA methylation at gDMRs/ICRs in the oocyte [START_REF] Borgel | Targets and dynamics of promoter DNA methylation during early mouse development[END_REF]Kobayashi et al., 2012a;[START_REF] Smallwood | Dynamic CpG island methylation landscape in oocytes and preimplantation embryos[END_REF][START_REF] Smith | A unique regulatory phase of DNA methylation in the early mammalian embryo[END_REF][START_REF] Wang | Programming and inheritance of parental DNA methylomes in mammals[END_REF]. Notably, abolition of oocyte transcription throughout the Zac1 locus has been shown to prevent DNA methylation acquisition at the intragenic Zac1

ICR [START_REF] Veselovska | Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape[END_REF]. At the Zdbf2 locus, the gDMR -that contains the Liz TSS-is located in the first intron of the Gpr1 gene, which is located about 50kb upstream of Zdbf2 and transcribed in the opposite direction. Gpr1 being highly genes with growth-related functions, the parental orientation of Zdbf2 expression supports the theory (although having post-natal rather than pre-natal growth-promoting effects), suggesting that genomic imprinting at the Zdbf2 locus might have evolved in the realm of the parental "thug-of-war".

Phenotypic outcomes

My work provided new insights into the physiological function of ZDBF2 in the brain.

By modulating the doses of Zdbf2 expression in the hypothalamus and the pituitary gland, in combination or individually, I was able to clearly define that the product of Zdbf2 acts on post-natal, but not pre-natal, growth in mice. Moreover, I observed that a reduction of Zdbf2 in the hypothalamo-pituitary axis is associated with both growth defect and a partial post-natal lethality phenotype: about 40% of the newborn mice do not survive the first week of life.

Growth phenotype

Using both Liz-LOF and Zdbf2-LOF mutant mice, I observed that Zdbf2 downregulation in the hypothalamus and the pituitary gland systematically results in growth reduction.

Inversely, increased dose of Zdbf2 in the Liz-GOF mice leads to general overgrowth.

Regarding the timing and localization of Zdbf2 expression in the anterior pituitary gland, it is tempting to propose that the product of Zdbf2 is involved in growth hormonemediated regulation of growth. Particularly, both growth reduction and overgrowth phenotypes start only after birth, affect all organs similarly and culminate at 2 weeks of age; these phenotypic characteristics are highly reminiscent of GH-deficient genetic mutants [START_REF] Snell | Dwarf, a New Mendelian Recessive Character of the House Mouse[END_REF][START_REF] Sornson | Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism[END_REF][START_REF] Wilson | Growth hormone and prolactin immunoreactivity in the pituitary gland of postnatal little (lit) mice[END_REF]. The fact that Zdbf2 is expressed in hypothalamic nuclei that are involved in stimulating pituitary hormone-producing cells also suggests a function of Zdbf2 in the hypothalamopituitary-GH signaling. However, immuno-histochemistry experiments from pituitaries at both E18.5 embryonic and post-natal P15 stages did not reveal any apparent defect in GH-producing cells. To further investigate a possible GH-linked phenotype in Zdbf2-LOF mutant, future analyses will include ELISA tests to assess the level of plasma circulating GH and IGF1, the downstream liver target of GH.

Zdbf2 expression is particularly high in the hypothalamus, suggesting that alternatively, Zdbf2 could be involved in hypothalamus-specific functions. Notably, the hypothalamus is involved in metabolic functions and behavior. In an attempt to identify candidate genes that up-or down-regulated upon Zdbf2 loss of function, I performed RNA-seq on 6 week-old hypothalamus from Liz-LOF males. Most of the differentially expressed genes from this sequencing appeared to be false positives: notably, misregulation of potentially interested genes involved in food behavior were not confirmed by RT-qPCR. Importantly, the hypothalamus has been shown as a "hot spot"

for imprinted gene function and a number of them are organized around imprinted gene networks [START_REF] Ivanova | Imprinted genes and hypothalamic function[END_REF][START_REF] Patten | Regulatory links between imprinted genes: evolutionary predictions and consequences[END_REF]. We thus investigated whether the downregulation of Zdbf2 in the Liz-LOF mutants had any effect on other imprinted genes expressed in this tissue (Figure 28). Our analysis did not reveal any significant change of imprinted gene expression, supporting that Zdbf2 is not involved in an imprinting network. Altogether, our results from the Liz and Zdbf2 LOF and GOF mutants did not allow us to understand the hypothalamic origin of the observed growth phenotypes.

In the literature, most of the reported overgrowth phenotypes are either due to the loss of function of a whole body growth-inhibiting factor or to adipocyte tissue misregulation, leading to obesity [START_REF] Bischof | Inactivation of the mouse Magel2 gene results in growth abnormalities similar to Prader-Willi syndrome[END_REF][START_REF] Curley | Increased body fat in mice with a targeted mutation of the paternally expressed imprinted gene Peg3[END_REF][START_REF] Dalgaard | Trim28 Haploinsufficiency Triggers Bi-stable Epigenetic Obesity[END_REF]. Alternatively, overexpression of GH itself or other genes of the GH pathway can also promote overgrowth, raising up to twice the weight of WT littermates [START_REF] Palmiter | Dramatic Growth of Mice that Develop from Eggs Microinhected with Metallothionein-growth Hormone Fusion Genes[END_REF][START_REF] Palmiter | Metallothionein-Human GH Fusion Genes Stimulates Growth of Mice[END_REF]. Strikingly, overgrowth phenotypes related to elevated levels of circulating GH have a typical onset after around two weeks of age, while Liz-GOF mutants have an increased body weight as early as 2 days after birth. Moreover, in Liz-GOF mutants, while biallelic expression of Zdbf2 is observed in the hypothalamopituitary axis of both sexes, the overgrowth phenotype is male-specific. Unfortunately, we do not have yet a clear explanation for this sex-specific phenotype. One hypothesis is that the male-specific overgrowth phenotype could be due to a slight increase in GH circulating levels. This would not be elevated enough to affect females but would enhance growth only in males, either by interfering with male hormones or through the effect of GH on muscle and skeletal development. Alternatively, the male-specific phenotype could be linked to Zdbf2 expression in the testis, suggesting that higher testis Zdbf2 levels could be involved in growth-related pathways.

Overall, the strongest phenotype we reported is the post-natal lethality observed upon loss of Zdbf2 expression, in both Liz-LOF and Zdbf2-KO animals. This phenotype is not fully penetrant but still consequent: 40% of the mutants do not survive the preweaning period and particularly die during the first week of post-natal life. Zdbf2 is far from being the first gene associated with early post-natal lethality, in combination with a growth defect [START_REF] Appelbe | Conditional deletions refine the embryonic requirement for Dlk1[END_REF][START_REF] Hsu | DAZAP1, an hnRNP protein, is required for normal growth and spermatogenesis in mice[END_REF][START_REF] Yamaguchi | Role of Tet1 in erasure of genomic imprinting[END_REF]. One hypothesis to explain this phenotype is simply that mutants being smaller, they are disadvantaged in the competition with their WT littermates for food access. The period directly following birth is particularly critical for survival since the pups need to adapt to a new environment. Alternatively, this phenotype could result from a functional effect of Zdbf2 in the hypothalamus and/or in the pituitary gland. To decipher between these two hypotheses, I will measure the survivability of littermates with identical Zdbf2 genotypes, either by crossing male Zdbf2-KO homozygotes with WT females or by removing WT pups from a paternal cross. This experiment should help us to understand if competition with WT littermates is involved in the lethality phenotype. Interestingly, the sDMR-KO mutants gave us unexpected information about Zdbf2dependent growth control. What comes out from all our data is a two-component system: on one hand, we reported a dual regulation of Zdbf2 in the pituitary and the hypothalamus and, on the other hand, the Zdbf2-associated growth phenotype seems to have tissue-specific origins. This may be responsible for the two phases of the phenotype in Zdbf2 deficient mice (both Liz-LOF and Zdbf2-KO) : (1) an early and minor growth reduction is triggered from the first day of life, in association with reduced survivability and (2) a "GH-like" growth reduction phenotype is then observed, culminating from 10 to 21 days of life and maintained in the adult. From this, several conclusions and hypotheses can be drawn about how tissue-specific Zdbf2 expression can result in this two-component phenotype.

Tissue-specific regulation: a two-component system

First, one dose of Zdbf2, either of paternal or maternal origin, in both the pituitary gland and the hypothalamus leads to normal post-natal growth (Figure 29A andE).

Second, deficient Zdbf2 expression in the pituitary gland is systematically associated with a growth reduction (Figure 29B, C and G). Importantly, the growth phenotype of the sDMR-MKO/Liz-LOF mutants-where Zdbf2 is not expressed in the pituitary but still expressed in the hypothalamus (Figure 29G) has only been assessed at 2 and 3 weeks of age so far. For now, we can thus only conclude that the expression of Zdbf2 in the pituitary acts on the second phenotypic component. To decipher if the pituitary-specific decrease of Zdbf2 is also affecting the first phenotypic component, I will investigate whether an early-life lethality phenotype exists in a cohort of sDMR-MKO/Liz-LOF mutants during the pre-weaning period. If these mutants do not present any lethality phenotype, it will be tempting to propose that Zdbf2 expression in the hypothalamus is vital immediately after birth.

Third, like the growth reduction phenotype, the overgrowth phenotype is also found when Zdbf2 is upregulated in the pituitary gland (sDMR-MKO animals upregulate Zdbf2 only in the hypothalamus and have a normal size) (Figure 29D and F). This observation may suggest that that the expression of Zdbf2 in the hypothalamus has no effect on the "GH-like", second growth phenotype. Even if we did not perform detailed morphological assessment, the sDMR-MKO mice appear phenotypically normal, raising the question of why Zdbf2 needs to be mono-allelically expressed in the hypothalamus. Indeed, it might be needed to have Zdfbf2 expressed in the hypothalamus but an increased dose does not seem to be physiologically impactful (Figure 29F). Altogether, the consequent number of mouse mutant models I generated were instrumental to reveal the dose-sensitive, parent-of-origin-independent and tissue-specific effect of Zdbf2 on post-natal growth in mice.

The molecular function of ZDBF2 in mice and humans

Although my work provided major insights into the biological role of ZDBF2, I was not able to reveal its precise molecular function. The only annotated domain of the protein is a DBF4-like zinc finger domain. The DBF4 protein, which is most likely the ancestral form ZDBF2, is an activator of the CDC7 serine/threonine kinase, which has a conserved role in cell cycle and DNA replication initiation from yeast to humans [START_REF] Hughes | Crystal structure of human CDC7 kinase in complex with its activator DBF4[END_REF]. Even though ZDBF2 function has probably diverged since its ancestral duplication from the DBF4 gene, we should consider that ZDBF2 might also be involved in cell cycle regulation. Indeed, a number of growth-control genes affect cell cycle duration [START_REF] Efstratiadis | Genetics of mouse growth[END_REF]. Moreover, it would be interesting to investigate if Zdbf2-related growth phenotypes are related to cell number variation.

The ZDBF2 protein is 2498-amino-acid-long and has been particularly difficult to study. Notably, we tried to generate a polyclonal anti-mouse ZDBF2 antibody, which finally failed to specifically detect the ZDBF2 protein in Western blot assays, even after immunoprecipitation. To investigate more closely the cellular localization of ZDBF2 and its interactors, I therefore generated a CRISPR-based knock-in mouse containing a HA-FLAG tag at the C-terminal end of the ZDBF2 protein. However, my attempts to visualize the ZDBF2-HA protein using immunofluorescence were not conclusive. More efforts should be dedicated to the biochemical study of the ZDBF2 protein in the future.

Importantly, the imprinted status of ZDBF2 is conserved in humans [START_REF] Duffié | The Gpr1/Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals[END_REF]. Available RNA-seq data from multiple human tissues reveal that ZDBF2 is highly expressed in the brain and in the pituitary gland (Figure 30). It is thus tempting to propose that the biological function of ZDBF2 in mice could be conserved in humans.

Recently, a number of genome-wide association studies (GWAS) studies have focused on identifying variants associated with height determination in humans [START_REF] Benonisdottir | Epigenetic and genetic components of height regulation[END_REF][START_REF] Garg | Detection of parent-of-origin specific 207 expression quantitative trait loci by Cis-association analysis of gene expression in trios[END_REF][START_REF] Wood | Defining the role of common variation in the genomic and biological architecture of adult human height[END_REF][START_REF] Zoledziewska | Height-reducing variants and selection for short stature in Sardinia[END_REF]. Notably, parent-of-origin models testing for variants associated with human height demonstrated that common variations alter human growth through parental imprinting [START_REF] Benonisdottir | Epigenetic and genetic components of height regulation[END_REF]. These data represent a starting point to investigate if ZDBF2 affects growth in the human population. However, most studies have been performed on adult individuals: we might not be able to associate ZDBF2 so height variations in this population if -as it is the case in mice-ZDBF2 mostly affects growth at puberty. Liz apparaît ainsi comme un « interrupteur épigénétique », essentiel à la programmation de l'activation du gène Zdbf2 dans le cerveau adulte et ce, dès les quatre premiers jours de développement de l'embryon. mission maternelle de la délétion est muette, puisque l'allèle maternel de Liz est normalement silencieux. L'utilisation parallèle de cellules souches embryonnaires (ES) en culture nous a permis de comprendre le mécanisme moléculaire par lequel Liz agit : la région en amont du promoteur de Zdbf2 est occupée par défaut par un bloc de marques chromatiniennes répressives de triméthylation de la lysine K27 de l'histone H3 (H3K27me3) déposée par le complexe polycomb1 [11]. La méthylation de l'ADN et celle de H3K27 sont connues pour leur relation antagoniste [11]. La méthylation de l'ADN déposée par Liz permet ainsi de dissoudre localement cette chape répressive dans l'embryon précoce : ceci est indispensable pour libérer le promoteur de Zdbf2, amorçant de manière préemptive son expression en vie post-natale (Figure 2).

  Structure and function of the mature pituitary gland 4.2.b Formation of the pituitary gland during development 4.3 GROWTH CONTROL IN MICE 4.3.a General principles of mouse growth control 4.3.b Hypothalamo-pituitary axis-dependent control of growth INTRODUCTION RESULTS DISCUSSION AND PERSPECTIVES INTRODUCTION 20

Figure 1 :

 1 Figure 1: Sciara flies and their cycle of reproduction.(A) Picture of a Sciara fly (fungus gnat) (B) Formation of male gametes during Sciara development. In the gastrula, one paternal X chromosome (Xp) is eliminated, then meiosis occurs in the pupa. During meiosis I, the paternal genome is eliminated. The maternal set of autosomes (Am) is then eliminated during meiosis II and the surviving maternal genome will be inherited as paternal in the sperm. (C) After fertilization, in the seventh cleavage, all somatic precursors lose two Xps in males or one Xp in females.Drawing adapted from «Imprinting and Paternal Genome Elimination inInsects » book chapter, Glenn Herrick and Jon Seger, 1999

Figure 2 :

 2 Figure 2: Genomic imprinting in the maize endosperm. (A) Scheme of the double fertilization happening in the female gametophyte of all angiosperm plants. One sperm cell fertilizes the egg, which will lead to the embryo proper, whereas the other sperm cell fertilizes the 2n polar nucleus to give rise to the endosperm. (B) rr/RR kernels exhibiting a mottled pigmentation. (C) RR/rr fully colored kernels.

Figure 3 :Figure 4 :

 34 Figure 3: Drawing of E17 T hp heterozygote embryos by Johnson. (A) Wild-type embryo.(B) embryo resulting from a cross between +/+ females and T hp /+ males. This type of embryos are viable, with varying degrees of shortening or kinky tail (blue triangle). (C) embryo resulting from a cross between T hp /+ females and +/+ males. Most of these embryos die during the second half of pregnancy. The ones that exceptionally survive until E17 are oedematous, have short kinky tail (red triangle) and exhibit a small post-axial polydactyly (small red triangle). Image from[START_REF] Johnson | Hairpin-tail: a case of post-reductional gene action in the mouse egg[END_REF]

Figure 5 :

 5 Figure 5: Scheme of embryology experiments made by Surani and Barton in 1983. (A) Non-modified fertilized egg. (B and C) Androgenetic (B) and gynogenetic (C) embryos generated by the removal of the maternal (B) or paternal (C) pronucleus from a fertilized egg, followed by cytochalasin B treatment to prevent cytokinesis and restore a diploid state. (D) Triploid fertilized egg, made of two maternal pronuclei and one paternal pronucleus. (E and F) Diploid (E) and dygynic (F) zygotes generated by the removal of the maternal (E) or paternal (F) pronucleus from a triploid egg.

Figure 6 :

 6 Figure 6: Scheme of embryology experiments carried out by Surani and colleagues and Solter and colleagues in 1984. (A) Biparental diploid control (1) and androgenetic conceptuses (2) generated by the nuclear transfer of a female (1) or a male (2) pronucleus into an haploid androgenetic embryo. The biparental diploid control gives rise to normal embryo and extra-embryonic tissues, whereas the embryo does not develop in the diploid androgenetic context. Image fromBarton and al. 1984. (B) Biparental diploid control (1) and gynogenetic conceptuses (2) generated by the nuclear transfer of a male (1) or a female (2) pronucleus into an haploid gynogenetic embryo. The biparental diploid control gives rise to normal embryo and extra-embryonic tissues, whereas extra-embryonic tissues are impaired in the gynogenetic context. Image from[START_REF] Surani | Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis[END_REF]

Figure 7 :

 7 Figure 7: Representation of the parental expression pattern at the Igf2r locus and the Igf2-H19 locus in mice. (A) Igf2r expression is restricted to the maternal chromosome. (B) Igf2 and H19 are two adjacent imprinted genes expressed from the paternal and the maternal allele, respectively.

  mechanistic regulation of imprinted regions, which I will address in the next chapter.

Figure 8 :

 8 Figure 8: Map of imprinted genomic regions and genes known to exhibit parent-of-origin expression. In yellow are represented the regions tested by Cattanach and colleagues for uniparental disomies leading to abnormal embryonic and/or post-natal phenotypes. Image from https://www.mousebook.org.

Figure 9 :

 9 Figure 9: DNA methylation in mammals. (A) Scheme of the transformation from a non-methylated cytosine to a methylated cytosine (B) Typical DNA methylation landscape in mammals. Repeated DNA sequences, intergenic and intragenic CpGs are usually methylated, whereas promoter CGIs are DNA methylation-free, in general. Black circle: methylated CpG; white circle: unmethylated CpG.

Figure 10 :

 10 Figure 10: Dnmt mutants in mice.(A) Wild-type embryo at E9.5 with Dnmt1-KO littermate. Picture from[START_REF] Lei | De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells[END_REF] (B) Wild-type embryo at E9.5 with Dnmt3a/3b double-KO littermate. Picture fromOkano et al., 1999. (C) Wild-type embryo at E12.5 with Dnmt3b-KO littermate. Picture fromOkano et al., 1999. (D) Wild-type mouse at 4 weeks of age with Dnmt3a-KO littermate. Picture fromOkano et al., 1999.(E) Wild-type embryo at E9.5 with maternal Dnmt3l-KO littermate. Picture from[START_REF] Bourc'his | Dnmt3L and the establishment of maternal genomic imprints[END_REF]

Figure 11 :

 11 Figure 11: Typical DNA methylation profiles carried by the sperm and oocyte genomes.The sperm is globally highly methylated with CpG methylation (about 80%) mostly localized in intergenic regions and transposons sequences. The oocyte genome is less methylated (about 40%), highly transcribed and acquires DNA methylation on gene bodies, including intragenic CpG islands.

Figure 12 :

 12 Figure 12: Dynamics of DNA methylation levels during mammalian embryo development. After fertilization, the paternal and maternal genomes undergo a wave of demethylation concomitant with the acquisition of pluripotency. Maternal and paternal genomes have different kinetics and mechanistic of demethylation. After this epigenetic reprogramming, post-implantation embryos go through genome-wide remethylation.

A

  common feature of Imprinting Control Regions (ICRs) is the post-fertilization maintenance of the parent-of-origin DNA methylation profiles inherited from the gametes. This property underpins the existence of mechanisms protecting the methylated allele of ICRs from the genome-wide demethylation occurring after fertilization.

Figure 13 :

 13 Figure 13: Protection of imprinted regions during mammalian embryo development.During the phase of epigenetic reprogramming, the methylated allele of ICRs has been proposed to be protected both by STELLA from active demethylation and by ZFP57/KAP1 from e passive demethylation. After implantation, ICRs are probably protected from remethylation by the binding of specific transcription factors. This figure shows the example of a maternally DNA methylated region; the same kind of mechanism applies for regions that inherit DNA methylation from the paternal allele. The grey line represents the global changes in genomic DNA methylation levels.

Figure 14 :

 14 Figure 14: Waves of DNA demethylation during mammalian epigenetic reprogramming.From E6.5, the embryo begins PGC (in green) differentiation in a process called germline specification. PGCs will then migrate to the embryonic endoderm, to the hindgut and finally towards the genital ridges, while undergoing passive and active genome-wide demethylation. By E13.5, the level of PGC DNA methylation is around 10%.

Figure 15 :

 15 Figure 15: Genomic imprinting during the mouse life cycle.Genomic imprinting is established in the male and female gametes during pre-and post-natal life, respectively, through the action of DNMT3A and the DNMT3L co-factor. Following fertilization, the methylated allele of ICRs is protected from epigenetic reprogramming by STELLA and the ZFP57/KAP1 complex. When DNA methylation is re-established at implantation, transcription factor binding could protect the unmethylated allele of ICRs. During germline differentiation, genomic imprinting is erased through active TET demethylation in both male and female germlines.

Figure 16 :

 16 Figure 16: Transient form of imprinting. (A) The initially unmethylated paternal allele gains DNA methylation after implantation and the region becomes biallelically methylated in all somatic tissues. (B) The initially methylated maternal allele loses DNA methylation after implantation and the region becomes biallelically unmethylated in all somatic tissues.

Figure 17 :

 17 Figure 17: Conservation of the gene structure at the Zdbf2 and Dbf4 locus in mice and humans. (A and B) The genomic organization of the Zdbf2 locus appears to be conserved in mice and humans. The only difference is the de novo insertion of the CPO gene in humans compared to the mouse. (C and D) Synteny the Dbf4 locus in humans and mice. Interestingly, Dbf4 is adjacent to Adam 22 whereas Zdbf2 is adjacent to Adam23 suggesting an ancestral duplication. (E) In humans, two DBF4 genes exist. DBF4B also lies next to an ADAM gene copy.

Figure 18 :

 18 Figure 18: The Gpr1-Zdbf2 locus in mice.(A) Schematic view of the Gpr1-Zdbf2 locus. The locus contains three transcripts: Gpr1 (purple), the canonical form of Zdbf2 (green), and the long isoform of Zdbf2, called Liz (blue). It also contains two DMRs: one of germline origin (gDMR), located in an intron of Gpr1 and coinciding with Liz TSS, and a somatically established DMR (sDMR) located about 7-12kb upstream from the canonical promoter of Zdbf2. CpG islands are indicated in light green. (B) Dynamic regulation of the Gpr1-Zdbf2 locus during mouse development. In the preimplantation embryo, Liz expression initiates from the paternal unmethylated allele of the gDMR. At implantation, imprinting of the gDMR disappears by gain of DNA methylation and the paternally methylated sDMR emerges. In the post-implantation embryo and onwards, Liz is shut down, the sDMR is paternally methylated and Zdbf2 is paternally expressed.

Figure 19 :

 19 Figure 19: Phylogenetic tree of Amiotes.Genomic imprinting emerged in Theria, at the same time as expansion of transposable elements (TEs), DNMT3L emergence and acquisition of the primitive placenta.

Figure 20 :

 20 Figure 20: The BWS locus in humans. Here are represented the two domains of the 11p15.5 region, containing two ICRs. Domain 1 contains the IGF2-H19 locus, while CDKN1C-KCNQ1 forms domain 2.

Figure 21 :

 21 Figure 21: Schematic coronal view of the mouse brain. The hypothalamus is highlighted in light blue. This coronal view focuses on the tuberal hypothalamic region (lateral area) and the third ventricle (3V), the median eminence (Me), the arcuate hypothalamic nucleus (ARC), the ventromedial and dorsomedial hypothalamic nucleus (VMH and DMH) are annotated. Image adapted from the Mouse Brain Atlas.

  and anterior lobes have common embryonic origin and form the adenohypophysis. The posterior lobe does not contain endocrine cells but only glial cells and axonal termini from the hypothalamus. The intermediate lobe is composed of one endocrine population, the melanotrophs, which secrete the melanocyte-stimulating hormone (MSH). It is noteworthy that the intermediate pituitary lobe disappears during embryogenesis in humans, which suggests its little functional importance in the mature gland. The only source of MSH in humans comes from the skin[START_REF] Rizzoti | Genetic regulation of murine pituitary development[END_REF].The anterior lobe part is the largest and is vascularized by capillaries that serve for the reception of hypothalamic inputs and the transmission of secreted hormones to the blood stream. The anterior lobe contains five types of specialized hormoneproducing cells (Figure22)[START_REF] Watkins-Chow | How many homeobox genes does it take to make a pituitary gland?[END_REF]. The corticotrophs produce the adrenocorticotrophic hormone (ACTH), which has metabolic functions such as cholesterol regulation through stimulation of the adrenal cortex. The thyrotrophs are the cells producing the thyroid-stimulating hormone (TSH) that impacts on whole body metabolism through thyroid gland signaling. The lactotrophs are prolactin (PRL)-secreting cells required for lactation during post-pregnancy period. The gonadotrophs include the follicle-stimulating hormone (FSH) and the luteinizing hormone (LH)-producing cells. These two hormones are produced at the onset of puberty, stimulated by the gonadotrophin-releasing hormone (GnRH) from the hypothalamus, and define the neuro-endocrine timing of puberty[START_REF] Ebling | The neuroendocrine timing of puberty[END_REF].Finally, growth-hormone (GH)-releasing cells are called somatotrophs and play major roles in pre-and post-natal growth regulation. Together, the five types of hormoneproducing cells of the anterior pituitary gland regulate positive and negative feedback controls from the hypothalamus to peripheral endocrine organs, through the trophic factors they secrete.

Figure 22 :

 22 Figure 22: Organization of the mature mouse pituitary gland.Adapted from[START_REF] Rizzoti | Genetic regulation of murine pituitary development[END_REF]

  5, while the onset of somatotroph differentiation starts at E13.5, concomitantly with the Pit-1 expression peak. The maturation of prolactin-secreting cells will begin only after birth. The differentiation of the corticotroph cells starts at E12.5, through the expression of Pomc, the precursor of ACTH. Of note, Pomc is also the precursor of MSH-secreting cells in the intermediate lobe of the pituitary. Finally, gonadotrophs (LH and FSH) differentiation follows the GnRH hypothalamic signal around E12.5. Overall, all the differentiating endocrine cells are present at E14.5 and are mature at E18.5, right before birth. However, the final differentiation signals take place in the post-natal pituitary gland as cells increase in size and receive hypothalamic inputs.

Figure 23 :

 23 Figure 23: Embryonic development of the pituitary gland.Adapted from[START_REF] Rizzoti | Genetic regulation of murine pituitary development[END_REF]

Figure 24 :

 24 Figure 24: Scheme of the GH-mediated growth control by the hypothalamo-pituitary axis.The production of GHRH from hypothalamic neurons stimulates the somatotroph cells from the anterior lobe of the pituitary gland, which in turns produce GH. GH then acts on multiple endocrine organs, and notably induces the production of IGF-1 by the liver to act on general body growth.

1 .

 1 discovery that some loci exist as transiently imprinted only during a short window of time in the pre-implantation embryo raises the possibility of revisiting the number of annotated imprinted regions and opens a new layer of investigation in the field. The starting point of my PhD work was to understand whether transient and early embryonic epigenetic events could have long-term functional relevance. To gain insight into this developmental problem, the analysis and functional characterization of transient genomic imprinting appeared as the perfect model. Despite the number of potential candidate loci in the mouse genome, the Liz/Zdbf2 locus has been the only region described and characterized as transiently imprinted in mice and humans so far. I focused my whole PhD work on the functional characterization of this locus, using in vivo techniques in mice. I was lucky enough to start my PhD right after what is-so far-the biggest discovery and revolution of the twenty-first century in molecular biology, also known as the famous "CRISPR Craze" (RE et al., 2013; Savell and Day, 2017). I was thus able to generate multiple CRISPR-based mouse mutants to gain a detailed overview of the function and regulation of Liz and Zdbf2. I gained major insights into 1. the role of transient imprinting at the Zdbf2 locus, 2. the biological relevance of having Zdbf2 imprinted in the adult mice and 3. the dual-component of Zdbf2 regulation. What is the role of transient and early Liz transcription in mouse embryos?The first goal of my work was to understand what could be the role of transient imprinting in mammals. This new form of imprinting was particularly intriguing: does it impact the early embryonic transcriptome? Could it have life-long consequences on genome regulation and phenotypes? I addressed these questions using the Liz/Zdbf2 locus as a case of study and the first part of my PhD work focused on understanding the role of the early transcription of Liz in the pre-implantation mouse embryo. Looking back in the time, we could have been confronted to the fact that Liz deletion had no major consequence, neither at the molecular nor at the phenotypic level, and this PhD manuscript would have probably been a complete different story. Fortunately, it was

3 . 1 .

 31 Zdbf2, as an imprinted gene in mice and humans? When I started my PhD, nothing was known about the molecular and biological function of Zdbf2 as a paternally expressed gene in mice and humans. Regarding the transiently imprinted status of Liz and the complex interplay orchestrated at the locus during mouse embryonic development, it was expected that Zdbf2 may encode a product with physiologically essential function. As a second goal of my PhD work, I attempted to precisely characterize the function of Zdbf2 in the adult mouse. Unfortunately, for this part of the work, we lacked biochemical tools to enlighten the precise molecular function of the ZDBF2 protein. However, my work focused on addressing the biological relevance of Zdbf2, which I demonstrated by genetic means to have an essential function in regulating post-natal growth. I managed to characterize the Zdbf2-associated phenotype in details, to decipher the importance of a very sensitive dose-dependent regulation of Zdbf2 and I have been able to generate an inversion of the parental origin of the imprint at the locus.The observation of the growth phenotype in our mutant mice was among the most exciting results of my PhD, since it revealed that "our" gene was actually doing something essential for mouse physiology. It was also the moment where my project started to shift towards growth regulation and the pituitary-hypothalamo axis, which I did not expect when I first started in Deborah's lab. However, even if there were a lot of jokes in the lab about how my project was becoming more endocrinology than epigenetics-related, I would not change the path that my PhD project took for anything in the world! For this second part, the results that I present in this thesis are still on-going work but close to a final version and represent a first draft of a manuscript that we plan to submit after the summer. What is the regulatory function of the somatic DMR at the locus? Finally, I added a third part of my PhD work, in which I attempted to address another layer of regulation at the Zdbf2 locus: the functional link between the DNA methylation at the somatic DMR and the activation of Zdbf2 promoter. This part is still unachieved but the preliminary results we obtained raise a lot of interesting questions and add unexpected new clues in our investigation of ZDBF2-mediated regulation of growth. Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth The achievement of the first goal of my PhD work relies on a close collaboration with Maxim Greenberg, a post-doctoral colleague from our laboratory. We started to teamup during my Master training, when I had the chance to discover the great mentor he was. In this study we investigated the potential of early embryonic epigenetic events in programming adult phenotypes. Transient imprinting constitutes a perfect model to address this question since it involves a transient transcriptional signal during mouse embryonic development, which is then switched off in an epigenetically locked state.We thus attempted to unravel the role of transient imprinting by taking advantage of the Liz/Zdbf2 locus in the mouse model.Our work was complementary: Max focused on the mechanistic analysis of the Liz/Zdbf2 locus using in cellula approaches in mouse embryonic stem cells (mESCs) whereas my contribution was in understanding the functional relevance of Liz through an in vivo genetic approach. Fortunately, all the molecular analyses we did were highly congruent between the mESCs and the in vivo model. To decipher the role and regulation of Liz, we generated mutant mESCs and mouse embryos lacking the expression of Liz. I was involved in the generation of the mutant mice using the CRISPR-Cas9 engineering in vivo. These mice have been produced with the precious help of the transgenesis platform of Institut Curie (Fatima El Marjou et Colin Jouhanneau) and they were the first CRIPSR mice ever generated in Paris! Back in February 2014, genotyping our first CRISPR founder mice ever, you can imagine our excitement over the remarkable efficiency of this deletion: 45% of the pups carried at least one affected allele (Figure25). I fully characterized the multiple CRISPRmediated deletions obtained after transgenesis and I followed two founder lines for further analyses. I then performed all the in vivo phenotypic analyses reported in our publication of the Liz-KO mice, both at the molecular and the physiological level.

Figure 25 :

 25 Figure 25: Scheme of CRISPR-mediated in vivo generation of the Liz-KO mutant embryos. The transgenesis part was performed by Fatima El Marjou and Colin Jouhanneau at the transgenesis platform of the Institut Curie.

Figure 1 A

 1 Figure 1 A cellular model of the dynamic regulation of the Gpr1/Zdbf2 locus. (a) Genomic organization of the Gpr1/Zdbf2 locus. The gDMR lies in the second intron of Gpr1 (red) and includes the TSS of Liz (blue). The sDMR is located ~10 kb upstream of the promoter of Zdbf2 (green) and is organized into three subregions (1, 2 and 3), which can be considered as single entities. CGIs are displayed in green. (b) Schematic of de novo DNA methylation upon epiblast differentiation, in vivo and upon in vitro conversion of 2i+vitC-grown ESCs into EpiLCs. (c) CpG methylation status of the gDMR and sDMR in 2i+vitC ESCs (day 0), and in day 4 and day 7 EpiLCs, by bisulfite cloning and sequencing. White circles, unmethylated CpGs; black circles, methylated CpGs. (d,e) Kinetics of de novo CpG methylation of the gDMR and sDMR (bisulfite pyrosequencing (BS-pyro)), as compared to the whole genome (LUMA quantification) (d), and Liz and Zdbf2 expression quantified by NanoString nCounter (e), over 7 d of EpiLC differentiation. Error bars, s.e.m. from three biological replicates. (f) Scatterplot for single-cell RT-qPCR analysis of Liz and Zdbf2 expression in day 4 EpiLCs. Values were normalized to the geometric mean of Actb, Rrm2 and Rplp0. The highest value for each transcript was set to 1.

Figure 2

 2 Figure 2 Liz transcription promotes sDMR methylation and Zdbf2 expression. (a) Graphic models of the two Liz-mutant cell lines. Top, deletion of the gDMR/Liz TSS ( gDMR/ Liz); bottom, interruption of Liz transcription by a tandem polyadenylation cassette (Liz::2×STOP). The location of RT-qPCR primers is featured. (b) Expression of spliced and unspliced Liz upon EpiLC differentiation. In comparison to the wild-type (WT) E14 line (blue), Liz (red) exhibits near depletion of detectable RNA; Liz::2×STOP (orange) displays intermediate reduction. (c) Stellaris RNA FISH of nascent Liz transcripts in Liz-mutant EpiLCs at day 2. Left, representative microscopy images; right, quantification of nuclei with two, one or no FISH signal. n is the number of counted nuclei. Note the absence of FISH signals in Liz nuclei and their lower intensity in Liz::2×STOP nuclei. Scale bars, 10 m. (d,e) Kinetics of de novo CpG methylation of the sDMR, as measured by BS-pyro (d), and Zdbf2 expression, as measured by RT-qPCR, upon EpiLC differentiation of Liz-mutant cells (e). Data are shown as means s.e.m. from at least three biological replicates.

Figure 3

 3 Figure 3 Zdbf2 is repressed by PRC2. (a) H3K27me3 ChIP-seq reads at the Gpr1-Zdbf2 locus at day 0, day 4 and day 7 of differentiation of wildtype and Liz cells. An H3K27me3 block extends over the sDMR-from an intergenic CGI to the Zdbf2 CGI promoter-in wild-type day 0 ESCs and is depleted upon EpiLC differentiation. Scale and coordinates are as in Figure 1a. The y-axis values for the H3K27me3 tracks are given in units of reads per million mapped reads. (b) H3K27me3 ChIP-qPCR validation. The value for each biological sample is relative to input and normalized to Pax5. (c) RT-qPCR of Zdbf2 in Eed -/-cells at day 0 and day 4. The J1 cell line was used as a control. (d) RT-qPCR of Zdbf2 upon deletion of Eed in Liz cells generated on the E14 background ( Liz; Eed -/-). (e) ChIP-qPCR analysis of H3K4me3 enrichment at the Zdbf2 promoter in wild-type and Liz cells across differentiation. All data are shown as means s.e.m. from three biological replicates, except for in e (two biological replicates). Statistical analyses were performed by two-tailed unpaired t test: ****P 0.0001.

Figure 4

 4 Figure 4 DNA methylation is required to derepress Zdbf2. (a,b) Time course of Liz (a) and Zdbf2 (b) expression upon EpiLC differentiation of DNMT triple-knockout cells (DNMT tKO; orange) and wild-type cells on the same J1 background (black), as measured by RT-qPCR. The spliced form of Liz was measured here; similar results were obtained with the unspliced form (data not shown). Data are shown as means s.e.m. from three biological replicates. (c) H3K27me3 ChIP-qPCR indicates H3K27me3 retention in DNMT triple-knockout cells as compared to wild-type cells across differentiation. ChIP primers amplify three regions within the sDMR, the intergenic CGI and the Zdbf2 CGI promoter. Values and error bars are as in Figure 3b.

Figure 5

 5 Figure 5 Liz RNA regulates sDMR and Zdbf2 in vivo. (a) RT-qPCR of Liz in single blastocysts descending from wild-type crosses and Liz +/-fathers and mothers. (b) BS-pyro shows reduced CpG methylation at the sDMR in E9.5 paternal-knockout (Pat KO ) embryos. (c) Zbdf2 transcripts are undetectable in E9.5 paternal-knockout embryos by RT-qPCR. Data are shown as means s.e.m. from n embryos. (d) Persistent methylation defect in tissues of Liz paternal-knockout and homozygous mice at 8 weeks. (e) Bisulfite cloning and sequencing of tail DNA from 8-week-old hybrid wild-type and paternalknockout mice (JF1 × Liz +/-cross). Red, maternal alleles; blue, paternal alleles. cross, informative JF1 SNP; hole, sequencing error. (f) X-gal staining of the brain of wild-type (left) and Zdbf2::lacZ (right) males at 6 weeks. C, cortex; Ce, cerebellum; H, hypothalamus; M, medulla; Ob, olfactory bulb; P, pituitary gland. Scale bar, 5 mm. (g) Zdbf2 expression is eliminated in the pituitary gland and hypothalamus in 8-week-old Liz paternal-knockout mice. (h) ChIP-qPCR detects higher H3K27me3 enrichment at the sDMR in the hypothalamus of 6-week-old Liz paternal-knockout mice than in wild-type littermates. (i) Allelic H3K27me3 enrichment in hypothalamus from 3-week-old hybrid mice, measured by ChIP-PCR pyrosequencing at the sDMR (top) and the Zdbf2 promoter (bottom). In, input. Statistical analyses were performed by two-tailed unpaired t test: ****P 0.0001, ***P 0.001, *P 0.05. Data are shown as means s.e.m. For d, g and h, at least three mice were analyzed for each genotype. For i, two Liz paternal-knockout and four wild-type littermates were analyzed.

Figure 6

 6 Figure 6 Liz paternal-knockout mice exhibit a lifelong growth defect. (a) Normalized body weight of Liz paternal-knockout mice relative to their wildtype littermates (100%) followed at different ages (1 to 210 d). Red, females; blue, males. The weight reduction is seen as early as 1 d.p.p. and amplifies within the first weeks of life. (b,c) Growth curves of female and male mice, comparing the body weights of the wild-type and Liz paternalknockout genotypes, through the first 3 weeks of life (b) and through 6 months (c). (d) Scatterplot comparing the body weights of 2-week-old wild-type and Liz paternal-knockout males (left) and females (right). Each point represents one individual. n is the number of mice analyzed. ****P 0.0001. (e) Representative photograph of a smaller Liz paternal-knockout male as compared to a wild-type littermate at 2 weeks. (f) Frequency of weight distribution for normalized weights of 2-week-old paternal-knockout mice relative to wild-type mice (100%) grouped into 10% bins. The weight reduction phenotype appears to be highly penetrant. The skewing toward lower weight is more important for females, as seen in a. For a-c, 20-40 mice were analyzed per genotype, depending on age and sex. Data are shown as means s.e.m.

Figure 7

 7 Figure7Model of Liz-dependent epigenetic programming of Zdbf2 expression and growth potential on the paternal allele. Top, early epigenetic programming: the sDMR and the promoter of Zdbf2 are covered by an H3K27me3 block at the blastocyst stage. At the exit from naive pluripotency in the epiblast, the burst of Liz transcription promotes DNA methylation in cis at the sDMR and eviction of H3K27me3 marks (left). In Liz knockout embryos (right), the lack of paternal Liz results in sDMR hypomethylation and H3K27me3 retention. Bottom, lifelong consequences on growth: following the epiblast stage, Liz is turned off, but the epigenetic signature it conferred in the early embryo is maintained in a lifelong manner, allowing Zdbf2 expression in the hypothalamus. Without prior Liz expression, Liz paternal-knockout mice fail to express Zdbf2 and exhibit reduced body size.

Figure 1 :

 1 Figure 1: Zdbf2-KO mice exhibit growth reduction and partial post-natal lethality phenotypes. (A) Graphical model of the deletion of Zdbf2 generated using two sgRNAs upstream and downstream of exon 6. This deletion generates a frame-shift leading to a loss-of-function protein. (B) Survivability curves from the first day of life up to 12 weeks of age comparing the rate of survival between WT and Zdbf2-KO males and females. The partial post-natal lethality phenotype appears to be specific to the pre-weaning period, from the first day to 3 weeks of age. (C) Normalized body growth of Zdbf2-KO mice to their WT littermates (100%) followed at different ages in males and females. The weight reduction is seen as early as 1 d.p.p. and amplifies within the first weeks of life. Data are shown as means ± s.e.m. from individuals from n= 27 litters. (D, E) Growth curves of female and male mice, comparing the body weights of the WT and Zdbf2-KO, through the first 3 weeks of life (D) and through 6 months (E). (F) Representative photography of a smaller Zdbf2-KO male as compared to a WT littermate at 2 weeks of age. (G) Half dot-half violin plots showing the weight distribution at 2 weeks of age in males (left) and females (right) between WT and Liz gain-of-function. **** p ≤ 0.0001, *** p ≤ 0.001. (H) Frequency of weight distribution for normalized weights of 2-week-old paternal-knockout mice relative to WT mice (100%) grouped into 10% bins. The weight reduction phenotype appears to be highly penetrant.

Figure 2 :

 2 Figure 40

Figure 3 :

 3 Figure 3: Liz RNA is necessary to induce DNA methylation and Zdbf2 expression at the locus. (A) Graphic model of the Liz gain-of-function deletion (line #379) obtained by the CRISPR-Cas9 engineering. The deletion is the result of only one cut of Cas9 at the left sgRNA, followed by NHEJ. The deletion includes the three ZFP57 binding sites (orange) and the annotated TSS of the Liz transcript (blue). (B) Bisulfite-pyrosequencing shows increased sDMR methylation in tissues from 6 week-old mice when the deletion is maternally transmitted. The paternal transmission of the deletion behaves as silent. (C) Bisulfite cloning and sequencing showing CpG methylation levels at the sDMR locus of hypothalamus DNA from 6 week-old hybrid WT (left) and Liz-GOF (right) mice (Liz-GOF +/-x JF1 cross). Red, maternal alleles; blue, paternal alleles. cross, informative JF1 SNP. (D) RT-qPCR measurement reveals a 1.6 fold-increase of Zdbf2 expression in the hypothalamus and pituitary gland of mice with a maternal transmission of the deletion at 3 weeks. (E) Allelic Zdbf2 expression in hypothalamus and pituitary gland from 3 week-old mice, measured by RT-pyrosequencing. Genomic DNA extracted from a C57Bl/6 x JF1 hybrid cross was used as a control for pyrosequencing bias. For C, D and E, data are shown as means ± s.e.m. from n individuals. Statistical analyses were performed by two-tailed unpaired t-test: **** p ≤ 0.0001, *** p ≤ 0.001, ** p≤ 0.01 * p ≤ 0.05.

Figure 4 :

 4 Figure 4: Liz gain-of-function mice exhibit male-specific overgrowth. (A) Normalized body growth of Liz gain-of-function mice to their WT littermates (100%) followed at different ages (1 to 84 days). The overgrowth is seen specifically in males, from 5 to 28 days. Data are shown as means ± s.e.m. from heterozygote individuals from n= 14 litters. (B, C) Growth curves of female and male mice, comparing the body weights of the WT and Liz gain-of-function, through the first 3 weeks of life (B) and through 10 weeks (C). (D) Half dot -half violin plots showing the weight distribution at 2 weeks of age between WT and Liz gain-of-function. *** p ≤ 0.001. (E) Representative photography of a bigger Liz gain-of-function male as compared to a WT littermate at 2 weeks. (F) Frequency of weight distribution for normalized weights of 2-week-old paternal-knockout mice relative to WT mice (100%) grouped into 10% bins. The male specific overgrowth phenotype appears to be penetrant. Females show a normal distribution of weight, confirming the sex-specific phenotype seen in A, B and C. For A -C, 10-30 mice were analyzed per genotype, depending on age and sex. Data are shown as means ± s.e.m.

Figure 5 :

 5 Figure 5: The dose of Zdbf2 and not its parental origin is important for proper post-natal growth. (A) Scheme of the cross made to obtain embryos with an inversion of the parental origin of Liz expression. Heterozygote females for the Liz-GOF deletion were crossed with heterozygote males for the Liz-LOF deletion (left) to obtain one quarter of embryos expressing one dose of Liz from the maternal allele (right, bottom). (B) Bisulfite-pyrosequencing shows an almost complete rescue of the Liz-LOF and Liz-GOF defects of sDMR methylation from tissues of Liz-GOF/LOF mice at 6 week-old. Data are shown as means ± s.e.m. (C) Bisulfite cloning and sequencing at the sDMR locus of hypothalamus DNA from a 6 week-old Liz-GOF/LOF mouse. The sDMR methylation level is similar to the WT situation (Figure3B). (D) Zdbf2 expression in the hypothalamus and the pituitary gland is shown for each of the four possible genotypes. The level of Zdbf2 in Liz-GOF/LOF mice almost completely rescues the defect seen in Liz-LOF and Liz-GOF mutants. Data are shown as means ± s.e.m. (E) Normalized body growth of Liz-LOF, Liz-GOF and Liz-GOF/LOF males to their WT littermates (100%) followed at 7, 14 and 21 days after birth. Liz-GOF/LOF adult mice exhibit a body weight similar to the WT showing a partial rescue of the growth reduction and overgrowth phenotype due to respectively the lack of Zdbf2 and the gain of Zdbf2 expression in the brain. Data are shown as means ± s.e.m. from individuals from n= 17 litters. (F) Representative photography of four male littermates from a Liz-GOF x Liz-LOF cross (as shown in A). For each animal, the genotype, the dose of Zdbf2 expression and the weight are indicated.

Figure S1 :Figure S2 :Figure S3 :

 S1S2S3 Figure S1: Phenotypical characterization of the Zdbf2-KO mouse line. (A, B) Sex ratios (A) and genotype ratios (B) observed at birth among Zdbf2 heterozygotes upon paternal and maternal transmission of the deletion after three backcrosses. Numbers of counted animals of each sex are indicated on the histograms. (C) RT-qPCR measuring Zdbf2 expression in 3 week-old hypothalamus reveals that the transcriptional activity of Zdbf2 is not affected by the deletion of the exon 6. (D) Bisulfite cloning and sequencing of the sDMR locus from hypothalamus DNA from 3 week-old hybrid WT (top) and Zdbf2-KO (bottom) mice (JF1 x Zdbf2-KO cross). Red, maternal alleles; blue, paternal alleles. cross, informative JF1 SNP. (E-G) Survivability curves as in Figure 1B but comparing WT and Liz-LOF males and females (E), WT and Zdbf2-∆exon6 males and females upon maternal transmission of the deletion. (F) and WT and Liz-∆gDMR males and females upon maternal transmission of the deletion (G). (H) Normalized body weight of Zdbf2-KO E18.5 embryos to their WT littermates (100%) shows no growth defect before birth. Data are shown as mean ± s.e.m. of n=3 litters. (I, J) Growth curves as in Figure 1D and Figure 1E, respectively, but comparing the mutants with a maternal transmission of the Zdbf2-∆exon6 deletion with their WT littermates. The maternal deletion behaves as a silent mutation with no growth-related phenotype in the progenies

Figure S4 :Figure S5 :

 S4S5 Figure S4: Zdbf2 is expressed in neuronal hypothalamic cells. (A, B) Expression of Zdbf2 in hypothalamic neuronal (A) and non-neuronal (B) cell clusters. These data have been extracted from publically available tracks of hypothalamic single-cell RNA-sequencing from Chen et al., Cell Reports, 2017.In red are indicated the three clusters where Zdbf2 is the most represented.

Figure S6 :Figure S7 :

 S6S7 Figure S6: Characterization of Liz-GOF mouse lines. (A) Graphic model of a Liz gain-of-function deletion obtained by an independent founder line (#397). As for the line #379, the deletion is the results of only one cut by Cas9 at the left sgRNA, followed by NHEJ and it includes the three ZFP57 binding sites (orange) and the annotated TSS of the Liz transcript (dark green). (B) Bisulfite-pyrosequencing from post-implantation embryos at E9.5 shows increase of sDMR CpG methylation only upon maternal transmission of the deletion, both in Liz-GOF #379 (left) and #397 (right) CRISPR lines. (C) Quantification of allelic sDMR CpG methylation measured by bisulfite cloning and sequencing from n WT and Liz-GOF adult tissues. The gain of sDMR methylation from the maternal allele in the mutant compared to their WT littermates is about 60%. (D) Screenshot of a UCSC session showing p300 (green), H3K27ac (red) and Oct4 (blue) ChIP-sequencing peaks around the Liz transcription start site region. The Liz-GOF deletion is highlighted in pink; the Liz-LOF deletion is in light blue. These data are coming from publically available tracks. (E,F) Bisulfite cloning and sequencing showing DNA methylation level at the gDMR (E) and sDMR (F) locus from blastocysts hybrid WT (left) and Liz-GOF (right) mice (Liz-GOF +/-x JF1 cross). Red, maternal alleles; blue, paternal alleles. cross, informative JF1 SNP. (G) ChIP-qPCR detects lower H3K27me3 enrichment at the sDMR and at Zdbf2 CGI in the hypothalamus of 3-week-old Liz-GOF than in WT littermates. For B,C and G, data are shown as means ± s.e.m. from n individuals

Figure 1 :

 1 Figure 1: Hypothalamus-specific gain of Zdbf2 expression in ∆sDMR mutant mice. (A) Schematic view of the Liz/Zdbf2 locus and the 8.3kb ∆sDMR CRISPR-generated deletion. The sDMR (black) is a tripartite DMR localized in an intergenic region 10-kb upstream of the canonical promoter of Zdbf2 (green). The 8.3-kb deletion englobes the entire tripartite DMR. (B) Histogram representing Zdbf2 expression from hypothalamus and pituitary of 3 week-old mice generated upon maternal, paternal and homozygous transmission of the sDMR deletion. Data are shown as means ± s.e.m. from n individuals. (C, D) Dot plots showing the relative expression of Zdbf2 measured on a cohort of individuals from the four types of genotype, in the hypothalamus (C) and the pituitary gland (D). For B-D, primers amplifying the exon1-3 of the Zdbf2 transcript were used. (E) Allelic expression of Zdbf2 in hypothalamus and pituitary gland from 3 week-old mice, measured by RT-pyrosequencing. Genomic DNA extracted from a C57Bl/6 x JF1 hybrid cross was used as a control for pyrosequencing bias. (F,G) Relative and allelic expression of Zdbf2 in hypothalamus (E) and pituitary gland (F) from 3 week-old mice. For each genotype, the analysis of n=4 individuals is represented; the dashed line indicates the average of their relative expression. For E-G, primers amplifying the exon7 of the Zdbf2 transcript were used.

Figure 2 :

 2 Figure 2: Increased Zdbf2 expression in the hypothalamus does not impact post-natal growth. (A-C) Dot plots representing the body weight of mutant mice with a maternally inherited deletion of the sDMR (sDMR-MKO) at 7 (A), 14 (B) and 21 (C) days post-partum. (D-F) Same data from mice with a paternally inherited deletion of the sDMR (sDMR-PKO) at 7 (D), 14 (E) and 21 (F) days post-partum. (G-I) Same data from mice homozygotes for the deletion of the sDMR (sDMRKO-KO) at 7 (G), 14 (H) and 21 (I) days post-partum.

Figure 3 :

 3 Figure 3: Pituitary-specific Zdbf2 loss-of-function is sufficient to induce growth deficiency. (A) Scheme representing the resulting progeny from the mating of a female carrying a sDMR deletion with a Liz-LOF male. For each of the four possible genotyped obtained from this cross, the expected allelism of Zdbf2 expression in both the hypothalamus (beige) and in the pituitary (orange) is indicated. (B) Histogram representing the body weight of males at 14 and 21 days post-partum. Data are shown as means ± s.e.m. from n individuals. (C) Dot plots representing the same data as in B from males at 14 days. Statistical analyses were performed by two-tailed unpaired t-test: ** p≤ 0.01 * p ≤ 0.05

Figure S1 :

 S1 Figure S1: View of ATAC-sequencing tracks at the Liz/Zdbf2 locus.For each tissues, we performed two replicates (Rep1 & 2). The regions deleted in the sDMR-KO mice is highlighted in light grey.

  The exact developmental regulation and biological function of Zdbf2 in mice was unknown when I started my PhD and my work precisely consisted in deciphering these questions. I demonstrated that Zdbf2 activation in post-natal brain is programmed in the early mouse embryo by the transient expression of Liz, which promotes an epigenetic switch at the locus. Probably one of the most fascinating findings of my work is that -even being present during only 4 days in the early embryo-Liz is strictly required to program post-natal growth. What made the study of the Zdbf2 locus particularly exciting to me was that I could work with all the aspects of molecular biology I like: developmental biology, genetic regulation and physiological relevance.Dissecting the growth-related function of Zdbf2 was sometimes challenging since it falls in a field that was not within the expertise of the laboratory. Using a panoply of CRISPR mutants, I used the power of genetics to play with the dose and allelism of Zdbf2 and assess phenotypic consequences. Functional understanding of developmental phenotypes is what I like but I have to admit that it is quite frustrating to wrap up this PhD thesis without knowing the molecular function of ZDBF2. In our laboratory, the transient imprinting at this locus has been uncovered and described by a former PhD, mechanistically studied by a post-doc and I partially achieved its functional characterization. Altogether this is more than 6 years of work on a single gene of the mouse genome. However-and this is the beauty of research-I have probably more unresolved questions than when I started my PhD. In the following parts of this discussion, I will develop some of them, starting with the analysis of Liz transient expression, then addressing the importance of the dose-regulation and arguing about the possible causes of the growth phenotype and finally, discussing the function of ZDBF2 in mice and humans.

Figure 26 :

 26 Figure 26: Molecular consequences of the ∆gDMR deletion in the male germline. (A) Scheme of the locus, centered around the tripartite sDMR and the Zdbf2 transcript. Sequences amplified for bisulfite cloning sequencing and RT-qPCR are indicated in orange. (B) DNA methylation has been assessed by bisulfite sequencing at the sDMR3 of the tripartite SMR in WT, heterozygote and homozygote sperm from mice carrying a deletion of the gDMR. (C) Zdbf2 expression arising from exon 1-3 (left) or exon 7 (right) was measured by RT-qPCR in testes from the same mutant mice. Data are shown as means ± s.e.m. from n individuals.

  Transient imprinting can disappear through either a gain of DNA methylation on the unmethylated allele, or a loss of methylation on the initially methylated allele. This raises several questions about the mechanism by which the methylation status changes after implantation. Why transient imprinted loci are not able to maintain their asymmetrical profile? What makes them different from the canonical imprinted ones?Which factors are involved in this transient phenomenon compared to what has been described at canonical imprints? During the global demethylation in the preimplantation embryo, the ZFP57/KAP1 complex protects the methylated allele at all known canonical ICRs and likely also transient ones. However, what protects the unmethylated copy from the genome-wide gain of DNA methylation after implantation is less described. Methylation-resistant CpGs are likely occupied by trans-acting factors and characteristic marks of active transcription, such as permissive histone modifications or long R-loop structures[START_REF] Ginno | R-Loop Formation Is a Distinctive Characteristic of Unmethylated Human CpG Island Promoters[END_REF][START_REF] Lienert | Identification of genetic elements that autonomously determine DNA methylation states[END_REF]. The transient ICRs that gain DNA methylation-as it is the case at the gDMR of the Liz/Zdbf2 locus-are probably ICRs that contain methylation-permissive CpGs. Conversely, the loss of DNA methylation in the context of the global remethylation of the genome must involve sequence-targeted mechanisms.

  about the regulation of the Zdbf2 locus. Using pyrosequencing-based DNA methylation assays on tail DNAs, I screened the original 20 founders carrying a positive CRISPR cut (45% of the total founder number) for their level of methylation at the sDMR. This was the first surprising result of my PhD and it paved the way of our dose-dependent growth regulation story. I expected half of the positive founders to have a silent effect (i.e. deletion on the maternal allele) and the other half showing a lack of DNA methylation at the sDMR (i.e. deletion of the paternal allele). I indeed observed these two patterns of sDMR DNA methylation but, in addition, I found a third group of founder mice that exhibited an unexpected hypermethylation status of the sDMR (Figure27).

Figure 27 :allelic

 27 Figure 27: Distribution of the level of sDMR methylation among the 44 CRISPR-generated founder mice.

Figure 29 summarizes

 29 Figure 29 summarizes the seven types of mutants I generated at the Liz/Zdbf2 locus, their consequences on Zdbf2 transcript levels in the hypothalamus and the pituitary gland and the phenotypic outcomes in terms of both growth and survivability.

Figure 30 :

 30 Figure 30: ZDBF2 expression from public RNA-seq datasets of different human tissues. As in mice, ZDBF2 is the most strongly expressed in brain-derived tissues, in particular the pituitary gland. Image from the GTex human tissues consortium (https://www.gtexportal.org/home/)

Figure 1 .Fécondation 1031 Figure 2 .

 110312 Figure 1. Dynamique de méthylation de l'ADNau cours du développement embryonnaire. À l'échelle globale du génome (A), les profils de méthylation de l'ADN hérités des gamètes sont remodelés par une phase de déméthylation puis de « re-méthylation », établissant les profils de méthylation somatique au moment de l'implantation. Les régions soumises à empreinte maternelle maintiennent les profils de méthylation hérités de l'ovocyte, toute la vie (B), ou seulement pendant le développement préimplantatoire (C). L'empreinte transitoire est annihilée soit par une perte de méthylation sur l'allèle maternel (cas 1), soit par un gain de méthylation sur l'allèle paternel (cas 2) au moment de l'implantation.

  empreinte paternelle somatique prend le relais au bout d'une semaine et perdure tout au long de la vie. Ce mode complexe de régulation pose la question du rôle de l'expression fugace de Liz sur la régulation à long terme du locus, via le dépôt d'une signature qui, elle, est indélébile.Liz, un « interrupteur épigénétique » dans l'embryon préimplantatoireAfin d'élucider le rôle de Liz chez la souris, nous avons supprimé sa transcription in vivo par la méthode d'édition CRISPR-Cas9[10]. En l'absence de Liz, les embryons sont incapables d'acquérir la marque de méthylation somatique en amont du promoteur de Zdbf2 et, plus tard au cours de la vie, d'activer l'expression de Zdbf2 dans le cerveau, alors que sa séquence génétique est parfaitement intacte (Figure2B). En accord avec le statut d'empreinte de Liz, ce défaut n'est observé que lors de la transmission paternelle de la délétion de Liz ; la trans-

  la synthèse de L. Lebreton et al., m/s n° 3, mars 2016, page 290

Table of content INTRODUCTION PREFACE 1. THE DISCOVERY OF GENOMIC IMPRINTING: AN HISTORICAL OVERVIEW

	1.1 PARENT-OF-ORIGIN EFFECT IN INSECTS
	1.1.a First observations
	1.1.b Other examples
	1.2 GENOMIC IMPRINTING IN PLANTS
	1.2.a Imprinting in the endosperm
	1.2.b Mechanisms of genomic imprinting in plants
	1.3 GENOMIC IMPRINTING IN MAMMALS
	1.3.a Parent-of-origin effects
	1.3.b Embryology experiments
	1.3.c Uniparental disomy experiments
	1.3.d First imprinted genes in mice
	1.3.e Nowadays
	2. EPIGENETIC CONTROL OF GENOMIC IMPRINTING IN MAMMALS
	2.1 DNA METHYLATION: GENERAL CONCEPTS
	2.1.a Transcriptional silencing
	2.1.b Maintenance through cell division
	2.1.c De novo methylation
	2.1.d De-methylation mechanisms
	2.1.e Parental-specificity at imprinting control regions (ICRs)
	2.2 MAINTENANCE OF ICRS IN THE EMBRYOS
	2.2.a Epigenetic asymmetry in the gametes
	2.2.b Epigenetic reprogramming of the early embryo
	2.2.c Protection of genomic imprints during pre-implantation development
	2.3 ESTABLISHMENT OF ICRS IN THE GERMLINE
	2.3.a Epigenetic reprogramming in the germline
	2.3.b Germline specification
	2.4 DIFFERENT TYPES OF GENOMIC IMPRINTING
	2.4.a Canonical imprinting
	2.4.

b Tissue-specific imprinting 2.4.c DNA methylation-independent transient imprinting 2.4.d DNA-methylation dependent transient imprinting

.4 Different types of genomic imprinting

	2
		KAP1
	STELLA	ZFP57 TGCCGC	P PROTECTION ROTECTION
			TF
	GENOMIC
	IMPRINTING
	DNMT3L DNMT3A
	ESTABLISHMENT STABLISHMENT
	DNMT3L DNMT3A		ERASURE RASURE TET
			57

  [START_REF] Greenberg | Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth[END_REF]. Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth, NatureGenetics 49: 110-118, doi:10.1038/ng.3718. * These authors equally contributed to this work.

	100/50/50 ng/uL
	Cas9 mRNA
	sgRNA 1	AAAAAAAA sgRNA 2	Embryo tranfer
		Cutting + NHEJ				CRISPR-KO 20/44	WT 24/44
	Zygote B6D2F1				45.4% CRISPR-generated deletions
	F1000Prime	recommendation,	15	Dec	2016	-
	F1000Prime.com/726971069#eval793526035

110 VOLUME 49 | NUMBER 1 | JANUARY 2017 NATURE GENETICS During early mammalian development, embryonic cells develop into a naive, pluripotent state before diversifying their functions. Dynamic and extensive changes of the transcriptional and chromatin landscape accompany these important fate transitions 1 . An outstanding question in developmental biology is whether such events, although transient in nature, can autonomously program patterns of gene expression and phenotypes later in life.

1,4 , Fatima El Marjou 2 , Marius Walter 1 , Aurélie Teissandier 1,3 & Déborah Bourc'his 1

Supplementary Table 1. List of oligonucleotides used in this study.

	RT-qPCR Primers
	Liz PreAmp
	Liz Exon 1
	Liz Exon 2-3
	Unspliced Liz
	Gpr1 Exon 2-3
	Zdbf2 Exon 1-3
	Rplp0
	Rrm2
	ß-Actin
	Hprt1
	ChIP Primers
	Intergenic CGI
	sDMR_A
	sDMR_B
	sDMR_C
	pZdbf2
	pOct4
	pßActin
	pPax5
	Liz Intron 2
	sDMR
	Zdbf2 Body
	ß-Actin Body
	Myglobin Body
	Sox9 Body
	pZdbf2 Pyrosequencing
	sDMR Pyrosequencing

  Does it have any homeostasis function apart from growth control? Interestingly, I reported a lethality phenotype in Zdbf2-LOF mice(Glaser et al., in preparation). This phenotype has been observed upon Zdbf2 downregulation (both in Zdbf2-KO and Liz-LOF mice) but not overexpression. The possibility that Zdbf2 input in the hypothalamus exerts essential function for post-natal survivability must be considered. The hypothalamus is notably involved in vital functions, such as post-natal thermogenesis through brown adipocytes tissues

Voir la Synthèse de N. Foray et al, m/s n° 4, avril 2013, page 397

  Ce travail collaboratif (LIMMS/CNRS-IIS UMI 2820) a été développé dans le cadre du projet SMMiL-E et a permis la conception et la réalisation des SNT.Les irradiations ont été conduites au centre de radiothérapie Oscar Lambret de Lille. Les études théoriques, comme le transfert de technologie SNT, ont été menées à l'IEMN UMR8520. The chemical basis of radiation biology.London : Taylor and Francis, 1987.modélisation met en évidence une corrélation entre les irradiations et la réduction de la rigidité de la fibre qui est cohérente. Cependant, elle ne permet pas, actuellement, de traduire les altérations mécaniques en une succession d'événements moléculaires, comme les cassures directes double brin, les cassures induites par des espèces oxydantes (générées dans la solution), ou la conformation de l'ADN.ConclusionCette étude[11] est la première observation en temps réel de la dégradation mécanique d'une fibre d'ADN sous rayonnement thérapeutique. Le protocole expérimental et la modélisation ouvrent la voie aux études fondamentales et cliniques des mécanismes de dégradation de l'ADN sous faisceaux ionisants en vue de l'amélioration des traitements des tumeurs par radiothérapie. Le faible coût et l'intégration à grande échelle des dispositifs MEMS à base de silicium, tels que le SNT permettent d'entrevoir des traitements de Épigénétique et reproduction chez les mammifères, Paris sciences et lettres, section recherche, CNRS, Inserm, Institut Curie, 26, rue d'Ulm, 75248 Paris Cedex 05, France. deborah.bourchis@curie.fr > Chez les mammifères, certains gènes font fi des règles mendéliennes et ne sont exprimés qu'en une seule « dose », à partir de l'allèle maternel ou paternel. Ce phénomène, connu sous le nom d'empreinte parentale, résulte de la très grande divergence des profils de méthylation transmis par l'ovocyte et le spermatozoïde [1, 2] (➜). (Figure 1A et B). L'empreinte gamétique maternelle, qui dépend de marques de méthylation héritées de l'ovocyte, a un rôle dominant sur le contrôle de la centaine de gènes soumis à empreinte (GSE) répertoriés chez la souris et chez l'homme. D'un point de vue fonctionnel, la dose mono-allélique des GSE est déterminante pour le développement in utero, notamment via les échanges entre la mère et le foetus, mais également après la naissance, pour le développement du cerveau et les fonctions métaboliques [4].

	radiothérapie spécifiques pour chaque
	patient [12] (➜).
	Une biopsie réali-
	sée chez le patient
	pourrait ainsi être
	mise en culture,
	l'ADN extrait placé dans la lyse cellulaire
	puis irradié afin d'évaluer l'efficacité
	potentielle de l'irradiation. ‡
	Measure of the biomechanical
	degradation of a DNA fiber under
	the influence of therapeutic X-rays
	REMERCIEMENTS
	fécondation, sauf pour les régions sou-
	mises à empreinte, qui ont la capa-
	cité exceptionnelle de conserver une
	mémoire parentale [3] L'empreinte parentale peut exister
		sous différentes formes
		Dans le cas de l'empreinte parentale
		classique, telle qu'on la connaît depuis
	Ces différences	sa découverte dans les années 1980 [5,
	sont générale-	6], les marques de méthylation diffé-
	ment effacées dans	rentielles parentales sont maintenues
	l'embryon après	invariablement tout au long de la vie

LIENS D'INTÉRÊT

Les auteurs déclarent n'avoir aucun lien d'intérêt concernant les données publiées dans cet article.

RÉFÉRENCES 1. Sonntag C. (➜)

Quatre petits jours pour définir la taille adulte

	Juliane Glaser, Deborah Bourc'his

NOUVELLE

(➜) Voir la Synthèse de C. Proudhon et D. Bourc'his, m/s n° 5, mai 2010, page 497

Programmation irréversible de la taille adulte dans l'embryon préimplantatoire

  Dans l'embryon, Liz orchestre le remplacement de marques polycomb (méthylation d'histones) par des marques de méthylation de l'ADN, ce qui est nécessaire à l'activation de Zdbf2. Mais quelle est la signification biologique d'un tel ballet épigénétique ? Bien qu'exprimé exclusivement dans l'embryon précoce, Liz n'a aucun effet sur celui-ci : les embryons dans lesquels Liz est muté sont viables, se développent normalement et ne présentent aucun défaut. De même, les souris adultes dérivées des embryons mutés pour Liz sont parfaitement viables et fertiles.

NOUVELLE La face cachée du poumon : une usine à plaquettes et une réserve de progéniteurs sanguins

  Nouvelles_Decembre.indd 1031 Nouvelles_Decembre.indd 1031 30/11/2017 12:04:23 30/11/2017 12:04:23 fortement exprimé dans l'axe hypothalamo-hypophysaire. En conclusion, Zdbf2 est un gène stimulateur de croissance post-natale dont l'expression est épigénétiquement programmée dès les premiers jours de développement. Compte-tenu de la conservation de l'empreinte transitoire du locus Zdbf2 chez l'homme, il est tentant de prévoir que des perturbations de ce locus dans les gamètes ou l'embryon, par des voies génétiques ou environnementales (techniques d'assistance médicale à la programmation ou régimes maternels sub-optimaux) pourraient avoir un impact sur la détermination de la taille dans les populations humaines. Smith ZD, Chan MM, Mikkelsen TS, et al. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 2012 ; 484 : 339-44. 2. Proudhon C, Bourc'his D. Évolution de l'empreinte parentale chez les mammifères : quelle ménagerie ! Med Sci (Paris) 2010 ; 26 : 497-503. Ces souris présentent cependant, dés la naissance, un défaut de croissance hautement pénétrant. Ce phénotype de petite taille concerne tous les organes. Il culmine à l'âge de deux semaines, avec une réduction d'environ 20 % du poids, et est maintenu chez la souris adulte [8]. L'expression éphémère de Liz dans l'embryon est donc nécessaire pour programmer à long terme le potentiel de croissance post-natal. Conclusions/Perspectives L'étude fonctionnelle du transcrit Liz, soumis à empreinte transitoire, fournit une des très rares démonstrations formelles de l'existence d'une programmation épigénétique précoce de la physiologie adulte. En l'absence de Liz dans l'embryon, Zdbf2 ne peut être activé dans le cerveau après la naissance et les souris présentent une forme de nanisme. Nos travaux en cours visent à tester si, à l'inverse, une double dose de Zdbf2 pourrait conduire à un phénotype de gigantisme. La fonction de la protéine ZDBF2 est inconnue, mais un rôle dans le contrôle endocrine de la croissance est suspecté car Zdbf2 est

			Université de Californie, San Francisco HSE
			1355A -513 parnassus ave, San Francisco,
	Emma Lefrançais, Mark Roberts Looney	CA-94143-0130, États-Unis.
			emma.lefrancais@ipbs.fr
	La circulation pulmonaire	milliards de plaquettes sont produites	par Wright, en 1906, a été rapidement
	est une usine à plaquettes	quotidiennement afin de maintenir un	admis [3], les questions du mécanisme
	Les plaquettes sont des cellules dépour-	niveau sanguin constant. Ces plaquettes	et du lieu de leur production sont res-
	vues de noyau qui circulent dans le	proviennent de larges cellules appelées	tées plus controversées. Les mégaca-
	sang et participent activement à la	mégacaryocytes, qui ont été décrites	ryocytes se développent dans la moelle
	coagulation, à l'intégrité vasculaire et	pour la première fois en 1890 par Howell	osseuse, et l'on suppose que la pro-
	à l'immunité. En moyenne, 150 à 400	[1, 2] (➜).	duction des plaquettes, aussi appelée
	milliards de plaquettes par litre de sang	Si le rôle du méga-	thrombopoïèse, s'y
	circulent chez l'homme (1 000 milliards	caryocyte dans la	produit également
	chez la souris). La durée de vie de ces	formation des pla-	chez l'adulte [4]
	cellules étant de quelques jours, cent	quettes proposé	(➜).

‡ Four little days to define adult height LIENS D'INTÉRÊT Les auteurs déclarent n'avoir aucun lien d'intérêt concernant les données publiées dans cet article. RÉFÉRENCES 1.

Dans sa globalité, mon travail de thèse met en lumière le rôle du locus soumis à empreinte parentale Liz/Zdbf2, de l'embryon précoce à la physiologie adulte. Notre découverte concernant le rôle de l'expression précoce et transitoire de Liz pour la programmation de la croissance adulte constitue la première preuve de l'importance de l'empreinte transitoire chez la souris. Ces résultats démontrent aussi une régulation précise de l'activation de Zdbf2 dans le cerveau après la naissance. En accord avec cette régulation complexe, j'ai pu caractériser de façon très détaillée le phénotype de croissance induit par une modulation de la dose de Zdbf2 dans l'axe hypothalamohypophysaire. Zdbf2 apparaît comme un gène contrôlant la croissance post-natale de façon dose-dépendante mais indépendamment de son origine parentale. La structure génique, l'empreinte ainsi que les sites d'expression de Zdbf2 sont conservés chez l'humain. Mes travaux de thèse peuvent donc conduire, à plus long terme, à l'étude de la protéine ZDBF2 chez l'humain qui pourrait potentiellement avoir un rôle similaire à celui identifié chez la souris dans le contrôle de la croissance post-natale.

size at birth but then completely stop growth at 2 weeks of age[START_REF] Fraichard | The T3R alpha gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production[END_REF].As a consequence, these mice do not exceed 6g of body mass and are not viable.Overall, the hypothalamo-pituitary-GH axis has been extensively described as a main regulator of post-natal growth in humans and rodents. To summarize, the action of GH on growth is mediated by the hepato-production of IGF1, which then plays a role on the whole body growth (Figure24).

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 93.3% 6.1% WT JxB sDMR, 3 weeks X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

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Les proté ines du groupe polycomb (PcG) assurent le dépôt de la mé thylation de l'histone H3(K27) sur les promoteurs inactifs.

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Acknowledgements

AUTHOR CONTRIBUTIONS

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

VOLUME 49 | NUMBER 1 | JANUARY 2017 NATURE GENETICS

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Supplementary Figure 1

Liz/Zdbf2 dynamics in vivo and in vitro.

(a) Schematic of Liz/Zdbf2 regulation during early development. Summary from Duffié et al. 18 . Liz transcript initiates from the unmethylated paternal gDMR in the preimplantation embryo. After implantation, Liz expression is turned off and the gDMR is methylated on both alleles. From this stage on, DNA methylation is present at the sDMR and Zdbf2 is expressed from its canonical promoter on the same allele from which Liz was previously expressed, i.e., the paternal allele. Nature Genetics: doi:10.1038/ng.3718

ABSTRACT

Genomic imprinting is a paradigm of epigenetic regulation, which results in the monoallelic and parent-of-origin expression of a subset of mammalian genes. This phenomenon has been long recognized as essential for embryogenesis and imprinted genes display a wide range of functions in development, behavior and physiology.

However, whether genomic imprinting has evolved as a gene dosage or a parental memory regulatory mechanism has not been clarified yet. Here, we describe the dosesensitive but parent-of-origin independent control of growth through Zdbf2. In mice and humans, Zdbf2 (DBF-type zinc finger-containing protein 2) is an imprinted gene whose mono-allelic paternal expression in the brain is epigenetically programmed during preimplantation development. Using mouse genetic mutants of loss-and gain-of function, we characterized the growth-promoting effect of Zdbf2 in the hypothalamo-pituitary axis. The dose of Zdbf2 is the key parameter: lacking Zdbf2 is deleterious and causes a growth reduction whereas an increased dose enhances growth. By generating a unique model of parental inversion in which Zdbf2 expression comes from the maternal allele, we demonstrated that the parental origin is not critical for growth control. This study provides an experimental in vivo example of the importance of imprinted gene dosage, but not parental memory, in the control of physiological functions in mice. Zdbf2-KO mice do not have functional Zdbf2 in the brain , which leads to a post-natal growth reduction phenotype. At the blastocyst stage, this mutation has no consequences on Liz transcription. Zero dose of Zdbf2 thus leads to a diminished growth. (C) Liz-GOF mice theoretically exhibit Liz bi-allelic expression during preimplantation embryogenesis. The adult mice show a bi-allelic expression of Zdbf2 in the brain and a male-specific overgrowth phenotype. Two doses of Zdbf2 thus leads to an increased growth. (D) By crossing the two Liz mutants we generated, we obtained mice with a parental inversion of the imprint at the locus, leading to mono-allelic maternal expression of Zdbf2 in the adult brain. These mice show a growth phenotype similar to WT mice, demonstrating the parent-of-origin independent control that the imprinted Zdbf2 gene exerts on post-natal growth. The Zdbf2-KO and Liz-GOF mutant mice lines were derived by CRISPR/Cas9

EXPERIMENTAL PROCEDURES

engineering in one-cell stage embryos, according to published protocols [START_REF] Wang | One-step generation of mice carrying mutations in multiple genes by CRISPR/cas-mediated genome engineering[END_REF]. Briefly, the Cas9 DNA was amplified by PCR from the pX330 plasmid and cloned into the T7 promoter-containing pCR2.1-XL plasmid. After linearization and phenol-chloroform purification, in vitro transcription (IVT) was performed using mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). The Cas9 mRNA was then purified with RNeasy Mini Kit (Qiagen). The two deletion-promoting sgRNAs were amplified by PCR with primers containing a 5' T7 promoter sequence. After gel purification, the T7-sgRNA PCR products were used as the template for IVT using MEGAshortscript T7 kit (Life Technologies) and then purified using MEGAclear kit (Life Technologies). The Cas9 mRNA and the sgRNAs were eluted in DEPC RNase-free water and their quality controlled by electrophoresis on a denaturing agarose gel.

Zygote injection of the CRISPR/Cas9 system was performed by the Transgenesis Platform of the Institut Curie. Eight week-old superovulated B6D2F1 (C57BL/6J X DBA2) females were mated to stud males of the same background. Cytoplasmic injection of Cas9 mRNA and sgRNAs (100 and 50 ng/ul, respectively) was performed in zygotes collected in M2 medium (Sigma) at E0.5, with well-recognized pronuclei.

Injected embryos were cultured in M16 medium (Sigma) at 37°C under 5% CO2, until transfer at the 1-cell stage the same day or at the 2-cell stage the following day in the infudibulum of the oviduct of pseudogestant CD1 females at E0.5. The flounder mice were then genotyped and two independent founders with the expected deletion were backcrossed to segregate out undesired genetic events, with a systematic breeding scheme of Zdbf2-KO heterozygous females with WT C557Bl6/J males and Liz-GOF heterozygous males with WT C557Bl6/J females to promote silent passing of the Total RNA was extracted using Trizol (Life Technologies). To generate cDNA, Trizolextracted total RNA was DNase-treated (Ambion), then reverse transcribed with SuperscriptIII (Life Technologies) primed with random hexamers. RT-qPCR was performed using the SYBR Green Master Mix on the Viia7 thermal cycling system (Applied Biosystem). Relative expression levels were normalized to the geometric mean of the Ct for housekeeping genes Rrm2 and ArpPO, with the ΔΔCt method.

Primers are listed in Supplementary Table 1. Statistical analyses were performed by a two-tailed unpaired t-test using GraphPad Prism6 software.

LacZ staining

Whole brains and pituitary glands were fixed in 4% paraformaldehyde (PFA) in PBS (pH 7.2) for overnight, washed in PBS, incubated in sucrose gradients and embedded in OCT for conservation at -80°C before cryosectioning and lacZ staining. LacZ staining was performed from frozen sections. Sections were first fixed 10 minutes in solution of Glutaraldehyde, then washed in wash solution (2mM MgCl2, 0.02% NP40, 0.01% C24H39NaO4 in PBS) and finally incubated at room temperature overnight in Xgal solution (5mM K4Fe(CN)63H20, 5mM K3Fe(CN)6, 25mg/mL X-gal in wash solution).

After several PBS washes, samples were mounted with an aqueous media and conserved at room temperature before imaging.

Histological analysis and RNA in situ hybridization

Embryos at E13.5 and E15.5 were embedded in paraffin and sectioned at a thickness of 5 μm. For histological analysis, paraffin sections were stained with Hematoxylin and Eosin. RNA in situ hybridization analysis on paraffin sections was performed following a standard procedure with digoxigenin-labeled antisense riboprobes as previously described [START_REF] Gaston-Massuet | Genetic interaction between the homeobox transcription factors HESX1 and SIX3 is required for normal pituitary development[END_REF]. The antisense riboprobes used in this study [Gh, Pomc1, LIM homeobox protein 3 (Lhx3) and Pit1] have been described.

Immuno-histochemistry on histological sections

Embryos were fixed in 4% (wt/vol) paraformaldehyde and processed for immunodetection as previously described [START_REF] Andoniadou | Sox2+stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential[END_REF]. Detection of hormones was carried out using antibodies for (α-ACTH) (10C-CR1096M1), α-TSH (NHPP AFP-1274789), α-LH (NHPP AFP-C697071P), and α-FSH (AFP-7798-1289) (Developmental Studies Hybridoma Bank) at a 1:1,000 dilution.

Statistical Analyses

Significance of obtained data was determined by performing two-tailed unpaired t-

DISCUSSION AND PERSPECTIVES

The Liz/Zdbf2 locus is an intriguing case of epigenetic and genetic regulation of growth in mice. The precise epigenetic setting orchestrated at the sDMR to activate Zdbf2 during embryogenesis suggested that this region may be a key player for the dosesensitive regulation of Zdbf2 in the hypothalamus and the pituitary gland. Moreover, the dose-response change in post-natal growth is consistent with an essential role of Zdbf2 in these two organs. Here, we reported that the deletion of the sDMR region, when maternally transmitted, results in an hypothalamus-specific increase of Zdbf2, and this does not impact post-natal growth. Conversely, mice displaying a decrease of Zdbf2 restricted to the pituitary gland are smaller than their WT and sDMR-KO littermates. While these data bring new clues in the complex investigation of ZDBF2 function, it also raises a number of additional questions.

In pluripotent embryonic cells, Zdbf2 is maintained in a repressive state through the occupancy of a block of H3K27me3 spreading from the sDMR to the Zdbf2 promoter, on the two parental alleles (Greenberg et al., 2017). From implantation and later in adult brain, the presence of DNA methylation on the paternal sDMR allele antagonizes polycomb proteins and allows the expression of Zdbf2. In the context of the absence of the sDMR region, in sDMR-MKO mice, we propose that the activation of Zdbf2 from the maternal copy in the hypothalamus is mediated through the lack of PRC2 binding. This will be tested by ChIP-qPCR from sDMR-MKO hypothalamus tissues in a next future. However, in the pituitary, the same 8.3-kb deletion of the sDMR is not sufficient to induce Zdbf2 maternal expression, suggesting that either PRC2

binding still occurs at the Zdbf2 promoter or that Zdbf2 repression is mediated through another mechanism in this tissue. Although the pituitary is a really small tissue, it contains enough cells to perform ChIP-qPCR and test for the presence of H3K27me3 in both WT and sDMR-KO animals. Alternatively, specific enhancers, localized outside of the sDMR deletion, could regulate the activity of Zdbf2 in the pituitary gland. To address this latter possibility, I tried to perform chromatin accessibility mapping by ATAC-seq in WT hypothalamus and pituitary gland but my results are so far inconclusive.

The second intriguing finding raised by these data is the absence of any growth outcome in sDMR-MKO mice. This result not only suggests a weak to nonexistent effect of the hypothalamic production of Zdbf2 in growth control but also raises the transcribed in the oocyte, it is very likely that the gDMR acquires DNA methylation as a consequence of Gpr1 transcriptional read-through. A deletion of the promoter of Gpr1, when inherited from the oocyte, should thus result in a DNA methylation-free gDMR and consequently, complete biallelic Liz expression in the post-fertilization embryo. Appropriately, Gpr1-KO mice were previously shown to be completely normal,

when not challenged by high fat diets [START_REF] Rourke | Gpr1 is an active chemerin receptor influencing glucose homeostasis in obese mice[END_REF]. I therefore recently generated ∆Gpr1 mice. Analyses of these mice are ongoing and will be instrumental for confirming the molecular and overgrowth phenotype we observed upon the previously generated, complex Liz-GOF model.

Dose-dependent regulation of growth through Zdbf2

Altogether, the Liz-GOF mutants allowed me to formally demonstrate the dosedependent regulation of growth through Zdbf2 expression. Upon a 1.6 fold increase of Zdbf2 dose, males exhibit a 10% overgrowth phenotype. Importantly, this very sensitive dose effect was confirmed in two other genetic models. First, we have obtained from the EUCOMM consortium a Zdbf2-LacZ reporter line, in which the transcription of Zdbf2 is only partially affected: while the complete Zdb2-LOF shows a maximum 15-20% growth reduction, these knock-in mice have only a 0.5 fold decrease of Zdbf2 expression in the hypothalamo-pituitary axis and consequently, show about 5-10% growth reduction.

The second consistent observation of the importance of Zdbf2 dose regulation came from the mice carrying an inversion of the parental origin of the imprint. These

Liz-LOF/Liz-GOF double mutants are the example of how beautiful mouse genetics can be. Instead of being paternally expressed, Zdbf2 is now maternally expressed in the hypothalamus and pituitary gland. Not only this enabled me to bring the last piece of evidence that the dose of Zdbf2 is instrumental in the control of post-natal body growth, but more importantly, it highlighted the parent-of-origin independent function of Zdbf2. This model of inversion of the parental origin is particularly relevant in the field of genomic imprinting and it would be interesting to apply this strategy at other imprinted loci. Although the parental origin of Zdbf2 expression does not matter for regulating growth, it is worth reminding that Zdbf2 is originally paternally expressed, which is of particular interest in regards to the parental conflict theory of genomic imprinting proposed by David Haig (Moore and Haig, 1991). Similarly to other imprinted Expression of imprinted genes in log(RPKM) and fold-change in the hypothalamus of WT and Liz-KO from males at 6 weeks. Importantly, the Zdbf2 gene is found as the most strongly downregulated gene in Liz-KO mice, as expected from the complete lack of activation of the canonical Zdbf2 promoter in these mutants. (D) Liz-GOF mice exhibit biallelic expression of Zdbf2 in both the hypothalamus and the pituitary gland and this results in a male-specific overgrowth phenotype.

Survivability phenotype

(E) When Zdbf2 is expressed at one dose, but from the maternal allele, in the hypothalamus and the pituitary gland, Liz-LOF/GOF mice have the same body weight as the wild-type. (F) sDMR-MKO mutants display an hypothalamus-specific biallelic expression of Zdbf2 and this is associated with normal body weight. (G) When crossing males Liz-LOF with females sDMR-MKO, the progeny should express Zdbf2 exclusively in the hypothalamus and from the maternal allele only , while pituitary Zdbf2 expression should be defective. This pattern of Zdbf2 expression results in a growth reduction similar to the one observed in Liz-LOF mutants.

ANNEXE