families. This experience would have been lonely without all of you. I've loved our adventures and will treasure them always. Cristi, mi Garbanza, you have been another light that has infused some sunshine into my life. I have so much fun with you, and I admire your sense of adventure. Carys, I have probably leaned on you most of all, and I am so grateful to you for everything. Friend, roommate, lockdown buddy, therapist, translator, and fantastic shopping companion -you've worn many hats, my dear. Thank you for being all of those things.

David, my partner, if ever there was someone who embodied the word "steadfast" it would be you. Your support of my aspirations has never wavered, even though it meant that we have had to be apart, and your resolute belief that I can do anything I set my mind to has bolstered my own confidence on numerous occasions. I love you, and I am beyond

patience, kindness, and a diplomacy that I aspire to, and I feel lucky to have had you as my PI.

To my fellow lab mates, past and present, I have immensely enjoyed getting to know you all (Djamila, Chongfa, Yongzheng, Laure, Benoit, Sébastien, Daniel, Stéph, Béa, and all of the master's students that have come through the lab). The environment of our lab has always felt friendly, and every member of the lab has always been quick to lend a hand. This atmosphere fostered by all of you made it easy to come to work every day. I must especially thank Stéphanie, Béatrice, and Laure for their help these last months while I was writing.

I must also thank those friends of mine that I refer to as "my army of post-docs" which include Michael, Matthew, Laura, and Daniel. I'm so thankful to have had you all to talk with, whether it was to commiserate or troubleshoot. I've definitely leaned on all of you when I needed some wisdom or encouragement, and it's meant a lot to me that you've all been so quick to give both. Mike, I'm glad you decided to induct me into the happy hour crew. It helped keep me sane, and it's been nice to feel like I have another network of buddies that I could turn to when I was struggling. I'm so happy for you and Laura and your growing family.

Matt, you are one of the most encouraging people I've encountered. You brought a lightness to the whole floor when you came, and I'm excited for you and your next chapter as well. I hope to see you when you make it State-side! Daniel, my grouchy but loveable Bubba, it's been so fun to share an office with you. I've learned a lot from the G.O.A.T. and will miss gossiping with you every day. I'm also excited for your next chapter at the shaker, and I hope all of your entrepreneurial dreams are realized.

The Fam (or Family) has also been essential for my life here. Carys, Cristi, Alex, Sergey, and André, I'm so glad to have found you all and made our own little family here when we were all so far from our own

Table of Contents

Table of Figures

ABSTRACT

Chlamydia trachomatis is an obligate intracellular bacterium that proliferates exclusively in human hosts.

Its genome has undergone intensive gene reduction and is missing many genes coding for essential metabolic pathways. As a consequence, the bacteria rely on their host cell to obtain several nutrients. Bacterial sequestration of metabolites shifts host cell metabolism which may impact many cellular processes. In this thesis, we sought to understand if a metabolic imbalance could account for epigenetic modifications in the host, and to understand its consequences.

During late C. trachomatis infection, we observed increased histone methylation at multiple histone lysine residues, despite the antagonistic nature of some of these modifications. Importantly, the level of hypermethylation was cell-dependent and all the histone marks tested were expressed at similar level for every individual cell, indicating co-regulation. We hypothesized that a decrease in the activity of histone demethylases, the enzymes responsible for removing methyl groups from the N-terminal tails of histones, might account for this hypermethylation phenotype. A large family of demethylases, the Jumonji domaincontaining (JmjC) demethylases, require oxygen, iron and -ketoglutarate as co-factors to carry out their function, all metabolites that are used by C. trachomatis. In support of our hypothesis, we observed that infected cells were sensitized to inhibition of this family of histone demethylases, compared to non-infected cells.

Our experiments suggest that iron availability is not limiting during infection. In contrast, supplying cell permeable dimethyl-ketoglutarate (DMKG) in the culture medium prevented hypermethylation of histones during late infection. This result indicates that histone hypermethylation is a consequence of a metabolic imbalance, likely caused by -ketoglutarate consumption, or its precursors, during infection. At the transcriptional level, we observed that DMKG supplementation affected about one third of the genes that are differentially expressed late in infection. DMKG supplementation resulted in gene up-regulation and in gene down-regulation in similar proportion, indicating that histone hypermethylation can have opposite consequences on gene expression, that need to be analyzed at the individual gene level. Finally, histone hypermethylation correlated with the presence of indicators of DNA damage in the nuclei of infected cells.

DMKG supplementation prevented DNA damage, confirming a link between these two phenotypes.

To conclude, we demonstrated that C. trachomatis infection generates a metabolic imbalance in the host, causing histone hypermethylation late in infection. This metabolic imprint of infection at the epigenetic level has an impact on the host transcriptomic response to infection and on the integrity of its DNA.

RÉSUMÉ

Chlamydia trachomatis est une bactérie intracellulaire obligatoire adaptée exclusivement à l'Homme.

Son génome, très réduit, a perdu plusieurs gènes codant pour des voies métaboliques essentielles. En conséquence, les bactéries dépendent de l'hôte pour obtenir plusieurs nutriments. La séquestration de métabolites par les bactéries crée des déséquilibres, avec des conséquences potentielles sur plusieurs processus cellulaires de l'hôte. Dans cette thèse nous avons entrepris de déterminer si un déséquilibre métabolique pourrait être à l'origine de modifications de l'épigénome de l'hôte, et de comprendre ses conséquences.

Nous avons observé que l'infection s'accompagne, tardivement, d'une augmentation de marques de méthylation sur plusieurs lysines, et ce en dépit du caractère antagoniste de certaines de ces marques. Le niveau de méthylation dépend des cellules, et pour une cellule donnée, toutes les marques testées sont exprimées à des niveaux similaires, indiquant un phénomène de co-régulation. Nous avons émis l'hypothèse qu'il puisse découler de l'inhibition d'enzymes à activité déméthylase. La grande famille des déméthylases Les Chlamydiacae ont perdu de nombreux gènes codant pour des fonctions métaboliques essentielles. C'est un trait commun chez les bactéries intracellulaires, et poussé à l'extrême chez intracellulaires obligatoire telles que les Chlamydiacae qui ne comptent plus qu'un petit millier de gènes. Le corolaire de cette situation est que les bactéries dépendent de l'hôte pour combler les « trous » dans des voies métaboliques essentielles, telles que la synthèse de nucléotides, ou de certains acide aminés. Les bactéries ont acquis un transporteur d'ATP, qui leur permet d'utiliser les ressources énergétiques de l'hôte. La croissance de l'inclusion nécessite elle un apport constant de lipides de l'hôte. Pour faire face à ces besoins, ainsi que pour contrecarrer les défenses innées que possèdent les cellules épithéliales pour éliminer les microbes qui les agressent, les bactéries utilisent des protéines qu'elles sécrètent dans l'inclusion ou dans le cytoplasme, appelées protéines Nous avons ensuite démontré que l'hyperméthylation des histones était liée à un changement métabolique de l'hôte sous l'effet de l'infection. Les cellules infectées sont sensibilisées à l'action d'un inhibiteur des déméthylases de la famille JmjC. L'activité de ces enzymes est dépendante de l'oxygène, du fer et de l'-ketoglutarate (KG), un métabolite engagé dans plusieurs voies, en particulier le cycle de Krebs dans les mitochondries. Nos résultats suggèrent que la concentration en fer dans les cellules infectées reste suffisante pour assurer l'activité des déméthylases de la famille JmjC. En revanche, l'KG semble limitant puisque l'addition d'un analogue qui traverse les membranes, le dimethyl-KG (DMKG), dans le milieu de culture, prévient l'hyperméthylation des histones. De façon surprenante nous avons observé que l'inhibition de l'enzyme qui convertit l'KG en succinyl-coA dans le cycle de Krebs, l'oxoglutarate dehydrogénase (OGDH) n'empèche pas l'hyperméthylation des histones, indiquant qu'elle ne permet pas de rétablir la concentration normale d'KG dans le cytoplasme et le noyau.

RÉSUMÉ DÉTAILLÉ

Ce résultat indique que l'acteur principal de la déplétion d'KG dans les cellules infectées est la consommation de ce métabolite, ou de ses précurseurs, par les bactéries. Plusieurs données de la littérature, et en particulier la consommation de glutamine, précurseur d'KG, par les bactéries, soutiennent cette hypothèse.

Nous avons ensuite caractérisé les conséquences de l'addition de DMKG dans le milieu de culture sur la réponse transcriptionnelle de l'hôte à l'infection. Chlamydia are intracellular bacteria that have long been shrouded in mystery. The name originally proposed for this pathogen, Chlamydozoa, is derived from the Greek word "Chlamys/khlamus" meaning "mantle" or "cloak" and referenced the mechanism in which the bacteria appear to hide themselves within an intracellular vacuole, that we now know as the inclusion [START_REF] Nunes | Evolution, phylogeny, and molecular epidemiology of Chlamydia[END_REF]. Descriptions of trachoma, the blinding disease caused by the ocular strains of Chlamydia trachomatis, have been described as early as 1550 BC in the Ebers Papyrus. Such archaic evidence of pathogenic Chlamydiae demonstrates its close link with early humans and suggests a coevolution of the two [START_REF] Bryan | Ancient Egyptian Medicine The Papyrus Ebers: Translated from the German Version[END_REF][START_REF] Clarke | Evolution of Chlamydia trachomatis[END_REF]. For such an ancient pathogen, it has not been well described until more recently. In fact, Chlamydiae were classified, incorrectly, as viruses until the 1960s due to their ability to pass through mesh filters, which were classically considered to retain bacteria, as well as their inability to grow outside of a host cell. It was characteristics such as: their replication by binary fission, their Gram-negative stain, their susceptibility to antibiotics, and the presence of both DNA and RNA in a membrane-less nucleoid that helped correctly categorize these pathogens as bacteria [START_REF] Moulder | The relation of the psittacosis group (Chlamydiae) to bacteria and viruses[END_REF].

Chlamydiae is a phylum united by obligate intracellular bacteria that undergo a unique biphasic developmental cycle. The phylum has been found to be genetically distinct and isolated from other free-living bacteria. Genomic analysis did not uncover any transposon homologs, bacteriophages, or any transformation or competence-related proteins similar to other bacteria. This finding, or lack thereof, supports the theory that the modern Chlamydiae did not undergo frequent genetic exchange with other free-living bacteria [START_REF] Stephens | Genome Sequence of an Obligate Intracellular Pathogen of Humans: Chlamydia trachomatis[END_REF]. Based on 16S rRNA-based phylogenetic analysis, the phylum, Chlamydiae, emerged about 2 billion years ago, with presentday pathogenic Chlamydiae having diverged about 700 million years ago [START_REF] Horn | Illuminating the evolutionary history of chlamydiae[END_REF]. The phylum counts a single class, Chlamydiia, which, in turn, houses the single order of Chlamydiales.

Variety arises at the family level, since the Chlamydiales order counted 8 families in 2013, and this number is increasing with the metagenomic data obtained from samples collected in various environments [START_REF] Fehr | Candidatus Syngnamydia venezia, a novel member of the phylum Chlamydiae from the broad nosed pipefish, Syngnathus typhle[END_REF][START_REF] Dharamshi | Marine Sediments Illuminate Chlamydiae Diversity and Evolution[END_REF]. Genomic sequence analysis of pathogenic Chlamydia and one of its Parachlamydia cousins, within the Chlamydiales order, revealed a high percentage of sequence similarity, despite a greater than two-fold difference in genome size. Many of the genes related to metabolism, invasion, and virulence have been shown to remain intact in both strains, suggesting that their last common ancestor, 700 million years ago, was already adapted for intracellular life [START_REF] Horn | Illuminating the evolutionary history of chlamydiae[END_REF]. The Chlamydiaceae family is housed within the order, Chlamydiales, and contains a single genus, Chlamydia. Whether the genus should be split into two, Chlamydia and Chlamydophila, was proposed based on 16S and 23S rRNA phylogenetic analysis (Everrett et al, 1999). However, the matter has since been hotly debated as the aforementioned study did not account for polymorphisms due to geographic isolation of the strains and ignored the otherwise high levels of gene homology and synteny across the strains. The community decided to come back to the use of the single genus, Chlamydia [START_REF] Stephens | Divergence without difference: Phylogenetics and taxonomy of Chlamydia resolved[END_REF]. There are sixteen species within the Chlamydia genus: C. pneumoniae, C. pecorum, C. suis, C. abortus, C. felis, C. caviae, C. psittaci, C. avium, C. ibidis, C. gallinacea C. muridarum, C. serpentis, C. poikilothermis, C.corallus, C. sanzinia, and the focus of this dissertation, C. trachomatis (Fig 1A) [START_REF] Phillips | Seventy Years of Chlamydia Vaccine Research -Limitations of the Past and Directions for the Future[END_REF]. Three of these species, C. psittaci, C. abortus, and C. felis, can be transmitted to humans zoonotically. C. pneumoniae and C. trachomatis are human pathogens that infect the lungs, and the eyes or urogenital tract respectively. Unlike C. trachomatis, C. pneumoniae infects a number of other animals, including amphibians and marsupials.

Chlamydia trachomatis consists of 15 serovars in which there is a high degree of identity and synteny across the genomes (Fig 1B). In addition to the ~10 6 bp circular chromosome, the Chlamydia trachomatis genome also contains a ~ 7.5 kb plasmid [START_REF] Stephens | Genome Sequence of an Obligate Intracellular Pathogen of Humans: Chlamydia trachomatis[END_REF]. It has been suggested that the plasmid plays a role in the virulence and tropism of a given strain. For example, the ocular strains of C. trachomatis (A-C) have a lower level of expression of the plasmid, than urogenital serovars (D-K, LGV L1 and L2) [START_REF] Ferreira | Assessment of the load and transcriptional dynamics of Chlamydia trachomatis plasmid according to strains' tissue tropism[END_REF]. Differential expression of various effectors, polymorphic membrane proteins (PMPs), and Inc (inclusion) proteins also play roles in virulence and tropism amongst the serovars, which we will now discuss in more detail [START_REF] Nunes | Evolution, phylogeny, and molecular epidemiology of Chlamydia[END_REF]. Treatments consists of oral azithromycin, (20 mg/kg up to 1g), given as a single dose, or a 6-week course of topical tetracycline. However, surgery is required to treat trichiasis and correct the inversion of the eyelashes (Taylor et al, 2014).

Multiple studies have shown that acquired immunity to reinfection is weak, short-lived, and often serovar specific, which makes vaccine development especially tricky [START_REF] Hu | Trachoma: protective and pathogenic ocular immune responses to Chlamydia trachomatis[END_REF].

Trachoma is classified, by the WHO, as a neglected tropical disease. To that end, in 1997 a resolution by the World Health Assembly, the governing body of the WHO, was established for the Global Alliance for the Elimination of Blinding Trachoma by the year 2020 (GET 2020). Public health initiatives using the "SAFE" strategy were implemented to help reduce the transmission of trachoma. SAFE stands for Surgery, Antibiotics, Facial cleanliness, and Environmental improvement. It involved offering surgery to individuals with trachomatous trichiasis to correct the inversion of the eyelid, and antibiotics to those with active trachoma. Facial cleanliness campaigns have been implemented to stress the importance of hygiene in the context of trachoma, as well as campaigns to ameliorate environmental factors that contribute to exposure to eye-seeking flies, such as improved water sources and waste management. In June of 2012, the Global Trachoma Mapping Programme was launched to map the global incidence of endemic trachoma using a population-based survey, as only about half of the at-risk areas were being covered by international trachoma control activities at the time (WHO). Despite the great strides made by both programs to eliminate trachoma, it is still endemic in 44 countries [START_REF] Burkhi | Trachoma: great gains, but elimination target missed[END_REF].

1.2.B Urogenital Serovars (D-K)

Urogenital infections of Chlamydia trachomatis, serovars D-K, are the leading bacterial cause of sexually transmitted infection worldwide. In 2016, there were 127 million new cases of urogenital Chlamydia infections globally, mostly in men and women ages . Serovars E and F cause up approximately 50 percent of all chlamydial urogenital infections, while serovars G, D, and J are more prevalent in rectal infections [START_REF] Nunes | Evolution, phylogeny, and molecular epidemiology of Chlamydia[END_REF].

Treatment for serovars D-K is usually a single dose of azithromycin, (1g), or 100 mg of doxycycline twice daily for two weeks. As most infections are asymptomatic, the current recommendations are for regular sexual health screenings to test for infection in tandem with partner alert systems, to help drive down the incidences of infection. There have been no cases antibiotic-resistance to Chlamydia infection, however recurrence is quite common, so posttreatment screenings are also recommended [START_REF] Workowski | Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines[END_REF].

Women with repeated C. trachomatis infections have been associated with having a higher risk of developing ovarian and cervical cancers [START_REF] Zhu | Chlamydia Trachomatis Infection-Associated Risk of Cervical Cancer: A Meta-Analysis[END_REF]. More studies have been conducted to determine by what mechanism infection may lead to cancer. Chlamydia-infected cells often display phenotypes associated with genomic instability, which makes them more vulnerable to becoming cancerous. Phenotypes include: multinucleation, dysregulation of centrosomes and spindle poles, and defects in chromosomal segregation [START_REF] Grieshaber | Chlamydia trachomatis causes centrosomal defects resulting in chromosomal segregation abnormalities[END_REF][START_REF] Johnson | Centrosome abnormalities during a Chlamydia trachomatis infection are caused by dysregulation of the normal duplication pathway[END_REF]Andreaolas et al, 2011). Further studies have shown that, while DNA damage is induced, DNA repair mechanisms are suppressed, which is also leads to genomic instability [START_REF] Chumduri | Chlamydia infection promotes host DNA damage and proliferation but impairs the DNA damage response[END_REF].

1.2.C LGV invasive Serovars (LGV L1-L3)

LGV serovars are endemic to subtropical regions of Asia, Africa, South America, or the Caribbean [START_REF] Herring | Lymphogranuloma venereum[END_REF]. However, there has been a rise in the incidence of LGV infections in North America and Europe in the population of men who have sex with men (MSM) (de Vreize et al,

2014).

LGV serovars (L1, L2, L2a, and L3) are much more invasive and cause lymphoma granuloma venereum. Infection often first presents with an ulcer at the site of inoculation, followed later by swelling at the site and a fever. As the bacteria invade the lymphatic system, more serious symptoms like bubo formation, fistulae, fibrosis, and rectal stenosis arise [START_REF] Herring | Lymphogranuloma venereum[END_REF]de Vreize et al, 2014). These serovars are usually treated by 100 mg of doxycycline administered twice daily for 3 weeks [START_REF] Workowski | Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines[END_REF]. Tissue damage from the lymphatic invasion can In the event of metabolic stress, RBs may convert to a persistent state, termed intermediate bodies (IBs), where the bacteria do not continue dividing, nor do they convert to EBs. This persistent state is reversed by removal of the stress stimuli, and then the IBs convert back to RBs, which in turn continue development until new EBs are ready to exit and infect new cells. Throughout development, the bacteria hijack and modulate host cell processes to their advantage, often through the secretion of effector proteins via a type three secretion system [START_REF] Abdelrahman | The chlamydial developmental cycle[END_REF][START_REF] Elwell | Chlamydia cell biology and pathogenesis[END_REF].

1.3.B Knock-knock -May I come in? (Entry)

Multiple mechanisms by which Chlamydiae may bind to a host cell have been uncovered over the years. While so many different interactions may seem to muddy the waters, they support the idea that chlamydial replication cannot take place extracellularly, and thus gaining entry into a host cell is a requirement. Utilizing a variety of mechanisms ensures that the bacteria can carry out their biphasic development, but also may play a role in tissue tropism [START_REF] Hegemann | Intracellular Pathogens 1: Chlamydiales[END_REF][START_REF] Elwell | Chlamydia cell biology and pathogenesis[END_REF].

The host proteins largely implicated in chlamydial entry are heparan sulfate proteoglycans (HSPGs). HSPGs are integral membrane proteins that are found in great numbers on host-cell membranes, and their extracellular domain consists of a negatively charged glycosaminoglycan (GAG). Though there has been variation between species and serovars, it has been shown that pretreatment of EBs with HS or heparin reduce their infectivity, indicating that there is a GAGspecific receptor on the EB surface that is necessary for attachment and subsequent invasion [START_REF] Zhang | Mechanism of C. trachomatis attachment to eukaryotic host cells[END_REF].

While the first interaction of EBs with the host cells is believed to be reversible electrostatic interactions with HSPGs, the second step is believed to be an irreversible binding stage with other host proteins, of which many have been implicated [START_REF] Dautry-Varsat | Recent insights into the mechanisms of Chlamydia entry[END_REF]. Among the host proteins identified, cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to bind chlamydial LPS, and CTFR KO mice are less susceptible to infection (Anjonuma et al, 2010). Platelet derived growth factor receptor β has also been implicated as a potential receptor for chlamydial adhesion, and a potential bridge that could act with HSPGs [START_REF] Elwell | RNA interference screen identifies Abl kinase and PDGFR signaling in Chlamydia trachomatis entry[END_REF].

Protein disulfide isomerase (PDI) has been shown to be important for adhesion and entry.

Interestingly, EB adhesion via PDI does not require the protein to be catalytically active, while entry does [START_REF] Abromaitis | Attachment and entry of Chlamydia have distinct requirements for host protein disulfide isomerase[END_REF]. Receptor tyrosine kinases, such as EphrinA2 receptor, have also been shown to be important for binding, invasion, and subsequent downstream signaling [START_REF] Subbarayal | EphrinA2 receptor (EphA2) is an invasion and intracellular signaling receptor for Chlamydia trachomatis[END_REF]. While the identification of these host proteins that bind EBs have been exciting additions to the understanding of Chlamydia adherence, for most of them a bacterial binding partner has yet to be determined.

There are several chlamydial proteins that are associated with adhesion. Proteins of the outer membrane complex, such as MOMP, OmcA, and OmcB have been implicated in chlamydial EB attachment. These proteins create a rigid structure via disulfide bonds and are important for maintaining structural integrity and osmotic stability of the EB [START_REF] Hegemann | Intracellular Pathogens 1: Chlamydiales[END_REF].

It has shown by electron microscopy that MOMP aggregates on epithelial cell surfaces. It was then determined that MOMP binding is inhibited when cells are pretreated with heparinidase, which indicated that heparan sulfate proteoglycans (HSPGs) are important for Chlamydia adhesion by MOMP [START_REF] Su | A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells[END_REF].

OmcB was also shown to be capable of binding human epithelial cells via HSPGs. OmcBexpressing yeast were pre incubated with soluble GAG derivatives and found that adhesion was diminished in OmcB-expressing yeast that had been pre-incubated with heparin. Heparin is an analog of heparan sulphate, thus this set of experiments determined that OmcB is not only capable of binding human cells, but that it binds via HS-like structures on host-cell surfaces. Experiments similar to those described above for the pmps, in which host cells were pre-incubated recombinant OmcB proteins from different Chlamydia species, and the OmcB from opposite strains had no effect on the infectivity of one another [START_REF] Moelleken | The Chlamydia outer membrane protein OmcB is required for adhesion and exhibits biovar-specific differences in glycosaminoglycan binding[END_REF].

Polymorphic membrane proteins (Pmps) are a family of chlamydial proteins that have also been implicated in adhesion. They represent 3-5% of the chlamydial genomes, which indicates that they must have an important functional role. Interestingly, the members of the pmp gene family show little similarity to one another, which points to roles in virulence and tropism [START_REF] Stephens | Genome Sequence of an Obligate Intracellular Pathogen of Humans: Chlamydia trachomatis[END_REF]. While a specific host binding partner has yet to have been identified, Pmp6, Pmp20, and Pmp21 from C. pneumoniae have been shown to encode adhesion domains and are capable of binding to human epithelial cells. Furthermore, pretreatment of cells with recombinant pmps has been shown to reduce infectivity, thus indicating that pmps are important for entry (Moelleken et al, 2010). It was later shown that pre-incubating host cells with recombinant pmps of C. trachomatis, also reduced infectivity of C. trachomatis, but did not affect infectivity of C. pneumoniae, and vice versa. This indicated that pmps have species-specific functions that are likely important for tropism. [START_REF] Becker | All subtypes of the Pmp adhesin family are implicated in chlamydial virulence and show species-specific function[END_REF].

1.3.C Teamwork makes the dream work (Entry with the help of T3SS effectors)

The entry of C. trachomatis into its host cell requires the cooperation or co-opting of host proteins classically implicated in endocytic events. The bacteria accomplish this through the secretion of bacterial proteins, termed effectors, that recruit and modulate the activity of host proteins. To that end, C. trachomatis employs the use of a type three secretion system (T3SS), commonly found in many pathogenic Gram-negative bacteria. The chlamydial T3SS is responsible for translocating bacterial effectors from the bacterial cytosol into the host cytosol. It is a needled-like structure composed of a basal core, which links the inner and outer bacterial membranes, the needle complex, which protrudes from the outer membrane of the bacteria, and the translocon, which inserts into the host membrane upon contact (Fig 6).

Initiation of injection occurs upon contact of the needle-tip complex with the host membrane [START_REF] Mueller | New frontiers in type III secretion biology: the Chlamydia perspective[END_REF][START_REF] Nans | Structure of a bacterial type III secretion system in contact with a host membrane in situ[END_REF].

The chlamydial T3SS is utilized throughout its development in both EBs and RBs. Pre-made effectors are injected upon the EB's contact with the host membrane to help modulate the host cytoskeleton, dampen the immune response, subvert vesicular trafficking and block fusion of the new inclusion with the phagolysosomeacidic compartments [START_REF] Bugalhão | The multiple functions of the numerous Chlamydia trachomatis secreted proteins: the tip of the iceberg[END_REF]. De novo synthesis of effectors in RBs is used to block fusion of the inclusion with endosomal compartments, modify further the inclusion membrane, promote immune evasion, inhibit cell death, acquire nutrients, as well as to make effectors ready for the next round of infection [START_REF] Mueller | New frontiers in type III secretion biology: the Chlamydia perspective[END_REF][START_REF] Bugalhão | The multiple functions of the numerous Chlamydia trachomatis secreted proteins: the tip of the iceberg[END_REF]. Overall, there are 80-100 of effectors secreted by Chlamydia trachomatis' T3SS, many of which have yet to be characterized.

One well studied example of an early effector is TarP (translocated actin-recruiting phosphoprotein). TarP is secreted upon the EB binding to the host cell and its interactions with host ARP2/3 and the GTPase Rac, modulate actin polymerization resulting in encapsulation of the EB [START_REF] Carabeo | Chlamydia trachomatis induces remodeling of the actin cytoskeleton during attachment and entry into HeLa cells[END_REF][START_REF] Carabeo | Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells[END_REF][START_REF] Lane | Chlamydial entry involves TARP binding of guanine nucleotide exchange factors[END_REF][START_REF] Jiwani | Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments[END_REF][START_REF] Cossé | One Face of Chlamydia trachomatis: The Infectious Elementary Body[END_REF]. It was recently shown to work in concert with a second effector targeting the actin cytoskeleton, TmeA. [START_REF] Keb | Chlamydia trachomatis TmeA Directly Activates N-WASP To Promote Actin Polymerization and Functions Synergistically with TarP during Invasion[END_REF]. We will mention a few other effectors as we move through the description of the developmental cycle.

1.3.D I think I'll make myself at home (Formation of the Inclusion Vacuole)

The EBs are initially engulfed in a plasma membrane-derived vesicle, termed the nascent inclusion. Nascent inclusions appear largely separated from endo-lysosomal traffic, and do not fuse with late endocytic compartments [START_REF] Scidmore | Restricted fusion of Chlamydia trachomatis vesicles with endocytic compartments during the initial stages of infection[END_REF]. Within a few hours they shuttle towards the microtubule organization center (MTOC) propelled by the microtubule motor protein, dynein.

The developmental regulation of infectious EBs to replicative RBs, coincides with a timed cascade of temporally regulated genes. The transition from EB to RB marks several differences in the bacterial forms. The chromatin of the nucleoid becomes decondensed from histone-like proteins, Hc1 and Hc2, as the bacteria becomes transcriptionally active [START_REF] Barry | Nucleoid condensation in Escherichia coli that express a chlamydial histone homolog[END_REF][START_REF] Brickman | Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity[END_REF]. There is also a reduction in disulfide bonds that crosslink the outer membrane proteins on the exterior of the bacteria [START_REF] Hackstadt | Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae?[END_REF][START_REF] Hatch | Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis[END_REF].

Among the early genes expressed by C. trachomatis (first detected ~1-2 hpi) are genes coding for proteins translocated into the inclusion membrane by the T3SS machinery, called inclusion proteins (Incs) (Belland et al, 2003). These proteins decorate the cytosolic surface of the inclusion and control its interactions with cellular compartments. For instance, several Inc proteins have been shown to interact RAB GTPases and SNARES proteins, two large families of proteins that play prominent roles in the regulation of membrane fusion [START_REF] Elwell | Chlamydia cell biology and pathogenesis[END_REF][START_REF] Hackstadt | Intracellular Pathogens 1: Chlamydiales[END_REF]. Soluble effector proteins are also implicated in redirecting host traffic to the inclusion, as recently illustrated by the role of TaiP in redirecting Rab6 positive vesicles towards the inclusion [START_REF] Hamaoui | The Chlamydia effector CT622/TaiP targets a nonautophagy related function of ATG16L1[END_REF].

As RBs replicate, with a doubling rate of ~2 h, the inclusion grows rapidly in size and by 24 hpi, its volume is usually bigger than that of the nucleus [START_REF] Shaw | Three temporal classes of gene expression during the Chlamydia trachomatis developmental cycle[END_REF]. It continues to grow, although at a slower rate, for another 24 h [START_REF] Lee | Replication-dependent size reduction precedes differentiation in Chlamydia trachomatis[END_REF]. The inclusion is supported and stabilized by an "cage" of cytoskeletal components composed of actin and septins, which are further stabilized by microtubules and intermediate filaments [START_REF] Al-Zeer | Chlamydia trachomatis remodels stable microtubules to coordinate Golgi stack recruitment to the chlamydial inclusion surface[END_REF][START_REF] Kumar | Actin and intermediate filaments stabilize the Chlamydia trachomatis vacuole by forming dynamic structural scaffolds[END_REF][START_REF] Zuck | Extrusions are phagocytosed and promote Chlamydia survival within macrophages[END_REF][START_REF] Tarbet | Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton[END_REF].

1.3.D The more the merrier -EBs to RBs and back again

The RBs asynchronously begin converting to EBs between 24-72 hpi [START_REF] Hackstadt | Intracellular Pathogens 1: Chlamydiales[END_REF][START_REF] Elwell | Chlamydia cell biology and pathogenesis[END_REF]. The trigger for this conversion remains a mystery, but it has recently been hypothesized that it could be size-dependent. Quantitative analysis of serial block-face scanning scavenged from the host. Below we will discuss some of the major metabolites and pathways that are exploited by C. trachomatis.

Glucose and Glycogen

C. trachomatis requires glucose from its host for energy and the biosynthesis of various metabolites. It's been shown that Chlamydia-infected cells have increased glucose uptake, and host glucose transporters GLUT1 and GLUT3 have been shown to be upregulated during infection demonstrating that C. trachomatis infection rewires host glucose metabolism [START_REF] Wang | Orchestration of the mammalian host cell glucose transporter proteins-1 and 3 by Chlamydia contributes to intracellular growth and infectivity[END_REF][START_REF] Maffei | Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells[END_REF]. The C. trachomatis genome is missing hexokinase-2, the enzyme necessary to convert glucose to glucose-6-phosphate (G6P), so it must import directly G6P from its host through chlamydial protein, UhpC [START_REF] Gehre | Sequestration of host metabolism by an intracellular pathogen[END_REF]. From there, it may be used to create glycogen, a glucose storage molecule, or as a substrate for other biosynthetic pathways.

Gehre et al., also demonstrated that host UDP-glucose, a building block of glycogen, could be transported into the inclusion lumen via the exploitation SLC35D2, a host transporter that is normally found on the ER and Golgi. Once in the inclusion lumen, chlamydial effector proteins GlgA and GlgB use it to synthesize glycogen for glucose storage [START_REF] Gehre | Sequestration of host metabolism by an intracellular pathogen[END_REF].

TCA cycle and its intermediates

The Tricarboxylic Acid cycle (TCA) is coupled with oxidative phosphorylation to generate energy in the host cell. The TCA cycle of C. trachomatis is missing three key enzymes, citrate synthetase, aconitase, and isocitrate dehydrogenase (Fig 9) [START_REF] Stephens | Genome Sequence of an Obligate Intracellular Pathogen of Humans: Chlamydia trachomatis[END_REF]. This suggests that C. trachomatis obtain certain TCA cycle intermediates from the host. It's been shown that bacteria can grow when the culture medium contained 20 mM -ketoglutarate, oxaloacetate, or malate as their sole carbon source, although the progeny was reduced by 100-fold compared what was obtained with 1 mg/ml glucose (Iliffe- [START_REF] Iliffe-Lee | Regulation of carbon metabolism in Chlamydia trachomatis[END_REF][START_REF] Nicholson | Chlamydia trachomatis lacks an adaptive response to changes in carbon source availability[END_REF][START_REF] Mehlitz | Metabolic adaptation of Chlamydia trachomatis to mammalian host cells[END_REF]. The mechanisms by which these TCA cycle intermediates are taken up by the bacteria remain elusive.

While it appears that C. trachomatis can make some amino acids de novo from host-derived glucose, (alanine, aspartate, and glutamate), they do require import of others from the host [START_REF] Karayiannis | Amino acid requirements of a Chlamydia trachomatis genital strain in McCoy cell cultures[END_REF][START_REF] Mehlitz | Metabolic adaptation of Chlamydia trachomatis to mammalian host cells[END_REF]. It was recently published that EBs take up glutamine and that its funneled through their TCA cycle to make diaminopimelate, a precursor of peptidoglycan. The authors also demonstrated that the host glutamine metabolism is reprogrammed through increased transcription of host glutamine transporter, SLC1A5, in response to the increased demand for glutamine that occurs during infection [START_REF] Rajeeve | Reprogramming of host glutamine metabolism during Chlamydia trachomatis infection and its key role in peptidoglycan synthesis[END_REF].

Lipids

Early studies suggested that C. trachomatis lipids are largely obtained from the host [START_REF] Wylie | Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis[END_REF]. However, it was shown more recently, that the bacteria have the capacity to produce most of their lipids [START_REF] Yao | Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis[END_REF]. They do import some eukaryotic lipids that are not normally produced in prokaryotes such as sphingomyelin [START_REF] Hackstadt | Lipid metabolism in Chlamydia trachomatisinfected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion[END_REF]. Host membrane trafficking is subverted during infection to enable expansion of the inclusion [START_REF] Elwell | Lipid acquisition by intracellular Chlamydiae[END_REF].

Other Metabolites

Several other small metabolites need to be obtained from the host. For instance, S-adenosyl-Lmethionine (SAM), a methyl donor for transmethylase reactions common in chromatin modifications, may be relevant for chlamydial reactions as a SAM/SAH transporter has been identified and characterized. Binet et al, found that CTL843 was capable of importing S-adenosyl-L-methionine (SAM) SAM and exporting the toxic byproduct of its methyl donation Sadenosylhomocysteine (SAH) [START_REF] Binet | Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter[END_REF]. Another illustration of the import of metabolites by the bacteria is provided by biotin, a cofactor necessary for a multitude of cellular processes, lipid metabolism among them. Biotin has been shown to be imported into the inclusion via a host transporter, the sodium multivitamin transporter (SMVT). Once in the inclusion lumen, biotin may be transported into the bacteria through the bacterial transporter BioY [START_REF] Fisher | Uptake of biotin by Chlamydia Spp. through the use of a bacterial transporter (BioY) and a host-cell transporter (SMVT)[END_REF]. [START_REF] Annunziato | DNA Packaging: Nucleosomes and Chromatin[END_REF]. The average human is made up of an estimated 3.72X10 13 cells, which puts the total length of our combined DNA, were we to line it up end-to-end, at 7.44X10 13 meters [START_REF] Bianconi | An estimation of the number of cells in the human body[END_REF]. That is long enough to travel to the sun and back just under 250 times. While the length is impressive, its shear bulk would certainly impede other cellular processes if left loose and unorganized. How do cells organize their DNA in such a way that it is severely compacted, but also easily accessed for transcription when the need arises?

The cell's answer to this organizational dilemma is to first package its genome into an ultracondensed fiber known as chromatin. Chromatin is comprised of an equal ratio of genomic DNA to specialized packing proteins. Its formation is not only the first step in the construction of chromosomes during mitosis, but also maintains the organization of DNA throughout the cell cycle [START_REF] Janssen | Heterochromatin: Guardian of the Genome[END_REF].

Chromatin is highly dynamic and constantly undergoing sub diffusive movement and reorganization. It can be sub-categorized as euchromatin or heterochromatin. Euchromatin contains gene-rich regions in which DNA is readily accessible for transcription. It is often referred to as "open" or "active" chromatin. The counter part of euchromatin is heterochromatin.

Commonly identified as "closed" or "inactive" chromatin, heterochromatin is comprised of DNA that is highly condensed, and is therefore less transcriptionally active [START_REF] Hauer | Chromatin and nucleosome dynamics in DNA damage and repair[END_REF].

The concepts of heterochromatin and euchromatin were first coined by German scientist, Emil Heitz in 1928 [START_REF] Heitz | Das Heterochromatin der Moose[END_REF]Berger, 2019). He observed in Pellia epiphylla, a type of liverwort moss, that some regions of the chromosomes were not visible, via his improved staining protocol, after telophase. This non-stained chromatin, he dubbed "euchromatin", and postulated correctly that it was associated with actively transcribed genes, and that the stained chromatin, which he termed 'heterochromatin', represented genetically inert regions (Fig 10) [START_REF] Heitz | Das Heterochromatin der Moose[END_REF][START_REF] Emil | Heitz and the concept of heterochromatin: longitudinal chromosome differentiation was recognized fifty years ago[END_REF]Allshire and Mahandi, 2018). Since then, it has been discovered that heterochromatin contains repetitive genetic elements such as satellite repeats and transposons. An important function of heterochromatin compaction is to ensure genetic stability through suppression of such elements. Heterochromatin has also been shown to be responsible for maintaining centromeres and telomeres during cell division, as well as directing cell-type-specific transcription via its spread or recession throughout the genome (Allshire and Madhandi, 2018).

In this section we will go over the basics of chromatin formation and introduce some of the proteins responsible.

2.1.B The Basic Unit of Chromatin: The Nucleosome

The basic unit of chromatin is the nucleosome. It's comprised of approximately 145-147 nucleotide base pairs of DNA wrapped 1.65 turns around a histone octamer. This octamer is comprised of two of each of the canonical core histones: H2A, H2B, H3 and H4 [START_REF] Klug | A low resolution structure for the histone core of the nucleosome[END_REF]. The core histones house a conserved hydrophobic histone fold domain that mediates the dimerization of H2A-H2B and H3-H4 [START_REF] Arents | The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix[END_REF][START_REF] Arents | The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization[END_REF]. Exposed positive charges on the outside of the histone core help stabilize histone-DNA interactions via ionic interactions with negatively charged phosphate groups of DNA [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 Å resolution[END_REF][START_REF] Luger | DNA binding within the nucleosome core[END_REF]. Histone H1, known as the "linker histone," binds the linker DNA that stretches between nucleosomes where it enters and exits the nucleosome. It has been shown to stabilize the structure of the nucleosome and aid in the formation of the secondary structure of chromatin known as the 30-nm chromatin fiber [START_REF] Woodcock | Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length[END_REF][START_REF] Kruithof | Single-molecule force spectroscopy reveals a highly compliant helical folding for the 30-nm chromatin fiber[END_REF][START_REF] Kalashnikova | Linker histone H1 and protein-protein interactions[END_REF][START_REF] Korolev | A systematic analysis of nucleosome core particle and nucleosome-nucleosome stacking structure[END_REF]. Interactions between nucleosomes mediated by post-translational modifications (PTMs) of the histones themselves, as well as non-histone chromatin associated proteins induce fiber-fiber interactions and looping that leads to further compaction (Fig 11).

Stability of the nucleosome core particle (NCP) determines DNA accessibility and can be influenced by several factors such as: nucleosome-DNA interaction, histone variants, DNA methylation and post-translational modifications of histone tails. It is important to note that DNA wrapped around histones is not necessarily unavailable for transcription. It has been shown that DNA can spontaneously, and reversibly, dissociate from its histones, giving transcription machinery accessibility [START_REF] Li | Rapid spontaneous accessibility of nucleosomal DNA[END_REF]. DNA binding affinity of the histone core can also be dependent on sequence [START_REF] Gottesfeld | Sequencespecific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides[END_REF].

Variant swapping a major-type histone with its non-canonical variant can induce further condensation or relaxation of the chromatin. For example, H2A.Z, a variant of H2A, has been shown to be enriched within euchromatin and acts to block the spread of heterochromatin into these regions undergoing active transcription [START_REF] Meneghini | Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin[END_REF]Chakravarthy et al, 2008).

Structural studies utilizing fluorescence resonance energy transfer (FRET) revealed that nucleosomes containing H2A.Z-H2B variant dimers are more stable than the canonical H2A-H2B containing nucleosome. This increased stability lends to this variant's ability mechanistically contribute to blocking heterochromatin spread [START_REF] Park | A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome[END_REF]Chakravarthy et al, 2008).

Modifications to the histones themselves are a major mechanism for influencing chromatin structure and function. They occur on the amino acid side chains of the eight lysine-rich tails that extend from the NCP via the N-terminal domains of histones H3 and H4 (Fig 11). These histone tails are largely responsible for interacting with neighboring nucleosomes and provide a binding site for non-histone proteins. Common PTMs to histone tails include but are not limited to acetylation, methylation, phosphorylation, ubiquitination, ADP-ribosylation, and SUMOylation.

Histone PTMs recruit chromatin remodeling machinery that work to either condense chromatin, rendering DNA transcriptionally inactive, or alternatively, to decondense chromatin for transcription. The different types of histone PTMs and their effect on chromatin remodeling will be further detailed later in the chapter. [START_REF] Luger | New insights into nucleosome and chromatin structure: an ordered state or a disordered affair?[END_REF][START_REF] Hauer | Chromatin and nucleosome dynamics in DNA damage and repair[END_REF].

2.1.C Chromatin Associated Proteins

While histones are key in the formation of nucleosomes, there are a number of other non-histone proteins that are important for influencing chromatin compaction.

The most abundant non-histone protein present in chromatin are the high mobility group proteins (HMG). They bind DNA and nucleosomes to modulate DNA accessibility for transcription machinery or other chromatin remodeling-complexes and can be subclassified into three subfamilies: HMGA, HMGB, and HMGN. Members of the HMG subfamilies have been shown to bind DNA in a structure-dependent manner and can influence chromatin state by competing with histone H1 to bind linker DNA, which then promotes loosening of the chromatin for transcription (Stross, 2010). They are also capable of bending and looping DNA to bring enhancer elements closer to transcriptional start sites [START_REF] Ozturk | HMGA proteins as modulators of chromatin structure during transcriptional activation[END_REF].

Heterochromatin protein 1 (HP1) has been well characterized for its architectural role in heterochromatin formation, chromosomal segregation, chromatid cohesion, DNA replication and repair, telomere maintenance, and transcriptional regulation. There are three isoforms in humans, HP1, HP1, and HP1, which recognize di-and trimethylation of histone H3, at lysine 9 (H3K9me2/3), and bind nucleosomes via their conserved chromodomains [START_REF] Kumar | Heterochromatin protein 1 (HP1): interactions with itself and chromatin components[END_REF]. HP1 isoforms interacts with themselves between nucleosomes to form higher-order chromatin structures. HP1 also interacts with histone methyltransferases, such as SUV39 as well as histone deacetylases to encourage heterochromatin spread (Janessen et al, 2018).

The dynamic activities of chromatin require rapid deposition and turnover of histones to keep up with transcriptional demands. A vast network of histone chaperones and co-chaperones are responsible for regulating the histone supply. Chaperones aid in the transport of histones to their new nucleosome, nucleosome assembly, and store of histones that are not DNA-bound by shielding them from non-DNA interactions. They also function to modulate histone turnover in which histones are evicted from the nucleosome at a given loci, and then rapidly replaced. Failure to maintain the balance of the histone pool could result in problems with centromere and telomere formation, DNA replication, and ultimately compromise genome integrity [START_REF] Hammond | Histone chaperone networks shaping chromatin function[END_REF].

2.1.D The Main Players in Chromatin Modulation: Writers, Readers, and Erasers

Whether a given gene is available for transcription depends on the state of the chromatin in which it is located. Transcription machinery is able to access the freely available, non-histone bound, euchromatin with ease. There are two major mechanisms employed to regulate chromatin accessibility. One such mechanism involves chromatin remodeling complexes that can slide or evict nucleosomes from the DNA, thus exposing DNA for transcription. The other mechanism for chromatin remodeling, and the focus of this thesis work, is the post-translational modification of histones. These PTMs recruit chromatin remodeling complexes capable of influencing the activity state of chromatin to make it more or less available for subsequent transcription [START_REF] Längst | Chromatin Remodelers: From Function to Dysfunction[END_REF].

Before we get into the mechanisms by which chromatin accessibility is modulated, we need to introduce the concept of readers, writers, and erasers in this context.

Proteins capable of recognizing chromatin landmarks are termed, "readers". Readers can recognize a multitude of chromatin targeting signals, such as histone modifications, DNA methylation, as well as DNA sequence/structure, non-coding RNAs, histone variants or DNA-bound interacting proteins. They may act within large protein complexes to recruit chromatin remodelers, or they can be a domain of protein that also contains a chromatin-modifying domain within the same protein [START_REF] Torres | Functional coupling between writers, erasers and readers of histone and DNA methylation[END_REF]. For example, Clr4, the yeast homolog of human histone lysine methyltransferase, Suv39h1/2, contains a 'reader' chromodomain at its N-terminus capable of recognizing H3K9 (the 9 th lysine residue the N-terminal tail of histone H3). Within the same peptide, Clr4 harbors a SET domain at its C-terminus with methyltransferase activity capable of adding methyl groups to H3K9 [START_REF] Nakayama | Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly[END_REF][START_REF] Zhang | Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin[END_REF]). This is an example of a protein that is both a "reader" and a "writer", which brings us to the ideas of "writers" and "erasers". "Writer" is the term used to describe proteins that add modifications to DNA or histone tails.

Writers include enzymes like methyltransferases, acetyltransferases, kinases, and ubiquitinases. "Eraser" describes proteins capable of removing modifications made by the writers. The most pertinent of example of an eraser for the scope of this work would be histone demethylases; enzymes that remove methyl groups to histone tails. Other well characterized erasers include deacetylases, phosphatases, and deubiquinases. (Fig 12) Readers identify chromatin landmarks, and in turn recruit writers or erasers to make modifications that influence chromatin accessibility.

Writers, readers, and erasers often work within larger chromatin remodeling complexes which also house chromatin remodeling enzymes to carry out more targeted activities. Such chromatin remodeling enzymes are ATPases capable of repositioning or evicting nucleosomes. For example, Chd3 and Chd4, from the CHD family of ATPase chromatin remodelers, work in concert with histone deacetylases, HDAC1 and HDAC2 in the NURD (nucleosome remodeling and deacetylase) complex to repress transcription during development [START_REF] Murawska | CHD chromatin remodelers and the transcription cycle[END_REF].

Thus, the activity of writers and erasers often depends on the PTM identified by a reader and the other enzymes with which they are complexed.

Histone Modification: An Overview

2.2.A Types of Histone Modification

There are a multitude of modifications that can be made to histones, and their mechanisms of influence on chromatin accessibility varies. In this section, we will give a brief overview of the following modifications: acetylation, phosphorylation, ubiquitination, and SUMOylation.

Methylation will be discussed in its own section later.

Acetylation

Acetylation and deacetylation involve the addition or removal of an acetyl group to the lysine residues of histone tails. The writers of histone acetylation are a group of enzymes called histone acetyltransferases (HATs), and the erasers are referred to as histone deacetylases (HDACs). active within larger protein complexes, which likely aids in directing substrate specificity. [START_REF] Bannister | Regulation of chromatin by histone modifications[END_REF].

Phosphorylation

Histone phosphorylation is performed by kinases that transfer a phosphate group from ATP to the hydroxyl group of the side chains of the target amino acids: serine, threonine, or tyrosine. This adds a negative charge to the histone tail that influences chromatin structure, resulting in a loosening of DNA. Phosphorylation can be reversed by phosphatase activity, though the mechanisms by which phosphatases, as well as kinases, are recruited to histones are not well understood.

Histone phosphorylation near the DNA entry/exit regions of the nucleosome has been shown to destabilize DNA binding and results increased accessibility for transcription. This DNAunwinding is further increased when phosphorylation works in tandem with histone acetylation [START_REF] Brehove | Histone core phosphorylation regulates DNA accessibility[END_REF]. In fact, histone phosphorylation is often associated with the recruitment of other histone modifying enzymes that encourage active transcription. For example, phosphorylation of H3T11 (histone H3, threonine 11) induced the activity of demethylase, JMJD2D, which is responsible for moving repressive methyl groups from H3K9 [START_REF] Metzger | Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation[END_REF].

Ubiquitination and SUMOylation

Addition of one or more ubiquitin polypeptides to histone tails is another major modification of histones that influences gene expression. While all histones may be affected by ubiquitination, this modification is best characterized by the study of mono-ubiquitination of H2A and H2B. Monoubiquitination of H2A (H2AK119ub1) is associated with transcriptional repression. Inversely, mono-ubiquitination of H2B (H2BK123ub1) is associated with gene activation. Both have been

shown to be important in regulating the DNA damage response [START_REF] Bannister | Regulation of chromatin by histone modifications[END_REF][START_REF] Meas | Histone ubiquitylation and its roles in transcription and DNA damage response[END_REF].

The addition of ubiquitin to its target, a lysine residue on histone tails, involves the sequential activity of three enzymes (Figure 14). Ubiquitin activating enzyme, E1, activates the ubiquitin residue in an ATP-dependent manner, and then loads it onto E2, a ubiquitin-conjugating enzyme.

E3, the ubiquitin-ligase, covalently ligates the ubiquitin to its target, and is largely responsible for substrate specificity. Ubiquitin residues may be removed by deubiquitinating enzymes (DUBs) [START_REF] Jeusset | Developing Targeted Therapies That Exploit Aberrant Histone Ubiquitination in Cancer[END_REF][START_REF] Vaughan | Chromatin Regulation through Ubiquitin and Ubiquitin-like Histone Modifications[END_REF].

SUMOylation, the addition of small ubiquitin-like modifiers (SUMO) to histone tails, has been shown to influence transcription. Covalent attachment of SUMO to the lysine residue of its target sequence occurs via a similar mechanism to that of ubiquitination, described above with the consecutive activity of E1, E2, and E3 enzymes (Fig 14) [START_REF] Boulanger | SUMO and Transcriptional Regulation: The Lessons of Large-Scale Proteomic, Modifomic and Genomic Studies[END_REF][START_REF] Vaughan | Chromatin Regulation through Ubiquitin and Ubiquitin-like Histone Modifications[END_REF]. SUMOylation of histones has been classically associated with transcriptional repression, but recently it's been suggested that it may have roles in active transcription of some genes [START_REF] Wotton | SUMO and Chromatin Remodeling[END_REF]. Histone methylation has many layers of complexity. It does not influence the charge of the nucleosome, thus its mechanism for influencing chromatin structure relies on the recruitment of other chromatin-modifying proteins. Lysines and arginines are the predominant amino acids targeted for methylation. Arginines can be mono-, and symmetrically or asymmetrically dimethylated, while lysines can be mono-, di-, or tri-methylated [START_REF] Bannister | Regulation of chromatin by histone modifications[END_REF].

Methylation

For the purpose of this work, we will focus on methylation of lysine residues.

Histone methylation may induce transcriptional repression or activation depending on the position of the modification within the gene region, which histone residue is methylated, or even how many methyl groups are on a given residue. Some histone methylation marks, especially when coexpressed, can be indicative of cellular processes. For example, methylation at H3K9, H3K27, and H4K20 are generally implicated in heterochromatin formation and subsequent transcriptional repression. Methylations at H3K4, H3K36 and H3K79 are associated with active transcription.

Dysregulated histone methylation, through unbalanced activity of histone lysine methyltransferases (HKMTs) and/or histone lysine demethylases (HKDMs) has been implicated in a number of cancers and neurodegenerative diseases, such as Alzheimer and Huntington diseases (Hyun et al, 2017). In the next section, we will introduce the enzymes responsible for methylation and demethylation of histone lysine residues and will discuss in more detail about the histone methylation marks most relevant for this work.

Writers and Erasers of Histone Lysine Methylation

2.3.A -HKMT Writers

As mentioned in the previous section, there are 24 different histone lysine methyltransferases.

With the exception of DOTL1, whose methyltransferase activity is catalyzed by its 7S (sevenbeta-strand) domain, the rest of the HKMTs contain SET domains [START_REF] Husmann | Histone lysine methyltransferases in biology and disease[END_REF].

The SET domain catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM)

to the -amino group of a lysine residue (Fig 15) [START_REF] Koturbash | Alterations in histone H4 lysine 20 methylation: implications for cancer detection and prevention[END_REF]. SAM is a metabolite synthesized from ATP and L-methionine and is a precursor for a multitude of cellular biochemical reactions including, transmethylation, and transsulfuration reactions, as well as polyamine synthesis [START_REF] Bottiglieri | S-Adenosyl-L-methionine (SAMe): from the bench to the bedside--molecular basis of a pleiotrophic molecule[END_REF][START_REF] Loenen | S-adenosylmethionine: jack of all trades and master of everything?[END_REF]. As the central player for so many metabolic processes, fluctuation in intracellular SAM levels can influence HKMT activity, thus linking metabolism to chromatin state. We will be discussing the association between metabolism and chromatin modification later on.

There is both redundancy and specificity of activity within these enzymes. Each HKMT is usually specific for methylating just one residue, though they may be able to influence more than one methylation state. However, there are often multiple HKMTs that can methylate at a given lysine residue. For example, SUV39H1 can only di-or tri-methylate H3K9, but H3K9 can also be dimethylated by G9a, GLP, and SETDB1, as well as di-and trimethylated by SUV39H2. While it may seem redundant, it is likely useful for targeting methylation to specific genomic regions in a context-dependent manner [START_REF] Husmann | Histone lysine methyltransferases in biology and disease[END_REF].

As discussed previously, HKMTs often work within larger protein complexes that modulate their target specificity. X-ray crystallography studies have shown that the specificity for multimethylation by different HKMTs is also dependent on which aromatic amino acid is present in its catalytic binding pocket [START_REF] Zhang | Structural basis for the product specificity of histone lysine methyltransferases[END_REF]. For example, if there is a phenylalanine in the G9a activity shifts to mono-and di-methylation. [START_REF] Collins | In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases[END_REF].

2.3.B -HKDM Erasers

Lysine-specific demethylases (LSD1/2)

LSD1 was the first histone demethylase ever to be characterized (Shi et al, 2004). LSD1 and its homolog LSD2 are FAD-dependent amine oxidases that utilize cofactor, flavin adenine The largest group of HKDMs are the jumonji domain-containing histone demethylases (JHDMs).

First discovered in 2006, they are capable of demethylating trimethylated lysine residues, unlike LSD1/2. This discovery was important as it demonstrated that histone methylation could be completely reversed and was more flexible than initially proposed [START_REF] Tsukada | Histone demethylation by a family of JmjC domain-containing proteins[END_REF][START_REF] Whetstine | Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases[END_REF]. The JHDMs belong to a superfamily of proteins called Fe 2+ -dependent dioxygenases (summarized in Table 1). These enzymes require ferrous iron (Fe 2+ ), molecular oxygen (O2), and -ketoglutarate (also called 2-oxoglutarate) as co-factors to perform their function.

Their mechanism of action, as depicted in Figure 16B, begins with the hydroxylation of the methyllysine group which uses O2 and -ketoglutarate to create a hydroxyl-methyllysine intermediate, and byproducts of carbon dioxide and succinate. This hydroxyl-methyllysine intermediate is inherently unstable and will spontaneously decompose to yield a demethylated lysine residue with formaldehyde as a byproduct [START_REF] Accari | Emerging Roles of JmjC Domain-Containing Proteins[END_REF]. By virtue of their metabolite co-factors, JDHM activity may be influenced by fluctuations in metabolism. The TCA intermediates, fumarate and succinate, and 2-hydroxyglutarate, a derivative, are competitive inhibitors of multiple -ketoglutarate -dependent dioxygenases which will be detailed in the next chapter [START_REF] Xiao | Inhibition of α-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors[END_REF]. JARID1A-D/KDM5A-D, and NO66 [START_REF] Højfeldt | Histone lysine demethylases as targets for anticancer therapy[END_REF]. The mechanism by which the JmjC demethylases work will be described in more detail later on in the chapter.

2.3.C Histone Lysine Methylation Modifications of Interest

Histone H3, Lysine 9 (H3K9)

Methylation of H3K9 is most associated with heterochromatin and gene silencing. Subsequent interactions with HP1 isoforms HP1/, who can read H3K9 methylation, result in heterochromatin spread between neighboring nucleosomes [START_REF] Lachner | Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins[END_REF][START_REF] Canzio | Mechanisms of functional promiscuity by HP1 proteins[END_REF]. It is especially important for the formation of pericentromeric heterochromatin and has been shown to play a role in alternative splicing (Hyun et al, 2017).

There are several HKMTs that methylate at this residue which include: SUV39H1, SUV39h2, G9a-GLP dimers, SETDB1, and the PRDM family of HKMTs. SETDB1 and SUV39H1/2 catalyze the formation of pericentromeric heterochromatin by trimethylation of H3K9me3 [START_REF] Rea | Regulation of chromatin structure by site specific histone H3 methyltransferases[END_REF].

Methylation via G9a-GLP heterodimers is associated with repressing gene expression from within euchromatin. [START_REF] Tachibana | G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis[END_REF][START_REF] Shinkai | H3K9 methyltransferase G9a and the related molecule GLP[END_REF].

Histone demethylases that remove methyl groups from H3K9 include members of the JmjC demethylases -2 (JHDM2/KDM3), the JHDM3/JMJD2/KDM4 family, and PHF8/KDM7.

Interestingly, PHF8 preferentially demethylates H3K9 from histones that already contain H3K4me3, thus revealing a mechanism in which demethylation supports transcriptional activation [START_REF] Horton | Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases[END_REF].

Histone H3, Lysine 27 (H3K27)

Trimethylation of H3K27 is associated with transcriptional repression and plays an important role in the stabilization of gene silencing; the most well characterized example of which is the inactivation X-chromosomes in mammalian cells in which H3K27me3 is highly enriched [START_REF] Silva | Establishment of histone h3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes[END_REF]. H3K27 methylation, and demethylation, also play a crucial role in directing cell differentiation in embryonic stem cells (ESCs) [START_REF] Margueron | The polycomb complex PRC2 and its mark in life[END_REF].

Mono-, di-, and trimethylation of this residue are carried out by the polycomb repressive complex 2 (PRC2), which is a multi-protein complex. The methyltransferase activity is carried out by the EZH2 subunit of PRC2, while the SUZ12 and EED subunits are capable of recognizing and binding the N-terminal domain of H3 and H3K27me3 respectively. This allows for the initiation and subsequent spread of H3K27me3 across a gene [START_REF] Margueron | The polycomb complex PRC2 and its mark in life[END_REF].

H3K27 demethylases, UTX/KDM6A, UTY/KDM5C, and JMJD3/KDM6B are all jumonji domain-containing [START_REF] Jeon | Writing, erasing and reading histone lysine methylations[END_REF]. They are all involved in gene derepression.

JMJD3/KDM6B demethylates H3K27me2/3 to activate transcription of development-related genes during endoderm development [START_REF] Jiang | Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway[END_REF]. Interestingly, UTX/KDM6A may be regulated by the H3K4 methyltransferase complex, MLL4 [START_REF] Issaeva | Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth[END_REF]). This is an example of crosstalk between activating (H3K4) and repressive (H3K27) histone modifications.

Histone H3, Lysine 79 (H3K79)

H3K79 methylation has been associated with active transcription. H3K79me2/3 are enriched in the coding regions of actively transcribed genes. Methylation at this residue is also important in the DNA damage response and is necessary for the recruitment of 53BP1 to repair sites [START_REF] Huyen | Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks[END_REF][START_REF] Jones | The histone H3K79 methyltransferase Dot1L is 116 essential for mammalian development and heterochromatin structure[END_REF].

DOT1L is the methyltransferase responsible for methylation at H3K79. Unlike the other HKMTs, DOT1L does not have a SET domain, and it has been shown to preferentially methylate nucleosomes that have been ubiquitinylated at H2B. H3K79me2/3 is enriched in actively transcribed genes [START_REF] Feng | Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain[END_REF][START_REF] Mcginty | Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation[END_REF]. Its role in transcription is supported by the activity of its HMKT, DOT1L. DOT1L is known to associate with transcription elongation factors, like ENL, or in complexes that contain transcription elongation factors [START_REF] Wood | DOT1L and H3K79 Methylation in Transcription and Genomic Stability[END_REF].

The identification of any H3K79 demethylase was unknown until recently. KDM2B was shown to interact with H3K79 via co-immunoprecipitation, and demethylase assays with recombinant H3K79me3 revealed that KDM2B is capable of demethylating H3K79 in vitro [START_REF] Kang | KDM2B is a histone H3K79 demethylase and induces transcriptional repression via sirtuin-1-mediated chromatin silencing[END_REF].

Methylation at H4K20 is associated with transcriptional repression and is implicated in DNA repair pathways, DNA replication, chromatin compaction, nucleosome turnover, and overall maintenance of genome integrity [START_REF] Jørgensen | Histone H4 lysine 20 methylation: key player in epigenetic regulation of genomic integrity[END_REF][START_REF] Shoaib | Histone H4K20 methylation mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing[END_REF].

Mono-, di-, and tri-methylation of H4K20 is catalyzed by different enzymes; SET8, SUV4-20H1, AND SUV4-20H2 respectively [START_REF] Jørgensen | Histone H4 lysine 20 methylation: key player in epigenetic regulation of genomic integrity[END_REF][START_REF] Jeon | Writing, erasing and reading histone lysine methylations[END_REF]. Interestingly, H4K20 methylation may be regulated by H3K9 methylation via a trans-tail mechanism in which the Cterminus of SUV4-20H2 interacts with HP1, which in turn recognizes and binds H3K9me2/3.

When H3K9 methylation is blocked, there is a global reduction of H4K20me3, suggesting that H4K20 methyltransferase activity is modulated by H3K9 methylation state [START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF].

PHD8 and PHF2 are the jumonji domain-containing demethylases capable of demethylating H4K20me1 and H4K20me3 respectively [START_REF] Jørgensen | Histone H4 lysine 20 methylation: key player in epigenetic regulation of genomic integrity[END_REF]. The PHD domain of PDH8 also interacts with H3K4me2 and H3K4me3, implicating the role of H4K20 demethylation in the activation of transcription [START_REF] Liu | PHF8 mediates histone H4 lysine 20 demethylation events involved in cell cycle progression[END_REF]). An alternative splice variant of LSD1/KDM1A, termed LSD1n, has been shown to demethylate H4K20me1/2 in vitro [START_REF] Wang | LSD1n is an H4K20 demethylase regulating memory formation via transcriptional elongation control[END_REF].

Influence of Metabolism on Histone Methylation

As discussed previously, histone modifying enzymes rely on common co-factors to carry out their function. Many of these cofactors, like SAM, acetyl-CoA, FAD, and -ketoglutarate are important metabolites or metabolic intermediates for multiple cellular processes, and their intracellular levels fluctuate with changes in metabolism (Fig 18). This is an important mechanism by which environmental changes can influence gene expression through chromatin modification [START_REF] Dai | The evolving metabolic landscape of chromatin biology and epigenetics[END_REF]. In this section, we will discuss relevant examples of how metabolism can influence chromatin structure and function through the co-factor requirements of histone methylases and demethylases.

S-adenosyl-L-methionine (SAM) is an important precursor for a number of intracellular process such as: synthesis of cysteine and glutathione via transsulfuration, synthesis of polyamines like spermine and spermidine, and transmethylation of a wide variety of substrates [START_REF] Finkelstein | Metabolic regulatory properties of S-adenosylmethionine and Sadenosylhomocysteine[END_REF][START_REF] Koturbash | Alterations in histone H4 lysine 20 methylation: implications for cancer detection and prevention[END_REF]. SAM is generated from dietary methionine in humans and fluctuations in amount of methionine consumed affect global histone methylation thus influencing gene expression [START_REF] Dai | The evolving metabolic landscape of chromatin biology and epigenetics[END_REF].

Dietary methionine restriction in mice, as well as human cancer cells lines, have been shown to reduce global methylation [START_REF] Mentch | Histone Methylation Dynamics and Gene Regulation Occur through the Sensing of One-Carbon Metabolism[END_REF][START_REF] Dai | Methionine metabolism influences genomic architecture and gene expression through H3K4me3 peak width[END_REF]. Subsequently, dietary methionine restriction can influence transcription in an anti-tumor capacity [START_REF] Gao | Dietary methionine influences therapy in mouse cancer models and alters human metabolism[END_REF].

Flavin adenine dinucleotide (FAD)

FAD(H2), derived from riboflavin (vitamin B2), is an important co-factor for TCA cycle reactions, as well as oxidative phosphorylation and fatty acid -oxidation [START_REF] Krautkramer | Metabolic regulation of histone posttranslational modifications[END_REF]. While FAD is manufactured in the mitochondria and the cytosol, there is also a pool maintained in the nucleus where it serves as a co-factor for histone demethylases, LSD1/2 [START_REF] Giancaspero | FAD synthesis and degradation in the nucleus create a local flavin cofactor pool[END_REF]. LSD1 is capable of demethylating H3K4, which acts to suppress transcription. It has been shown that LSD1-mediated transcriptional repression is dependent on FAD. When FAD is limited in adipocytes, LSD1 activity is inhibited, resulting in transcriptional activation of genes related to energy expenditure and mitochondrial metabolism via maintenance of H3K4me3 at promoter regions [START_REF] Hino | FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure[END_REF].

TCA cycle intermediates: -ketoglutarate, succinate, and fumarate

The largest family of histone lysine demethylases are the JmjCcontaining -ketoglutarate dependent dioxygenases. These enzymes require oxygen, iron, and -ketoglutarate-KG as cofactors to carry out their function.

-ketoglutarate is a TCA cycle intermediate produced from isocitrate via the activity of one of three isocitrate dehydrogenase (IDH) isoforms. IDH1 produces -ketoglutarate in the cytosol, while IDH2/3 produce -ketoglutarate in the mitochondria. -ketoglutarate can also be produced from glutamine. Mutations of TCA cycle enzymes or dysregulation of glutaminyolysis results in -ketoglutarate depletion leading to inhibition of JmjC demethylases, and subsequent hypermethylation of histones, as reported in different pathological contexts described below.

Glutamine deficiency in a melanoma model showed decreased JMJD3/KDM6B activity, resulting in hypermethylation of H3K27 that induced cell dedifferentiation and rendered the cells less sensitive to chemotherapeutics (Pan et al, 2016). -ketoglutarate analogs succinate, fumarate, and 2-hydroxyglutarate (2-HG), produced by succinate dehydrogenase (SDH), fumarate hydratase (FH), and mutated isoforms of IDH respectively, act as competitive inhibitor JmjC demethylases in vitro [START_REF] Krautkramer | Metabolic regulation of histone posttranslational modifications[END_REF]. Tumor-derived loss-of-function mutations in FH and SDH result in an accumulation of succinate and fumarate and a depletion of -ketoglutarate, which results in inhibition of JmjC demethylases and increased histone methylation [START_REF] Xiao | Inhibition of α-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors[END_REF][START_REF] Letouzé | SDH mutations establish a hypermethylator phenotype in paraganglioma[END_REF]. Mutations in IDH1/2 found in gliomas and acute myeloid leukemias produce 2hydroxyglutarate (2-HG) from -ketoglutarate and were shown to inhibit demethylase activity resulting in increased histone methylation at repressive residues and blocking of cell differentiation [START_REF] Ward | The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate[END_REF][START_REF] Lu | IDH mutation impairs histone demethylation and results in a block to cell differentiation[END_REF].

-ketoglutarate has also been shown to have important roles in ESC differentiation and autophagy through its activity as a demethylase co-factor [START_REF] Carey | Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells[END_REF][START_REF] Teslaa | α-Ketoglutarate Accelerates the Initial Differentiation of Primed Human Pluripotent Stem Cells[END_REF]. This finding supports the afore mentioned dedifferentiation phenotypes observed in cancers with mutations that deplete intracellular -ketoglutarate.

Iron

Iron is an important metal for countless cellular processes and has been well characterized as a necessary co-factor for JmjC demethylases. However, there is little in the literature characterizing the effect of iron modulation on histone methylation and subsequent chromatin state. It's been demonstrated that iron chelation induces increased sensitivity of breast cancer cell line, MCF-7, to chemotherapy by inhibiting demethylase activity [START_REF] Pogribny | Modulation of intracellular iron metabolism by iron chelation affects chromatin remodeling proteins and corresponding epigenetic modifications in breast cancer cells and increases their sensitivity to chemotherapeutic agents[END_REF]. Mitochondria biogenesis was also shown to be impacted by iron depletion via gene repression mediated by histone methylation [START_REF] Rensvold | Iron Deprivation Induces Transcriptional Regulation of Mitochondrial Biogenesis[END_REF]. Recently, one study observed that iron-dependent demethylation of H3K9 is important for cyclin E1 mediated B-cell proliferation (Jiang et al, 2019).

Illustration of epigenetic modifications by metabolism: the influence of the microbiota

Fluctuations in metabolism can shift chromatin remodeling activity and subsequently, transcription. The influence of gut microbiota on metabolism has been well characterized as a means of influencing chromatin landscape through shifting metabolism. Microbes within the microbiome have their own metabolism that work to serve their own needs but are also tied in with that of the host's metabolism. Gut microbiota may influence host chromatin state through multiple avenues including: de novo synthesis of metabolites, conversion of host dietary nutrients into other active compounds, co-metabolism of shared dietary substrates, and direct competition between the host and microbiota for metabolic intermediates (Kratukramer et al, 2017).

De novo synthesis of B-vitamins by the microbes of the gut are an interesting example of how the microbiome are involved in chromatin remodeling. Humans do not efficiently make enough Bvitamins necessary for daily function, but readily absorb those made by their microbiota (Said, 2011). Gut bacteria are capable of de novo synthesis of B-vitamins which play important roles as co-factors for histone modifying enzymes as well as for the cycles that support their activity.

Among them are riboflavin (B2), niacin (B3), panthothenic acid (B5), pyridoxine (B6), folate (B9), and cobalamine (B12) [START_REF] Leblanc | Bacteria as vitamin suppliers to their host: a gut microbiota perspective[END_REF]. As mentioned previously, riboflavin is the precursor for the formation of FAD, the co-factor required for the activity of demethylases LSD1/2. Folate (B9), pyridoxine (B6), and cobalamine (B12) are all crucial to the methionine and folate cycles, which are necessary for restoration of SAM, the methyl donor for methyltransferase reactions [START_REF] Mccaddon | Assessing the association between homocysteine and cognition: reflections on Bradford Hill, meta-analyses, and causality[END_REF]. Vitamins B3 and B5 are important precursors for NAD and Acetyl-CoA, which are required for histone deacetylation and acetylation respectively [START_REF] Kirkland | Niacin status impacts chromatin structure[END_REF][START_REF] Leonardi | Biosynthesis of Pantothenic Acid and Coenzyme A[END_REF].

The degradation of dietary substrates like polysaccharides, complex starches, proteins, and amino acids by the gut microbiome into products like short and branched chain fatty acids, and amines are another mechanism by which they may influence chromatin activity. In a colorectal cancer mouse model, the mice were colonized with a minimal microbiome in addition to high butyrate producer, Butyrivibrio fibrisolvens, and fed a high fiber diet. Donohoe et al observed that there was an increase in butyrate production from the microbiome's break-down of fiber, which resulted in HDAC inhibition and increased acetylation of Histone H3 subsequently influencing apoptosis and cell proliferation. These in vivo results were consistent with findings of higher butyrate and histone acetylation levels in human colorectal cancer samples, suggesting that diet can influence the outcome of chromatin regulation and gene expression in the context of disease [START_REF] Donohoe | A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota-and butyrate-dependent manner[END_REF].

The idea that diet and the microbiome are linked to the chromatin landscape was further demonstrated by Kratukramer et al in which it was demonstrated that mice with different gut microbiome compositions resulted in differences in the epigenetic states of liver, colon, and adipose tissue. Germ-free mice fed on a diet that included SCFAs normally produced by gut bacteria, recapitulated the histone PTM profile of conventionally raised mice, suggesting that the metabolites produced by the gut microbiota are important for regulating chromatin remodeling and gene transcription [START_REF] Krautkramer | Diet-Microbiota Interactions Mediate Global Epigenetic Programming in Multiple Host Tissues[END_REF].

These data and other support the model that gut microbiota serve as environmental sensors that influence host chromatin through their metabolic response to external stimuli. Metabolites secreted by the microbes of the gut during environmental changes are absorbed by the host and act as effectors that can modulate chromatin state and ultimately influence host gene expression.

Next, we will explore some examples in which bacteria can directly modulate chromatin remodeling.

Modulation of histone PTMs by bacteria

While the shifting of host metabolism and availability of metabolite pools can influence chromatin state through its chromatin modifying enzymes, pathogenic bacteria may do so through the secretion of virulence factors and toxins. Such effectors have been termed, "nucleomodulins," and are capable of localizing to the host nucleus to manipulate nuclear activities. Nucleomodulins may manipulate transcription by directly transforming host chromatin, or through the modulation or mimicry of host chromatin regulatory factors [START_REF] Bierne | When bacteria target the nucleus: the emerging family of nucleomodulins[END_REF][START_REF] Hamon | Revealing eukaryotic histone-modifying mechanisms through bacterial infection[END_REF][START_REF] Hanford | Bacterial nucleomodulins: A coevolutionary adaptation to the eukaryotic command center[END_REF]. Here we will discuss a few examples of nucleomodulins that influence histone modifications and subsequent transcription, that were all discovered at the Institut Pasteur in Paris.

An interesting example of a bacterial effector indirectly influencing histone modifications is the action of LntA (Listeria nuclear targeted protein A) in Listeria monocytogenes. LntA has been shown to encourage gene expression through interactions with its host binding partner, BAHD-1.

BAHD-1 (bromo adjacent homology domain-containing-1) acts as a scaffold for a larger chromatin remodeling complex composed of both histone deacetylases (HDAC1/2), and histone methyltransfersaes (G9a and SETDB1). BAHD-1-mediated binding of its complex to interferonstimulated gene (ISG) promoters induces heterochromatin formation and gene silencing via histone deacetylation, and subsequent methylation in those regions. LntA binding to BAHD-1 inhibits BAHD-1 binding to ISG promoters, thus maintaining transcription in those regions [START_REF] Bierne | When bacteria target the nucleus: the emerging family of nucleomodulins[END_REF][START_REF] Bierne | Targeting host epigenetic machinery: The Listeria paradigm[END_REF].

Contrasting to LntA, AnkA of A. phagocytophilum has been shown to bind DNA directly and recruit HDACs for directed deacetylation and promotes silencing of genes related to granulocyte response [START_REF] Rennoll-Bankert | Chromatin-bound bacterial effector ankyrin A recruits histone deacetylase 1 and modifies host gene expression[END_REF]. Recently, a non-invasive strain of Streptococcus pneumoniae has been shown to induce changes in methylation of H3K27me2 at the IL-11 promoter region through the recruitment of JmJC demethylase, KDM6B. The authors demonstrated that inhibition of KDM6B activity during infection with non-invasive S. pneumoniae induced disease in vivo. Conversely, the addition of exogenous IL-11 during infection with a normally invasive strain S. pneumoniae decreased bacterial burden and hampered bacterial invasion in vivo. While the mechanism by which KDM6B activity is induced by the bacteria under these conditions is not yet known, it is an interesting example of how commensal bacteria influence host chromatin remodeling [START_REF] Connor | The histone demethylase KDM6B fine-tunes the host response to Streptococcus pneumoniae[END_REF].

There are also examples of bacterial derived effectors that are capable of directly modifying histones. Among them is NUE (Nuclear effector), a T3SS effector secreted by Chlamydia trachomatis. NUE localizes to the host-cell nucleus through a nuclear localization signal in its Cterminus. It contains a domain homologous to the eukaryotic SET domain, capable of methyltransferase activity, and has been shown to automethylate, as well as transfer methyl groups to H2B, H3, and H4 in vitro [START_REF] Pennini | Histone methylation by NUE, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis[END_REF]. While the in vivo targets of NUE activity have yet to have been characterized, their potential for directly regulating host chromatin was an exciting discovery.

Legionella pneumophila, Paris and Philadelphia strains also secrete SET-domain containing effectors via their type 4 secretion system, termed RomA and LegAS4 respectively. After localizing to the host-cell nucleus, RomA has been shown to methylate H3K14, which has not been shown to be methylated in mammalian cells under normal conditions. Methylation of H3K14 by RomA inhibits transcription and blocks the innate immune response [START_REF] Sanulli | Legionella pneumophila effector RomA uniquely modifies host chromatin to repress gene expression and promote intracellular bacterial replication[END_REF].

LegAS4 also methylates H3K14 and has also been shown to methylate H3K4 to promote transcription of rDNA through associations with HP1 [START_REF] Li | SET-domain bacterial effectors target heterochromatin protein 1 to activate host rDNA transcription[END_REF].

Consequences of Chlamydia trachomatis infection on host transcription and chromatin

Consequences of infection on gene expression

The first microarray study of the consequences of infection on host gene transcription was published 20 years ago [START_REF] Hess | The reprogrammed host: Chlamydia trachomatis-induced up-regulation of glycoprotein 130 cytokines, transcription factors, and antiapoptotic genes[END_REF]. Since then, several microarray or RNA-seq data have been made available [START_REF] Hess | More than just innate immunity: comparative analysis of Chlamydophila pneumoniae and Chlamydia trachomatis effects on host-cell gene regulation[END_REF][START_REF] Porcella | Transcriptional profiling of human 123 epithelial cells infected with plasmid-bearing and plasmid-deficient Chlamydia trachomatis[END_REF][START_REF] Zadora | Integrated Phosphoproteome and Transcriptome Analysis Reveals Chlamydia-Induced Epithelial-to-Mesenchymal Transition in Host Cells[END_REF][START_REF] Hayward | Early Transcriptional Landscapes of Chlamydia trachomatis-Infected Epithelial Cells at Single Cell Resolution[END_REF] . As expected, infection induces important changes in the transcriptional profile of the host, which evolves with the time of infection.

Early microarray studies by Hess et al. indicated that Chlamydia infection induces increased expression in antiapoptotic genes, glycoprotein 130 cytokines , and transcription factors associated with growth (EGR1, FRA1, JUN) [START_REF] Hess | The reprogrammed host: Chlamydia trachomatis-induced up-regulation of glycoprotein 130 cytokines, transcription factors, and antiapoptotic genes[END_REF]. They demonstrated that these changes in host cell transcription were due to active infection that is Chlamydia specific [START_REF] Hess | More than just innate immunity: comparative analysis of Chlamydophila pneumoniae and Chlamydia trachomatis effects on host-cell gene regulation[END_REF].

Simultaneous RNA-seq of host cells and Chlamydia trachomatis by Humphreys et al. revealed transcriptional evidence of innate immune system dampening through modulation of expression of antimicrobial peptides, and mucins. Early host expression of collagen formation-related proteins, Tenascin C and Gremlin-1 are hypothesized to contribute to tissue damage and fibroid formation associated with persistent infection.

They also found that during immediate-early infection, that there was differential expression of linker histone, histone H1 variants. H1.0, the H1 variant most expressed in differentiated cells, was down-regulated compared to upregulated variants H1.4 and H1.5, which are expressed mostly during S-phase [START_REF] Pan | Role of H1 linker histones in mammalian development and stem cell differentiation[END_REF]. The core histones H2A and H3 are also up regulated, suggesting that modulation of chromatin dynamics due to infection begin as early at 1 hpi (Humphreys et al, 2013).

In 2015, Porcella et al. performed a microarray study on two genomically isogenic strains of Chlamydia trachomatis, one with the chlamydial plasmid and one without, to determine if it contributes to virulence. At 24 and 48hpi time points, both strains induced increased expression of pro-inflammatory cytokines (GM-CSF, MCSF, IL-1, IL-6, and IL-8), apoptotic proteins (CASP1, CASP9, FAS, IL-24), the T-cell chemokine response, and immune suppression (PD-L1), as well as genes involved in cell growth, metabolism, and fibrosis. However, the strain containing the chlamydial plasmid had significantly increased levels of expression of these genes relative to the plasmid-free strain. These results indicated that the chlamydial plasmid acts as a virulence factor that mediates inflammatory response [START_REF] Porcella | Transcriptional profiling of human 123 epithelial cells infected with plasmid-bearing and plasmid-deficient Chlamydia trachomatis[END_REF].

A recent single cell RNA-Seq study of early infection (3, 6, and 12 hpi), supports previous studies indicating that infection induces upregulation of inflammatory cytokines, signal transduction, and fatty acid metabolism. Genes involved in cell cycle progression, centrosome maturation, mitotic and centrosomal proteins, and the p53-related DNA damage response were down regulated at these very early times of infection [START_REF] Hayward | Early Transcriptional Landscapes of Chlamydia trachomatis-Infected Epithelial Cells at Single Cell Resolution[END_REF].

Analysis of the phosphoproteome carried out by Zadora et al. indicated that the MAPK signaling cascade is increased during infection, which leads to the induction of genes implicated in the epithelial-to-mesenchymal transition. This study is interesting in that it provides a mechanism by which C. trachomatis infection may induce pro-carcinogenic transcription [START_REF] Zadora | Integrated Phosphoproteome and Transcriptome Analysis Reveals Chlamydia-Induced Epithelial-to-Mesenchymal Transition in Host Cells[END_REF].

Global consequences of Chlamydia trachomatis infection on host chromatin

The consequences of Chlamydia infection on host-cell chromatin had not been explored until more recently. The concept chlamydial effectors had the potential to directly modify host chromatin was introduced with the discovery of NUE in 2010 [START_REF] Pennini | Histone methylation by NUE, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis[END_REF] 65 largest proportion of differential chromatin accessibility, with 3128 differentially accessible genomic regions, against 1358 ones at 24 hpi. Overall, the trend was towards a more open chromatin in intergenic and intragenic regions of late infected cells, compared to mock-infected controls. However, when focusing on promoter and enhancer regions, the inverse trend was observed, with more occurrence of decrease in accessibility [START_REF] Hayward | Chromatin accessibility dynamics of Chlamydia-infected epithelial cells[END_REF].

A recent study in a male model of infection demonstrated that Chlamydia infection induces DNA damage in testicular cell lines and that DNA was globally hypomethylated [START_REF] Bryan | DNA damage contributes to transcriptional and immunological dysregulation of testicular cells during Chlamydia infection[END_REF].

Questions addressed by this thesis

During C. trachomatis infection, the host cell undergoes major changes to its transcriptome. Many of these changes are due to the break in homeostasis imposed by the needs of developing bacteria (i.e., metabolites, lipids, nucleotides etc.), as well as the innate response of the host to fight the infection. It is also likely that some of the transcriptional regulation is controlled by the bacteria, either directly (e.g., through the secretion of chromatin modifiers) or indirectly (e.g., by interfering with signaling cascades that ultimately result in gene transcription). In agreement with these major changes in the host transcriptional program there is evidence that chromatin is remodeled during C. trachomatis infection, which is unsurprising as chromatin state informs downstream transcription.

Since C. trachomatis has the capacity to directly control histone methylation through T3SS effector, NUE, and potentially by other effectors that have yet to have been characterized, I began this work by investigating the evolution of several histone marks over the course of infection. It was observed that all histone methylation marks tested were co-regulated during late infection, with an overall increase in said marks in infected cells.

In the following section, I will present the experimental work aimed at addressing two questions:

1. What is the mechanism responsible for hypermethylation of histones during C. trachomatis infection?

2. What are the consequences of this hypermethylation on the host response to infection at the transcriptomic level? Data omitted, but not forgotten Before we dive into the method section, I would like to acknowledge that this dissertation does not cover the entirety of the work done during my thesis. I began my work on a different project in which I was attempting to characterize a putative nuclear effector of Chlamydia trachomatis, named CT790.

The preliminary results performed by immunofluorescence analysis of transfection of CT790 with an amino-terminal FLAG tag had indicated that the effector localized to the nucleus in Hela cells, a result which I confirmed. CT790 had also been observed in the host cell nucleus during infection with bacteria expressing CT790 with a carboxy-terminal FLAG tag, which suggested that it may be secreted. However, I could not reproduce this result. It is possible that the signal was just outside the limit of the sensitivity of this assay, but this was definitively not encouraging. Furthermore, secretion assays to determine if the putative effector was secreted using the bacterial type III secretion system were initially inconclusive. After some optimization, that assay determined that the effector was not secreted by the bacterial T3SS. This does not rule out the possibility that it is secreted by other means, but again this was a disappointing result.

In parallel to these experiments, a yeast Two-hybrid screen was performed using CT790 as bait, to try to identify a host binding partner. After many attempts to verify the host candidates from the screen by co-immunoprecipitation, I finally confirmed one. However, I was unable to confirm any interaction with the endogenous proteins or determine if either protein had an effect on the other in vitro during infection.

An antibody had been made, in rabbits, against CT790 and was used for much of the characterization. This was our most encouraging result, since the antibodies stained the nuclei of 10-20% of the infected cells at late time points of infection. However, based on additional control experiments we later came to the conclusion that, in addition to its ability to recognize CT790, the antibody was cross-reacting with an epitope that was only present in the nuclei of infected cells, not uninfected cells, but that was not CT790.

I also attempted to make a knock-out mutant strain of C. trachomatis for CT790 using the Targetron Gene Knock-out system that had been modified for use in Chlamydia. Chlamydia is notoriously difficult to manipulate genetically, and after much effort on my part, and the part of our collaborators who also attempted to make the KO strain, we were unable to obtain the mutant.

While it might mean that the protein is essential for C. trachomatis, not having a mutant severely limited any other study we could do to characterize the role of this putative effector.

In view of the inability to obtain a CT790 KO mutant, and of the artefactual reaction of our antibody, we decided to shift our focus to the project detailed in this manuscript concerning increased histone methylation during C. trachomatis infection. While I was initially disappointed to set the effector project aside after all of time I had dedicated to it, it was ultimately the best choice we could have made. In the end, it was wiser to pursue another equally interesting scientific question, and I am looking forward to sharing those results with the reader in this dissertation. As the data regarding CT790 remain inconclusive at this point, we have decided to omit it from this manuscript.

Cells and Bacteria

HeLa cells (ATCC) were grown in Dulbecco's modified Eagle's medium with Glutamax (DMEM, Invitrogen), supplemented with 10 % (v/v) heat-inactivated fetal bovine serum (FBS) (D'Eletto et al, 2009).

Primary cells were isolated from endocervix biopsies of female patients and were cultivated in keratinocyte-SFM medium (Thermo Fisher Scientific) containing 50 mg.L -1 of bovine pituitary extract (Thermo Fisher Scientific) and 5μg.L -1 of epidermal growth factor (EGF) human recombinant (Thermo Fisher Scientific) (Wu et al, in preparation). All cell cultures were maintained at 37 °C, in 5 % CO2 atmosphere.

C. trachomatis serovar LGV L2 strain 434, GFP-expressing L2 (L2 IncD GFP) and C. muridarum strain MoPn were propagated on HeLa cells, purified on density gradients as previously described and stored at -80 °C [START_REF] Scidmore | Cultivation and Laboratory Maintenance of Chlamydia trachomatis[END_REF][START_REF] Vromman | Quantitative Monitoring of the Chlamydia trachomatis Developmental Cycle Using GFP-Expressing Bacteria, Microscopy and Flow Cytometry[END_REF].

Immunofluorescence 100,000 HeLa cells, primary cells, or MEFs were seeded per well in a 24 well-plate. The following day cells were infected as indicated. Cells were treated at either 2 hpi or 24 hpi with 2,2'-Byridyl (Sigma-Aldrich D216305), iron (III) citrate (Sigma-Aldrich F5879), or DMKG (Dimethyl aketoglutarate Sigma-Aldrich 349631). 48 hpi, cells were fixed in 4% PFA for 20-30 minutes, quenched in 50mM NH4Cl, and then washed in 1XPBS. Coverslips were permeabilized with 0.15% Triton X in PBS for 10 minutes, and then blocked for 1 hour in blocking buffer (10% FBS, 3% BSA, and 0.1% Triton X in 1XPBS). Samples were incubated for 1 hour in primary antibodies diluted 1:500 in blocking buffer, washed 3X in 1XPBS, and then incubated for 1 hour in secondary antibodies and Hoechst diluted in blocking buffer. Antibodies used may be found in Table 2.

Samples were washed 3X in 1XPBS and then mounted on slides in mowiol. Microscopy was performed on an Axio observer Z1 microscope equipped with an ApoTome module (Zeiss, Germany) and a 63X Apochromat lens. Images were taken with an ORCAflash4.OLT camera (Hamamatsu, Japan) using the ZEN software from Zeiss. Images were analyzed with ImageJ (Rasband, 1998(Rasband, -2018) )

Progeny assay

Progeny assays were performed in HeLa cells in which 100,000 cells were seeded in each well of a 24-well plate and were infected with L2 IncD GFP bacteria at a MOI = 0.20 the following day.

Either 2 or 24 hpi after the initial infection, cells were treated with 5 mM DMKG (Dimethyl aketoglutarate Sigma-Aldrich 349631, diluted to 500 mM stock in DMSO) or an equal volume of DMSO as a control.

Forty hpi, cells were detached and fixed in 2 % PFA in PBS prior to flow cytometry analysis in order to evaluate the first round of infection. In duplicate wells, cells were detached, lysed using glass beads and the supernatant was used to infect new untreated cells) plated the day before (100 000 cells/well in a 24-well plate), in serial dilution. The next day, 3 wells per condition with an infection lower than 30 % (checked by microscopy) were detached and fixed as described above, before analysis by flow cytometry and determination of the bacterial titer [START_REF] Käser | Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria[END_REF].

Statistical analysis was performed in GraphPad Prism version 9.0.

Nuclear Isolation

For each condition, a 10 cm dish was seeded with 2.5 million HeLa cells. Cells were infected MOI

Histone extraction

Nuclei from nuclear isolation were resuspended in RIPA buffer (50mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM DTT, 1X cOmplete EDTA-free protease inhibitor cocktail from Roche) and incubated on ice for 10 minutes. Samples were then digested with 0.0025 units/l of MNase with 1 mM CaCl2 at 37 C for 15 minutes, vortexing often.

Next, samples were sonicated using Bioruptor Pico sonication device on HIGH, at 30 second intervals for 8 minutes. Finally, samples were centrifuged for 10 minutes at 13,200 rpm to pellet debris, and the supernatants were stored at -80 C.

Western blot

Nuclear samples from nuclear isolation were boiled for 10-15 minutes in 1X Lammeli buffer (300 mM Tris-HCl pH 6.8, 10% SDS, 50% Glycerol, 0.05% Bromophenol blue, 5% mercaptoethanol) Samples were then subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to polyvinylidene difluoride (PVDF)

membranes. The membranes were then blocked in 1X phosphate buffered saline (PBS) + 5% milk for 45 minutes-l hour. After blocking, the membrane underwent immunoblotting with primary antibodies diluted in 1XPBS + 5% milk for one hour. Between the primary and secondary antibody incubations, the membranes were washed in three-ten-minute intervals in 1XPBS + 0.1% Tween-20. Secondary antibodies, mouse-HRP (Amersham cat# NA 931) or rabbit-HRP (Amersham cat# NA 9340), were diluted in 1XPBS+5% milk and left to incubate for one hour. Incubation of the secondary antibody was followed by the wash cycle carried out previously. Blots were developed using Immobiolon HRP Substrate (Merck), and the signal was detected using the PXi Touch (Syngene).

qRT-PCR

A 24 well-plate was seeded with 50,000 HeLa cells/well. After cells had adhered, they were transfected with Lipofectamine RNAiMAX (Invitrogen) following the manufacturer's recommendation, using 10 nM of siRNA. The transfection incubated for 30 hours, and then the cells were retransfected again the same way and infected with LGLV2 at MOI = 1. 48hpi, RNA was extracted using RNeasy Mini Kit (Qiagen) with DNase treatment (DNase I, Roche). RNA concentrations were measured with a spectrophotometer NanoDrop (Thermo Fisher Scientific).

Reverse transcription (RT) was performed on 500 ng of each sample using the M-MLV Reverse Transcriptase (Promega) and quantitative PCR (qPCR) undertaken on the complementary DNA (cDNA) with LightCycler 480 system using LightCycler 480 SYBR Green Master I (Roche).

Primers used are listed in Table 3, their specificity was ensured through the analysis of melting curves and their efficiency tested using serial dilutions of samples encoding the genes of interest.

For cDNA, data were analyzed using the Delta-Delta Ct method with either 36B4 or ACTIN as a control [START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C(T) method[END_REF].

These data were obtained in HeLa cells, the epithelial cell line most commonly used in the Chlamydia field. However, their cancerous origin and passaging over multiple generations call for validation of the results in another cellular background. To that end, we looked at histone methylation in late-infected and non-infected primary epithelial cells isolated from the ectocervix vitro, but its targets in vivo remain unknown [START_REF] Pennini | Histone methylation by NUE, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis[END_REF]. Through chemical mutagenesis, the Validvia lab has obtained and isolated several mutant strains of C. trachomatis [START_REF] Kokes | Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia[END_REF]. One of these strains contained a nonsense mutation in NUE, which we designate here as L2 NUE*. To determine whether NUE could be responsible for the histone hypermethylation observed during late infection, cells were infected with L2 NUE*, or with the parental strain used in the mutagenesis strategy, as a control (Fig 25). The level of H3K9me3 and H4K20 me3 methylation in cells infected with L2 NUE* was similar to that observed in cells infected with the parental strain, indicating that hypermethylation of histones during late infection is independent of NUE activity.

II. Infected cells are sensitized to histone demethylase activity

The observation that several histone marks were simultaneously up-regulated late in infection suggested a common mechanism. Histone methylation and demethylation are achieved by dedicated enzymes, histone methyltransferases and histone demethylases, respectively. We sought to determine if the increase in histone methylation during infection was due to increased histone methyltransferase activity, or rather a consequence of decreased histone demethylase activity. It As an alternative approach to determine whether iron was the limiting factor for demethylase activity, we used the iron chelator, 2,2bipyridyl (Bpdl). Bpdl has been shown to sequester iron in infected cells, and to delay chlamydial development [START_REF] Thompson | An optimal method of iron starvation of the obligate intracellular pathogen, Chlamydia trachomatis[END_REF]. Bacterial development was strongly inhibited at this concentration of iron chelator, indicating that the iron concentration was below what it would have been normally in infected cells (Fig 27B). However, histone methylation was not detected in cells infected for 48 h in the presence of 50 M Bpdl, indicating that even the level of iron limitation reached in these conditions was not sufficient to induce histone hypermethylation. We concluded from these experiments that a decrease in iron availability in the cytoplasm and nucleus cannot account for the histone hypermethylation observed late in infection.

IV. DMKG supplementation during infection prevents histone hypermethylation

Next, we wanted to determine if increasing the intracellular level of -ketoglutarate would affect histone methylation. -ketoglutarate is not cell permeable on its own, so to increase its intracellular levels, it may be added in the form of an ester such as Dimethyl-ketoglutarate (DMKG). After entering the host cell cytosol, DMKG is hydrolyzed by host cell esterases into -ketoglutarate and has been shown to increase intracellular levels of -ketoglutarate [START_REF] Baracco | α-Ketoglutarate inhibits autophagy[END_REF]. One interesting category among those transcripts that did not code for genes are several tRNAs, which seem to be downregulated in infection, and recovered by DMKG treatment.

Analysis of the 115 protein-coding genes was carried out using several protein pathway enrichment algorithms such as: WikiPathways, STRING, Kegg, Reactome and GeneOntology. All of them indicated that the p53 transcriptional network was differentially regulated by the addition of DMKG. We thus chose 2 of the 4 genes highlighted in this network to validate the microarray data by RT-qPCR: GADD45A (Growth arrest and DNA damage-inducible protein GADD45 alpha) and PMAIP1 (PMA-induced protein 1, also called NOXA). Both genes were up-regulated by infection, with an additional boost upon DMKG supplementation, confirming the micro-array data (Fig 34).

Finally, we hypothesized that if DNA damage and histone hypermethylation were linked, preventing histone hypermethylation should also affect DNA damage. Indeed, we found that the H2AX signal was greatly reduced in cells supplemented with 5 mM DMKG (Fig 37 A&B). This observation indicates that DMKG supply as an important consequences not only on the epigenome of the host, but also on chromatin integrity

II. Histone hypermethylation is caused by reduced demethylase activity

The methylation state of a given histone residue can be altered by histone methyltransferases and histone demethylases. We first investigated whether the increased methylation could be due to the action of a bacterial protein. One such chlamydial effector, NUE, has been shown to possess histone methyltransferase activity in vitro [START_REF] Pennini | Histone methylation by NUE, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis[END_REF]. However, NUE did not appear to be the likely culprit behind this hypermethylation phenotype as cells infected with a strain of C. trachomatis containing a nonsense mutation in NUE (L2 NUE*) displayed similar levels of histone methylation to that of infection with the wild-type strain (Fig 25).

There are a number of histone methyltransferases and demethylases and each enzyme is usually only capable of changing the methylation state of one histone residue. Therefore, it is not likely that the hypermethylation observed during late infection is the result of a singular enzyme. Because the upregulation of methylation occurs at multiple marks simultaneously, it was therefore logical to investigate the role of co-factors that are shared between histone methyltransferases or histone demethylases. It has been shown that S-adenosyl-L-methionine, the common co-factor for histone methyltransferases, is consumed by the bacteria during infection, therefore it is unlikely that the hypermethylation phenotype observed is a result of increased histone methyltransferase activity [START_REF] Binet | Identification and characterization of the Chlamydia trachomatis L2 S-adenosylmethionine transporter[END_REF].

We turned our attention to histone demethylases and sought to determine whether infected cells are sensitive to demethylase activity. JIB-04, a JmjC-domain inhibitor that targets the large JmjC domain-containing family of demethylases, was used to treat infected and non-infected cells [START_REF] Wang | A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth[END_REF][START_REF] Parrish | The Jumonji-domain histone demethylase inhibitor JIB-04 deregulates oncogenic programs and increases DNA damage in Ewing Sarcoma, resulting in impaired cell proliferation and survival, and reduced tumor growth[END_REF]. In uninfected cells, there was no detectable increase in histone methylation. However, it was observed that at the same concentrations JIB-04 induced increased histone methylation in infected cells compared to their untreated controls (Fig 26). These results indicate the methylation state of C. trachomatis-infected cells is held in balance, and that infected cells are sensitized to demethylase activity.

III. Shifting Metabolites during infection influences histone modification

The largest family of histone lysine demethylases are the jumonji domain-containing demethylases. They require oxygen, iron, and -ketoglutarate, a TCA cycle intermediate, as cofactors to carry out their function.

We first investiaged the role of iron on histone demethylase activity, as iron is known to be necessary for normal C. trachomatis development [START_REF] Raulston | Response of Chlamydia trachomatis serovar E to iron restriction in vitro and evidence for iron-regulated chlamydial proteins[END_REF]. Supplementing infected cells with iron (Fe 3+ ) citrate resulted in a decrease in observable histone methylation (Fig 27A).

However, it is important to note that iron is also a necessary co-factor for many of the TCA cycle enzymes responsible for generating intermediates like -ketoglutarate [START_REF] Oexle | Iron-dependent changes in cellular energy metabolism: influence on citric acid cycle and oxidative phosphorylation[END_REF]Tesler et al, 2019). To determine whether iron was limiting, infected cells were treated with iron chelator, Bpdl, which has been shown to effectively sequester iron during C. trachomatis infection to the point that it halts bacterial development [START_REF] Thompson | An optimal method of iron starvation of the obligate intracellular pathogen, Chlamydia trachomatis[END_REF]. It was observed that Bpdl treatment of non-infected cells was not enough to induce histone methylation, but the same concentration was enough to inhibit bacterial development (Fig 27B). These results indicated that iron is not the limiting factor for demethylase activity during C. trachomatis infection.

-ketoglutarate is an energy-generating TCA cycle intermediate that has been implicated in a number of other roles, such as: amino acid formation, signaling, and tissue repair [START_REF] Zdzisińska | Alpha-Ketoglutarate as a Molecule with Pleiotropic Activity: Well-Known and Novel Possibilities of Therapeutic Use[END_REF]. C. trachomatis have an incomplete TCA cycle and must rely on their host to acquire TCA cycle intermediates to generate energy (Fig 9) [START_REF] Stephens | Genome Sequence of an Obligate Intracellular Pathogen of Humans: Chlamydia trachomatis[END_REF]. While glucose is their preferential carbon source, Ilfee-Lee and McClarty demonstrated that the bacteria are capable of utilizing -ketoglutarate as a carbon source by glucose starving the host cells and supplying only -ketoglutarate, among other gluconeogenic carbon sources (Iliffe- [START_REF] Iliffe-Lee | Regulation of carbon metabolism in Chlamydia trachomatis[END_REF].

While their study indicated that -ketoglutarate was a poor substitute for glucose as a carbon source, it is worth noting that they did not use a cell permeable source of -ketoglutarate, which could explain why bacterial development was low. To determine if increasing intracellular levels of -ketoglutarate could enhance demethylase activity, dimethyl-ketoglutarate (DMKG) was added to the culture media. DMKG is a cell permeable ester form of -ketoglutarate that, once it enters the cytosol, is rapidly hydrolyzed by host cell esterases and has been used to increase intracellular levels of -ketoglutarate [START_REF] Baracco | α-Ketoglutarate inhibits autophagy[END_REF]. After optimizing the doses of DMKG of the demethylase inhibitor JIB-04. We observed that the negative effect of DMKG on bacterial growth persisted in this experimental set-up, which preserved histone hypermethylation (data not shown). This result suggests that the detrimental effect of DMKG is exerted at the metabolic level, rather than via its consequence on chromatin structure.

IV. -ketoglutarate depletion in infection is likely driven by bacterial metabolism JmjC histone demethylases are influenced by metabolites other than their -ketoglutarate cofactor. They may be regulated by fellow TCA cycle intermediates, succinate and fumarate, which act as competitive inhibitors of -ketoglutarate, thus rendering these demethylases sensitive to intracellular levels of all three metabolites [START_REF] Xiao | Inhibition of α-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors[END_REF]. Interestingly, it has been shown that fumarate and succinate levels are increased during C. trachomatis infection (Fig 38) [START_REF] Rother | Combined Human Genomewide RNAi and Metabolite Analyses Identify IMPDH as a Host-Directed Target against Chlamydia Infection[END_REF]. One caveat of this study is that the location (cytoplasm, inclusion lumen, bacteria?) of these compounds is not known. Still, it supports the hypothesis that the ratio of succinate + fumarate over -ketoglutarate increases in the host cytoplasm and could account for the demethylase inhibition. To investigate whether decreasing intracellular succinate would restore JmjC demethylase activity, OGDH of the OGDH complex was silenced with siRNA (Fig 31). The OGDH complex is responsible for conversion of -ketoglutarate to succinyl-CoA, which is further converted into succinate. Since silencing OGDH was sufficient to increase -ketoglutarate levels in stem cells and reduced H3K27me3 levels [START_REF] Baksh | Extracellular serine controls epidermal stem cell fate and tumour initiation[END_REF], we opted for a similar strategy. Though the siRNA reduced expression of OGDH (Fig 31B), it did not prevent hypermethylation of histones during late infection (Fig 31A). These data suggests that -ketoglutarate that transits through the mitochondrial TCA cycle only marginally contributes to the -ketoglutarate pool in infected cells and is not sufficient to replenish what is consumed by the bacteria. It has been well established that the bacteria readily take up glutamine, a precursor of -ketoglutarate through glutaminolysis [START_REF] Rajeeve | Reprogramming of host glutamine metabolism during Chlamydia trachomatis infection and its key role in peptidoglycan synthesis[END_REF]. Our current hypothesis is that glutamine rerouting towards the inclusion acts as a sink that deprives host enzymes of -ketoglutarate, and thereby limits the enzymes that depend on this co-factor. Of note, -ketoglutarate itself, might be diverted, as metabolic labelling of purified bacteria suggested that several bacterial amino acids can be made de novo from host TCAderived dicarboxylates such as malate, oxaloacetate or -ketoglutarate [START_REF] Mehlitz | Metabolic adaptation of Chlamydia trachomatis to mammalian host cells[END_REF]. In support of this hypothesis, it is important to note that the truncated TCA cycle of the bacteria starts with -ketoglutarate dehydrogenase, as it lacks the upstream enzyme, isocitrate dehydrogenase (Fig 9). It would thus ideally be fed with direct -ketoglutarate import from the host. Interestingly, the enzymes of this truncated TCA cycle are much more abundant in EBs than in RBs [START_REF] Saka | Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms[END_REF], indicating that this need for -ketoglutarate increases after RB to EB conversion. This timing fits well with our observation that histone hypermethylation occurs late in infection.

V. C. trachomatis infection and DNA damage

Consistent with the study by Chumduri et al, we observed an increase in phosphorylation of DNA damage mark, H2AX, during late infection (Fig 35A) [START_REF] Chumduri | Chlamydia infection promotes host DNA damage and proliferation but impairs the DNA damage response[END_REF]. Remarkably, we made the observation that cells with increased DNA damage also displayed histone hypermethylation (Fig 35A&B). Furthermore, DMKG supplementation prevented phosphorylation of H2AX during late infection in a similar fashion to methylation at histone lysine residues (Fig 37A&B). Taken together, these data establish a link between histone hypermethylation and DNA damage. There are two non-exclusive hypotheses that could account for this link: histone hypermethylation might favor DNA damage, and/or it might impair DNA damage repair (DDR). In support for this second scenario, a recent article showed that the increase in succinate to -ketoglutarate ratio resulted in aberrant hypermethylation of H3K9 at loci surrounding DNA breaks. This hypermethylation masks a local H3K9 trimethylation signal that is essential for the proper execution of DDR (Sulkowski et al, 2020). Chumduri et al reported that the DDR machinery was blocked in infected cells, with an absence of p-ATM or 53BP1 staining in H2AX positive nuclei (Chumduri et al). We did not reproduce these observations, as we observed increased phosphorylation of H2AX in nuclei of infected cells with increased p-ATM or 53BP1 (Fig 36). However, the pattern of distribution of these different markers shows some heterogeneity, and we are currently developing image analysis tools to quantify these images and determine whether DNA damage repair is defective in cells displaying histone hypermethylation. There was no overall shift in transcription in which a global increase or decrease in gene regulation was detected. These results may have been anticipated based on the methylation marks we observed (Fig 21A). Some of these marks are associated with gene expression (H3K4me3 and H3K79me3) and others with gene repression (H3K9me3 and H4K20me3), and the resulting transcriptome seems to be a balance of these antagonistic marks [START_REF] Jeon | Writing, erasing and reading histone lysine methylations[END_REF]. Thus, restoring demethylase activity through DMKG supplementation, would have mixed effects transcriptionally that will depend on the histone residue in question and its location on the chromosome. There were 115 protein-coding genes found to be differentially transcribed during infection with DMKG supplementation. They were analyzed with protein pathway enrichments algorithms: STRING, Kegg, Reactome, and GeneOntology.

VI. DMKG supplementation changes infection transcriptome

One of the pathways indicated from the protein pathway enrichment algorithms was the p53 transcriptional network in which GADD45A, CCNG2, PMAIP1, and SESN2 were differentially regulated. The p53 transcriptional network is comprised of pathways involved in DNA damage response, apoptosis, and cell cycle regulation; all pathways known to be dysregulated during C. trachomatis infection. qRT-PCR analysis of GADD45A and PMAIP1 confirmed the microarray results and displayed that while both genes undergo increased transcription during infection, that DMKG supplementation during infection increases their transcription even further (Fig 34).

Interestingly, it has been shown that intracellular levels of -ketoglutarate are linked to p53 activity [START_REF] Morris | α-Ketoglutarate links p53 to cell fate during tumour suppression[END_REF]. Morris et al, demonstrated that restoring p53 expression in p53 deficient cells or supplementing them with -ketoglutarate induced similar transcriptional profiles, with a prominence for the p53 signaling network. Human cells infected with C. trachomatis undergo a very pronounced reduction in p53 expression (Siegl et al, 2014;[START_REF] González | Chlamydia infection depends on a functional MDM2-p53 axis[END_REF].

Our system, in which we supplement p53-low cells (as a consequence of infection) with DMKG, thus shows similarity with the study by Morris et al, which also supplemented p53-deficent cells with DMKG. In both cases, the p53 transcriptional network stands out among the differentially regulated pathways. The molecular mechanism behind this observation remains unclear at this point.

It is also interesting to note at this point that GADD45A, which we confirmed to be upregulated in infection by qRT-PCR, promotes DNA demethylation [START_REF] Wang | LSD1n is an H4K20 demethylase regulating memory formation via transcriptional elongation control[END_REF]. DNA hypomethylation was recently described in testicular cells infected by C. trachomatis, and GADD45 upregulation could be implicated (Bryan et al, 2020).

MAPK signaling was also implicated by the protein pathway enrichment analysis, in which the genes FGF2, EPHA2, EFNA1, HSPA1A, CACNB2, and DUSP6 are differentially regulated.

Several reports have shown that the MAPK pathway is upregulated during the infectious cycle and is important for optimal bacterial development (Buchholz and Stephens, 2007;[START_REF] Ajonuma | CFTR is required for cellular entry and internalization of Chlamydia trachomatis[END_REF]Olive et al, 2014;[START_REF] Zadora | Integrated Phosphoproteome and Transcriptome Analysis Reveals Chlamydia-Induced Epithelial-to-Mesenchymal Transition in Host Cells[END_REF]. Differential regulation of the MAPK signaling cascade influences cell survival and could be advantageous for the bacteria. One remaining question is whether histone hypermethylation is implicated in the regulation of this pathway, or whether DMKG plays a role through another independent mechanism. To address this question, we plan to measure the level of these transcripts in infected cells treated with a low dose of the JmjC demethylase inhibitor, JIB-04. Indeed, we have observed that JIB-04 exacerbates histone hypermethylation in infected cells (Fig 26). Thus, any gene regulation that is driven by histone hypermethylation should be amplified by JIB-04 treatment.

Interestingly, over half of the DREs found to be differentially regulated during infection with DMKG supplementation were non-protein-coding RNAs, the majority of which were tRNAs.

Importantly, infection alone already seems to decrease tRNA levels, a finding that has never been reported. These results need to be verified by Northern blot. If true, this loss in tRNA could explain the block in protein translation recently reported in C. trachomatis infected cells [START_REF] Ohmer | Infection of HeLa cells with Chlamydia trachomatis inhibits protein synthesis and causes multiple changes to host cell pathways[END_REF], and it will be important to understand its molecular basis.

Concluding remarks

This work demonstrated that C. trachomatis induces hypermethylation of histones during late infection at multiple residues simultaneously. This phenotype is a result of decreased histone lysine demethylase activity due to decreased intracellular levels of their shared co-factor, -ketoglutarate.

Demethylase activity of -ketoglutarate-dependent JmjC demethylases can be restored by increasing intracellular levels of -ketoglutarate via the supplementation of infected cells with DMKG, a cell permeable form of -ketoglutarate. DMKG supplementation results in decreased histone methylation during late infection and circumvents DNA damage. Increased intracellular -ketoglutarate also changes the transcriptome of infected cells with differential regulation of the p53 transcriptional network, and MAPK signaling. Overall, this works demonstrates that shifts in metabolism and nutrient availability caused by an intracellular microbe impacts the epigenome of its host, with broad consequences on chromatin integrity and gene expression.

Figure 1 .

 1 Figure 1. Phylogenetic tree of Chlamydia genus .......................................................................... Figure 2. Status of Global Trachoma Endemicity (2019) ............................................................. Figure 3. Regional estimates of new cases of the four curable STIs ............................................Figure 4. Evidence of biphasic development ................................................................................ Figure 5. Schematic of chlamydial developmental cycle ............................................................. Figure 6. Cryo-EM of chlamydial T3SS ....................................................................................... Figure 7. Schematic of mechanisms by which Chlamydia may exit the cell ............................... Figure 8. Scanning-EM of extrusion events ................................................................................. Figure 9. TCA cycle of C. trachomatis.........................................................................................Figure 10. Illustration of Heitz's chromatin staining of Pellia epiphylla chromosomes .............. Figure 11. Schematic of chromosome formation .......................................................................... Figure 12. Schematic describing histone 'writers' and 'erasers' .................................................... Figure 13. Schematic summarizing the activity of histone acetylases (HATs) and histone deacetylases (HDACs) .................................................................................................................. Figure 14. Scheme representing the ubiquitination and deubiquitination processes of histone H2A ....................................................................................................................................................... Figure 15. Schematic of SET domain-containing histone methyltransferase activity .................. Figure 16. Schematic of histone demethylase mechanisms of action ........................................... Figure 17. Summary of methyltransferases and demethylases for the major histone lysine methylation sites ........................................................................................................................... Figure 18. Schematic summarizing the metabolites involved in histone methylation and demethylation ................................................................................................................................ Figure 19. Schematic demonstrating how the methionine and folate cycles interact to restore methionine levels ..........................................................................................................................Figure 20. Infection with C. trachomatis (Ctr) induces DNA damage but blocks DNA repair mechanisms ................................................................................................................................... Figure 21. Immunostaining of histone modifications in HeLa cells infected with C. trachomatis for 48 hours ................................................................................................................................... Figure 22. Immunostaining of histone C. trachomatis infection time course .............................. Figure 23. Histone methylation of patient-derived ectocervical epithelial cells during C. trachomatis infection ..................................................................................................................... Figure 24. Histone methylation in cells infected with C. muridarum .......................................... Figure 25. Immunostaing of HeLa cells infected for 48 hours with C.trachomatis L2 WT or C. trachomatis L2 NUE* ................................................................................................................... Figure 26. JIB-04 treatment induces increased methylation during infection ..............................Figure 27. Effect of iron on histone methylation during infection ...............................................Figure 28. Effect of DMKG supplementation on histone methylation during infection ..............Figure 29. Effect of DMKG supplementation on infectious progeny ..........................................Figure 30. Effect of DMKG supplementation added 24 hpi on histone methylation ...................Figure 31. Silencing OGDH does not affect histone methylation during late infection ............... Figure 32. Scheme for microarray preparation ............................................................................. Figure 33. Microarray analysis of DMKG supplementation during infection .............................. Figure 34. qRT-PCR verification of microarray data ...................................................................

Figure 4 .

 4 Figure 1. Phylogenetic tree of Chlamydia genus .......................................................................... Figure 2. Status of Global Trachoma Endemicity (2019) ............................................................. Figure 3. Regional estimates of new cases of the four curable STIs ............................................Figure 4. Evidence of biphasic development ................................................................................ Figure 5. Schematic of chlamydial developmental cycle ............................................................. Figure 6. Cryo-EM of chlamydial T3SS ....................................................................................... Figure 7. Schematic of mechanisms by which Chlamydia may exit the cell ............................... Figure 8. Scanning-EM of extrusion events ................................................................................. Figure 9. TCA cycle of C. trachomatis.........................................................................................Figure 10. Illustration of Heitz's chromatin staining of Pellia epiphylla chromosomes .............. Figure 11. Schematic of chromosome formation .......................................................................... Figure 12. Schematic describing histone 'writers' and 'erasers' .................................................... Figure 13. Schematic summarizing the activity of histone acetylases (HATs) and histone deacetylases (HDACs) .................................................................................................................. Figure 14. Scheme representing the ubiquitination and deubiquitination processes of histone H2A ....................................................................................................................................................... Figure 15. Schematic of SET domain-containing histone methyltransferase activity .................. Figure 16. Schematic of histone demethylase mechanisms of action ........................................... Figure 17. Summary of methyltransferases and demethylases for the major histone lysine methylation sites ........................................................................................................................... Figure 18. Schematic summarizing the metabolites involved in histone methylation and demethylation ................................................................................................................................ Figure 19. Schematic demonstrating how the methionine and folate cycles interact to restore methionine levels ..........................................................................................................................Figure 20. Infection with C. trachomatis (Ctr) induces DNA damage but blocks DNA repair mechanisms ................................................................................................................................... Figure 21. Immunostaining of histone modifications in HeLa cells infected with C. trachomatis for 48 hours ................................................................................................................................... Figure 22. Immunostaining of histone C. trachomatis infection time course .............................. Figure 23. Histone methylation of patient-derived ectocervical epithelial cells during C. trachomatis infection ..................................................................................................................... Figure 24. Histone methylation in cells infected with C. muridarum .......................................... Figure 25. Immunostaing of HeLa cells infected for 48 hours with C.trachomatis L2 WT or C. trachomatis L2 NUE* ................................................................................................................... Figure 26. JIB-04 treatment induces increased methylation during infection ..............................Figure 27. Effect of iron on histone methylation during infection ...............................................Figure 28. Effect of DMKG supplementation on histone methylation during infection ..............Figure 29. Effect of DMKG supplementation on infectious progeny ..........................................Figure 30. Effect of DMKG supplementation added 24 hpi on histone methylation ...................Figure 31. Silencing OGDH does not affect histone methylation during late infection ............... Figure 32. Scheme for microarray preparation ............................................................................. Figure 33. Microarray analysis of DMKG supplementation during infection .............................. Figure 34. qRT-PCR verification of microarray data ...................................................................

Figure 28 .

 28 Figure 1. Phylogenetic tree of Chlamydia genus .......................................................................... Figure 2. Status of Global Trachoma Endemicity (2019) ............................................................. Figure 3. Regional estimates of new cases of the four curable STIs ............................................Figure 4. Evidence of biphasic development ................................................................................ Figure 5. Schematic of chlamydial developmental cycle ............................................................. Figure 6. Cryo-EM of chlamydial T3SS ....................................................................................... Figure 7. Schematic of mechanisms by which Chlamydia may exit the cell ............................... Figure 8. Scanning-EM of extrusion events ................................................................................. Figure 9. TCA cycle of C. trachomatis.........................................................................................Figure 10. Illustration of Heitz's chromatin staining of Pellia epiphylla chromosomes .............. Figure 11. Schematic of chromosome formation .......................................................................... Figure 12. Schematic describing histone 'writers' and 'erasers' .................................................... Figure 13. Schematic summarizing the activity of histone acetylases (HATs) and histone deacetylases (HDACs) .................................................................................................................. Figure 14. Scheme representing the ubiquitination and deubiquitination processes of histone H2A ....................................................................................................................................................... Figure 15. Schematic of SET domain-containing histone methyltransferase activity .................. Figure 16. Schematic of histone demethylase mechanisms of action ........................................... Figure 17. Summary of methyltransferases and demethylases for the major histone lysine methylation sites ........................................................................................................................... Figure 18. Schematic summarizing the metabolites involved in histone methylation and demethylation ................................................................................................................................ Figure 19. Schematic demonstrating how the methionine and folate cycles interact to restore methionine levels ..........................................................................................................................Figure 20. Infection with C. trachomatis (Ctr) induces DNA damage but blocks DNA repair mechanisms ................................................................................................................................... Figure 21. Immunostaining of histone modifications in HeLa cells infected with C. trachomatis for 48 hours ................................................................................................................................... Figure 22. Immunostaining of histone C. trachomatis infection time course .............................. Figure 23. Histone methylation of patient-derived ectocervical epithelial cells during C. trachomatis infection ..................................................................................................................... Figure 24. Histone methylation in cells infected with C. muridarum .......................................... Figure 25. Immunostaing of HeLa cells infected for 48 hours with C.trachomatis L2 WT or C. trachomatis L2 NUE* ................................................................................................................... Figure 26. JIB-04 treatment induces increased methylation during infection ..............................Figure 27. Effect of iron on histone methylation during infection ...............................................Figure 28. Effect of DMKG supplementation on histone methylation during infection ..............Figure 29. Effect of DMKG supplementation on infectious progeny ..........................................Figure 30. Effect of DMKG supplementation added 24 hpi on histone methylation ...................Figure 31. Silencing OGDH does not affect histone methylation during late infection ............... Figure 32. Scheme for microarray preparation ............................................................................. Figure 33. Microarray analysis of DMKG supplementation during infection .............................. Figure 34. qRT-PCR verification of microarray data ...................................................................

Figure 29 .

 29 Figure 1. Phylogenetic tree of Chlamydia genus .......................................................................... Figure 2. Status of Global Trachoma Endemicity (2019) ............................................................. Figure 3. Regional estimates of new cases of the four curable STIs ............................................Figure 4. Evidence of biphasic development ................................................................................ Figure 5. Schematic of chlamydial developmental cycle ............................................................. Figure 6. Cryo-EM of chlamydial T3SS ....................................................................................... Figure 7. Schematic of mechanisms by which Chlamydia may exit the cell ............................... Figure 8. Scanning-EM of extrusion events ................................................................................. Figure 9. TCA cycle of C. trachomatis.........................................................................................Figure 10. Illustration of Heitz's chromatin staining of Pellia epiphylla chromosomes .............. Figure 11. Schematic of chromosome formation .......................................................................... Figure 12. Schematic describing histone 'writers' and 'erasers' .................................................... Figure 13. Schematic summarizing the activity of histone acetylases (HATs) and histone deacetylases (HDACs) .................................................................................................................. Figure 14. Scheme representing the ubiquitination and deubiquitination processes of histone H2A ....................................................................................................................................................... Figure 15. Schematic of SET domain-containing histone methyltransferase activity .................. Figure 16. Schematic of histone demethylase mechanisms of action ........................................... Figure 17. Summary of methyltransferases and demethylases for the major histone lysine methylation sites ........................................................................................................................... Figure 18. Schematic summarizing the metabolites involved in histone methylation and demethylation ................................................................................................................................ Figure 19. Schematic demonstrating how the methionine and folate cycles interact to restore methionine levels ..........................................................................................................................Figure 20. Infection with C. trachomatis (Ctr) induces DNA damage but blocks DNA repair mechanisms ................................................................................................................................... Figure 21. Immunostaining of histone modifications in HeLa cells infected with C. trachomatis for 48 hours ................................................................................................................................... Figure 22. Immunostaining of histone C. trachomatis infection time course .............................. Figure 23. Histone methylation of patient-derived ectocervical epithelial cells during C. trachomatis infection ..................................................................................................................... Figure 24. Histone methylation in cells infected with C. muridarum .......................................... Figure 25. Immunostaing of HeLa cells infected for 48 hours with C.trachomatis L2 WT or C. trachomatis L2 NUE* ................................................................................................................... Figure 26. JIB-04 treatment induces increased methylation during infection ..............................Figure 27. Effect of iron on histone methylation during infection ...............................................Figure 28. Effect of DMKG supplementation on histone methylation during infection ..............Figure 29. Effect of DMKG supplementation on infectious progeny ..........................................Figure 30. Effect of DMKG supplementation added 24 hpi on histone methylation ...................Figure 31. Silencing OGDH does not affect histone methylation during late infection ............... Figure 32. Scheme for microarray preparation ............................................................................. Figure 33. Microarray analysis of DMKG supplementation during infection .............................. Figure 34. qRT-PCR verification of microarray data ...................................................................

Figure 30 .

 30 Figure 1. Phylogenetic tree of Chlamydia genus .......................................................................... Figure 2. Status of Global Trachoma Endemicity (2019) ............................................................. Figure 3. Regional estimates of new cases of the four curable STIs ............................................Figure 4. Evidence of biphasic development ................................................................................ Figure 5. Schematic of chlamydial developmental cycle ............................................................. Figure 6. Cryo-EM of chlamydial T3SS ....................................................................................... Figure 7. Schematic of mechanisms by which Chlamydia may exit the cell ............................... Figure 8. Scanning-EM of extrusion events ................................................................................. Figure 9. TCA cycle of C. trachomatis.........................................................................................Figure 10. Illustration of Heitz's chromatin staining of Pellia epiphylla chromosomes .............. Figure 11. Schematic of chromosome formation .......................................................................... Figure 12. Schematic describing histone 'writers' and 'erasers' .................................................... Figure 13. Schematic summarizing the activity of histone acetylases (HATs) and histone deacetylases (HDACs) .................................................................................................................. Figure 14. Scheme representing the ubiquitination and deubiquitination processes of histone H2A ....................................................................................................................................................... Figure 15. Schematic of SET domain-containing histone methyltransferase activity .................. Figure 16. Schematic of histone demethylase mechanisms of action ........................................... Figure 17. Summary of methyltransferases and demethylases for the major histone lysine methylation sites ........................................................................................................................... Figure 18. Schematic summarizing the metabolites involved in histone methylation and demethylation ................................................................................................................................ Figure 19. Schematic demonstrating how the methionine and folate cycles interact to restore methionine levels ..........................................................................................................................Figure 20. Infection with C. trachomatis (Ctr) induces DNA damage but blocks DNA repair mechanisms ................................................................................................................................... Figure 21. Immunostaining of histone modifications in HeLa cells infected with C. trachomatis for 48 hours ................................................................................................................................... Figure 22. Immunostaining of histone C. trachomatis infection time course .............................. Figure 23. Histone methylation of patient-derived ectocervical epithelial cells during C. trachomatis infection ..................................................................................................................... Figure 24. Histone methylation in cells infected with C. muridarum .......................................... Figure 25. Immunostaing of HeLa cells infected for 48 hours with C.trachomatis L2 WT or C. trachomatis L2 NUE* ................................................................................................................... Figure 26. JIB-04 treatment induces increased methylation during infection ..............................Figure 27. Effect of iron on histone methylation during infection ...............................................Figure 28. Effect of DMKG supplementation on histone methylation during infection ..............Figure 29. Effect of DMKG supplementation on infectious progeny ..........................................Figure 30. Effect of DMKG supplementation added 24 hpi on histone methylation ...................Figure 31. Silencing OGDH does not affect histone methylation during late infection ............... Figure 32. Scheme for microarray preparation ............................................................................. Figure 33. Microarray analysis of DMKG supplementation during infection .............................. Figure 34. qRT-PCR verification of microarray data ...................................................................

Figure 31 .

 31 Figure 1. Phylogenetic tree of Chlamydia genus .......................................................................... Figure 2. Status of Global Trachoma Endemicity (2019) ............................................................. Figure 3. Regional estimates of new cases of the four curable STIs ............................................Figure 4. Evidence of biphasic development ................................................................................ Figure 5. Schematic of chlamydial developmental cycle ............................................................. Figure 6. Cryo-EM of chlamydial T3SS ....................................................................................... Figure 7. Schematic of mechanisms by which Chlamydia may exit the cell ............................... Figure 8. Scanning-EM of extrusion events ................................................................................. Figure 9. TCA cycle of C. trachomatis.........................................................................................Figure 10. Illustration of Heitz's chromatin staining of Pellia epiphylla chromosomes .............. Figure 11. Schematic of chromosome formation .......................................................................... Figure 12. Schematic describing histone 'writers' and 'erasers' .................................................... Figure 13. Schematic summarizing the activity of histone acetylases (HATs) and histone deacetylases (HDACs) .................................................................................................................. Figure 14. Scheme representing the ubiquitination and deubiquitination processes of histone H2A ....................................................................................................................................................... Figure 15. Schematic of SET domain-containing histone methyltransferase activity .................. Figure 16. Schematic of histone demethylase mechanisms of action ........................................... Figure 17. Summary of methyltransferases and demethylases for the major histone lysine methylation sites ........................................................................................................................... Figure 18. Schematic summarizing the metabolites involved in histone methylation and demethylation ................................................................................................................................ Figure 19. Schematic demonstrating how the methionine and folate cycles interact to restore methionine levels ..........................................................................................................................Figure 20. Infection with C. trachomatis (Ctr) induces DNA damage but blocks DNA repair mechanisms ................................................................................................................................... Figure 21. Immunostaining of histone modifications in HeLa cells infected with C. trachomatis for 48 hours ................................................................................................................................... Figure 22. Immunostaining of histone C. trachomatis infection time course .............................. Figure 23. Histone methylation of patient-derived ectocervical epithelial cells during C. trachomatis infection ..................................................................................................................... Figure 24. Histone methylation in cells infected with C. muridarum .......................................... Figure 25. Immunostaing of HeLa cells infected for 48 hours with C.trachomatis L2 WT or C. trachomatis L2 NUE* ................................................................................................................... Figure 26. JIB-04 treatment induces increased methylation during infection ..............................Figure 27. Effect of iron on histone methylation during infection ...............................................Figure 28. Effect of DMKG supplementation on histone methylation during infection ..............Figure 29. Effect of DMKG supplementation on infectious progeny ..........................................Figure 30. Effect of DMKG supplementation added 24 hpi on histone methylation ...................Figure 31. Silencing OGDH does not affect histone methylation during late infection ............... Figure 32. Scheme for microarray preparation ............................................................................. Figure 33. Microarray analysis of DMKG supplementation during infection .............................. Figure 34. qRT-PCR verification of microarray data ...................................................................

Figure 35 .Table 1 . 2 .

 3512 Figure 35. C. trachomatis infection does not inhibit DNA repair machinery .............................. Figure 36. DNA damage occurs in infected cells with increased histone methylation ................ Figure 37. Effect of DMKG supplementation on DNA damage during infection........................ Figure 38. Levels of TCA cycle intermediates during C. trachomatis infection ..........................

  d'histones contenant un domaine Jumonji (déméthylases JmjC) utilisent l'oxygène, le fer et l'ketoglutarate, métabolites qui sont tous utilisés par C. trachomatis. L'observation que les cellules infectées sont sensibilisées à un inhibiteur de cette famille d'enzymes soutient l'hypothèse d'un fonctionnement réduit de ces enzymes dans les cellules infectées. Nos expériences indiquent que a disponibilité en fer n'est pas limitante durant l'infection. En revanche, l'apport de dimethyl-ketoglutarate (DMKG), un substitut d'-ketoglutarate, permet d'abolir l'hyperméthylation des histones. Ce résultat indique que ce phénotype est le résultat d'un déséquilibre métabolique, probablement causé par la consommation bactérienne d'-ketoglutarate ou de ses précurseurs durant l'infection. L'apport en DMKG modifie le niveau de transcription d'environ un tiers des gènes qui montrent une expression différentielle tard dans l'infection. Les conséquences sont à la fois négatives et positives, et ce dans des proportions similaires, indiquant que l'hyperméthylation des histones peut avoir des conséquences opposées sur l'expression des gènes, qui doit être analysée au niveau individuel. Enfin, nous avons observé que l'hyperméthylation des histones corrélait avec la présences d'indicateurs de cassures de l'ADN dans les noyaux des cellules infectées. L'apport en DMKG résulte en une perte de ces cassures, confirmant le lien entre ces deux phénotypes. En conclusion, nous avons démontré que l'infection par C. trachomatis crée un déséquilibre métabolique chez l'hôte, à l'origine d'hyperméthylation des histones. Cette empreinte métabolique de l'infection au niveau épigénétique a des conséquences sur la réponse génétique de l'hôte à l'infection et sur l'intégrité de son ADN.

  effectrices. Le principal mécanisme de sécrétion des Chlamydiaceae est appelé système de sécrétion de type 3, il est commun à plusieurs bactéries à Gram négatif pathogènes. Les protéines effectrices permettent aux bactéries de manipuler l'hôte et de détourner certaines fonctions cellulaires à leur profit. Une des protéines effectrice de C. trachomatis, NUE, possède un domaine également trouvé dans des enzymes capables de méthyler des histones. NUE possède effectivement une activité histone methyl transférase in vitro, suggérant que par son intermédiaire C. trachomatis puisse modifier l'épigénome des cellules qu'elle infecte.L'information génétique des cellules hôtes est renfermée dans le noyau, au sein de la chromatine, terme qui désigne l'ensemble de l'ADN et des protéines associées. La lecture de cette information est conditionnée à son accessibilité, elle-même gouvernée par la composition de la chromatine. L'élément de base en est le nucléosome, constitué de 146 paires de bases d'ADN enroulées autour d'un assemblage de 2x4 molécules d'histones (H2A, H2B, H3 et H4). Les histones et l'ADN sont susceptibles de subir des modifications chimiques réversibles qui influencent l'accessibilité de l'ADN, et donc l'expression génique. Certaines bases de l'ADN peut être modifiées par méthylation, tandis que les histones sont l'objet d'une variété de modifications, telles que la méthylation, l'acétylation, la phosphorylation, l'ubiquitinylation, surtout concentrées dans la partie N-terminale des histones, appelées queues d'histones. La combinatoire des modifications dites « épigénétiques » affectant les histones constitue un deuxième code, dit « code histones », qui se superpose au code génétique. Cette combinatoire est complexe car plusieurs résidus peuvent être modifiés, avec plusieurs types de modification. Les résidus lysines sont abondants dans les queues d'histones, et sont sujets à un grand nombre de modifications différentes, surtout des acétylations et des méthylations. Ces dernières peuvent concerner un, deux, ou trois groupe méthyl, ajoutant à la complexité de la combinatoire. De ces modifications, ainsi que de l'association d'autres protéines à l'ADN, et des modifications de l'ADN lui-même, va dépendre le degré de condensation de la chromatine. Elle est traditionnellement décrite comme euchromatine, peu condensée, accessible, et hétérochromatine, plus dense, moins accessible, mais ces termes ne rendent pas compte de différences locales qui gouvernent l'expression des gènes concernés. Les modifications épigénétiques sont réversibles, elles sont effectuées par des enzymes qualifiées d' « écrivaines » pour celles qui inscrivent les marques épigénétiques (methyl transférases, acetyl transférases ...) et d' « effaceuses » pour celles qui les retirent (demethylases, deacetylases...). Les méthylations d'histones sont assurées par plusieurs familles d'enzymes écrivaines histone methyl transférases, qui toutes utilisent la S-adenosylmethionine comme cofacteur. Leur déméthylation est assurée par plusieurs histones demethylases, regroupées en deux familles. La plus vaste, appelée famille Jumonji C (JmjC), contient des deoxygenases dépendante de l'-ketoglutarate, tandis que les deux enzymes connues de la deuxième famille utilisent la flavine adénine dinucléotide (FAD) comme co-facteur. Selon la lysine methylée, le type de méthylation (mono, di, tri), et la position de l'octamère d'histone par rapport au gène (enhancer, promoteur...) la méthylation peut faciliter l'accès de la machinerie de transcription ou au contraire la rendre plus difficile, ayant donc des effets antagonistes sur l'expression génique. Naturellement, l'infection par C. trachomatis entraine une modification importante de l'expression des gènes de l'hôte, impliquant certainement des modifications épigénétiques. Une partie de cette réaction constitue une « réponse » de l'hôte à l'infection. Par exemple la reconnaissance de déterminants « étrangers » déclenche l'activation de voies de défenses, en particulier inflammatoires, visant à l'élimination de l'intrus. En outre, la cohabitation avec les bactéries en division nécessite que l'hôte adapte son métabolisme pour faire face aux demandes accrues en nutriment. Une partie de ces modifications pourraient aussi être sous le contrôle des bactéries, par analogie à ce qui a été documenté dans le cas de la cohabitation microbiote/tissu, où le métabolisme du microbiote influence l'épigénome du tissu à son contact. De plus, la découverte d'une enzyme capable de méthyler les histones parmi les protéines sécrétées de C. trachomatis suggère que les bactéries aient la capacité de modifier directement les histones. Une étude précédente a montré que globalement plusieurs lysines des histones 3 et 4 sont methylées au cours de l'infection. Nous démontrons ici que cette hyperméthylation est un phénomène tardif du cycle infectieux, et concerne tous les résidus lysines, de façon corrélée. Nous avons alors chercher à comprendre les raisons de cette hyperméthylation des histones, et ses conséquences. Nous avons observé que toutes les marques de méthylation d'histones que nous avons testées étaient détectées de façon concomitante dans certaines cellules infectées, phénomène que nous désignons sous le terme d'hyperméthylation des histones. Il apparait tardivement dans le cycle infectieux, après l'initiation de la conversion des RBs en EBs. Initialement observé dans la lignée de cellules épithéliales d'origine cancéreuse HeLa, cette observation a également été faite dans des cellules épithéliales primaires issues de l'ecto-col. Elle n'est pas limitée à C. trachomatis, puisque l'espère C. muridarum, adaptée aux espèces murines, induit également une hyperméthylation des histones. Enfin, cette hyperméthylation n'est pas l'oeuvre de la methyl transferase bactérienne NUE, puisqu'elle est aussi observée lorsque les cellules sont infectées par une souche mutante n'exprimant pas NUE.

Figure 1 .

 1 Figure 1. Phylogenetic tree of Chlamydia genus Figure 1 (A) Phylogenetic tree of Chlamydia genus with natural hosts shown for each species. (B) C. trachomatis strains segregated by tissue-tropism based on the analysis of 819 genes which were conserved across the serovars. (From Nunes and Gomes, 2014).

1. 2

 2 Diseases, Treatments and Sequelae 1.2.A Trachoma (A, B, Ba, and C) Ocular infection of C. trachomatis serovars A, B, Ba, and C are the leading infectious causes of blindness globally. The WHO estimates that active trachoma affects up to 21 million people, with about 2.2 million who are blind or visually impaired. At least 7.3 million individuals suffer from potentially blinding trichiasis, an inversion of the eyelashes such that they scratch and damage the cornea. Trachoma is most prevalent in Africa, but also affects poor and remote areas in Asia, the Middle East, and Australia (Fig 2) (WHO).

Figure 6 .

 6 Figure 6. Cryo-EM of chlamydial T3SS Figure 6 Cryo-electron tomography of chlamydial T3SS in contact with host cell membrane during early infection in which an EB has made contact with a HeLa cell. Schematic of T3SS structure in which the injectosome reaches through the inner membrane (IM), outer membrane (OM) of the bacteria and makes contact with the host cell membrane (HCM). (Adapted from Nans et al, 2015)

35 2 .

 2 Chromatin remodeling and its influence on gene expression 2.1 Chromatin Structure and Function 2.1.A Overview of Chromatin Function: Heterochromatin vs. Euchromatin A human diploid cell contains approximately 6 billion nucleotide base pairs, which adds up to about 2 meters worth of DNA per cell

Figure 10 .Figure 10

 1010 Figure 10. Illustration of Heitz's chromatin staining of Pellia epiphylla chromosomes

Figure 11 .

 11 Figure 11. Schematic of chromosome formation Figure 11 Schematic of chromosome formation. Free DNA associates with histone core to form nucleosomes that further compact into the chromatin fiber, which condenses even further into a chromosome.

Figure 12 .Figure 12

 1212 Figure 12. Schematic describing histone 'writers' and 'erasers' Figure 12 Readers recognize post-translational modifications made to histone tails of H3 and H4, among other chromatin associated landmarks. Writers add modifications like methyl, acetyl, and phosphate groups. Erasers are capable of removing modifications made by the writers.

Figure 13 .Figure 13

 1313 Figure 13. Schematic summarizing the activity of histone acetylases (HATs) and histone deacetylases (HDACs) Figure 13 Schematic summarizing the activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs utilize Acetyl-CoA as a co-factor to attach an acetyl group to lysine residues of histone tails. HDACs remove acetyl groups, forming acetate. (From Sanaei[START_REF] Sanaei | Histone Deacetylases and Histone Deacetylase Inhibitors: Molecular Mechanisms of Action in Various Cancers[END_REF]

Figure 14 .Figure 14

 1414 Figure 14. Scheme representing the ubiquitination and deubiquitination processes of histone H2A Figure 14 Scheme representing ubiquitination and deubiquitination process of H2A.Ubiquitin activating enzyme, E1, attaches ubiquitin to ubiquitin-conjugating enzyme, E2. Ubiquitin ligating enzyme, E3 then ligates ubiquitin from E2 to the target mark. Ubiquitin can be removed by deubiquitinating enzyme (DUB) activity.

Figure 15 .

 15 Figure 15. Schematic of SET domain-containing histone methyltransferase activity Figure 15 Schematic of SET domaincontaining histone methyltransferase activity. HMTs remove a methyl group from S-adenosyl-L-methionine and transfer it to the amino group of the target lysine residue (From Koturbash et al, 2012).

  dinucleotide (FAD), to demethylate H3K3me1/2 or H3K9me1/2 via their amine-oxidase (AO)like domains. As depicted in Figure16A, LSD1 utilizes FAD to oxidize the methyl group on the methylated lysine which forms an imine intermediate. This intermediate is then hydrolyzed which results in a now demethylated lysine residue, and formaldehyde as a byproduct. Reduced FAD (FADH -) is reoxidized by molecular oxygen to replenish FAD and produces peroxide as a byproduct[START_REF] Koturbash | Alterations in histone H4 lysine 20 methylation: implications for cancer detection and prevention[END_REF][START_REF] Maiques-Diaz | LSD1: biologic roles and therapeutic targeting[END_REF].

Figure 16 .Figure 16

 1616 Figure 16. Schematic of histone demethylase mechanisms of action Figure 16 Schematic of histone demethylase mechanisms of action. A) LSD1 oxidizes the methyl group on the methylated lysine with FAD to form an imine intermediate. Hydrolysis of the imine intermediate results in formation of a demethylated lysine residue, and formaldehyde. B) JHDM hydroxylates the methyllysine group using O2 and -ketoglutarate which creates a hydroxylmethyllysine intermediate, and byproducts of carbon dioxide and succinate. This hydroxyl-methyllysine intermediate decomposes to yield a demethylated lysine residue and formaldehyde. (From Koturbash et al, 2012)

Figure 17 .

 17 Figure 17. Summary of methyltransferases and demethylases for the major histone lysine methylation sites Figure 17 Schematic summarizing methyltransferases (writers) and demethylases (erasers) for the major histone lysine methylation sites. Level of methylation associated with each is displayed by a single circle (me1), double circles (me2), and triple circles (me3). (From Hyun et al, 2017)

  Transmethylation reactions involving SAM, like the methylation of histone lysine residues, creates S-adenoysl-L-homocysteine (SAH) as a byproduct. SAH competitively inhibits histone methyltransferases and must thus be rapidly converted back into SAM else risk blocking methyltransferase activity. The balance of SAM and SAH is maintained by the methionine and folate cycles (Fig 19).

Figure 19 .Figure 19

 1919 Figure 19. Schematic demonstrating how the methionine and folate cycles interact to restore methionine levels Figure 19Schematic demonstrating how the methionine and folate cycles interact to restore methionine levels. Dietary methionine and ATP are necessary for the formation of SAM, which is converted to SAH after donating its methyl group. SAH is converted to homocysteine, which can either be further converted to glutathioine, or back to methionine. Methionine synthetase (MS), which is B12-depedent, catalyzes the formation of methionine by removing a methyl group form methyl-tetrahydrofolate to homocysteine (methyl-THF).(From McCaddon and Miller, 2015)

Figure 20 .Figure 20

 2020 Figure 20. Infection with C. trachomatis (Ctr) induces DNA damage but blocks DNA repair mechanisms Figure 20 Infection with C. trachomatis (Ctr) induces DNA damage but blocks DNA repair mechanisms. (A) End1 E6/7 cells 36 hpi with or without etoposide treatment were stained for phospho-H2AX to indicate DSBs and phospho-ATM to indicate recruitment of DNA repair machinery (B) End1 E6/7 cells 36 hpi with or without etoposide treatment were stained for phospho-H2AX to indicate DSBs and 53BP1 to indicate recruitment of DNA repair machinery (From Chumduri et al, 2013)

2

  with C. trachomatis LGV L2. 24 hpi, cell culture media was exchanged, and cells were treated with either 5 mM DMKG or an equal volume of DMSO. 48 hpi, cells were collected fractionation and resuspended in 574.6 l of Hypotonic Buffer (10 mM Tris pH 7.65, 1.5 mM MgCl2, 10 mM KCl, 0.2% NP-40, 0.6mM PMSF, 1X Protease inhibitor cocktail (Sigma)). Cells were lysed using a Dounce homogenizer, and then 192 l of Sucrose buffer (20 mM Tris-HCl ph 7.65, 15 mM KCl, 60 mM NaCl, 0.34 M sucrose, 150 M Spermine, 50M Spermidine, 0.6mM PMSF, 1X Protease inhibitor cocktail (Sigma)) was added to the samples. Sample were centrifuged 500xg for 10 minutes. The supernantant (cytosolic fraction) was reserved, and the pellet was resuspended in 5 ml of Wash buffer (1 mM Tris-HCl pH 7.5, 5 mM MgCl2, 60 mM KCl, 0.5 mM DTT, 15 mMNaCl, 300 mM sucrose). Nuclei were centrifuged at 1700xg for 10 minutes and the nuclei was discarded. Nuclei were resuspended in 500 l of 1XPBS to be used for other experiments.

(

  Fig 23A). Hypermethylation of H3K9 and H4K20 was observed in infected primary cells, compared to non-infected cells, confirming the observation made in the HeLa cell line.Finally, to know whether these observations were specific to the human adapted strain C. trachomatis, we infected HeLa cells for 48 h with C. muridarum. We observed that this mouse adapted strain also elicited hypermethylation of H3K4 and H4K20, showing that histone hypermethylation is not specific of C. trachomatis infection (Fig24).

Figure 24 .Figure 23 .Figure 23 Figure 24

 24232324 Figure 24. Histone methylation in cells infected with C. muridarum

Figure 25 .Figure 25

 2525 Figure 25. Immunostaing of HeLa cells infected for 48 hours with C.trachomatis L2 WT or C. trachomatis L2 NUE* Figure 25 Immunostaining of HeLa cells infected for 48 hours with C. trachomatis L2 WT or C. trachomatis L2 NUE*

Figure 27 .Figure 27

 2727 Figure 27. Effect of iron on histone methylation during infection

Figure 37 .Figure 37

 3737 Figure 37. Effect of DMKG supplementation on DNA damage during infection

(

  Fig 28A), it was determined that 5 mM DMKG was sufficient to reduce histone methylation during late infection by(Fig 28B). Note that reversion of histone hypermethylation by supplying DMKG was also reported in other studies. In epidermal stem cells, DMKG addition resulted in reduced trimethylation of H3K27me3[START_REF] Baksh | Extracellular serine controls epidermal stem cell fate and tumour initiation[END_REF]. In human embryonic stem cells engaged into neuroectodermal differentiation, DMKG supplementation caused a decrease in H3K4, H3K9, H3K27, H3K36 trimethylation marks[START_REF] Teslaa | α-Ketoglutarate Accelerates the Initial Differentiation of Primed Human Pluripotent Stem Cells[END_REF]. DMKG supplementation affected bacteria development. During the initial experiments, DMKG was added 2 hpi and cells were processed 48 hpi (Fig 28). It was noted that the inclusions in the DMKG-treated cells were smaller than those of the infected control, so progeny assays were performed to determine if DMKG treatment was affecting infectious progeny (Fig 29A). It was found that addition of DMKG at 2 hpi induced a significant decrease in infectious progeny, that could be partially rescued by moving the addition time to 24 hpi (Fig 29B). Addition of DMKG 24 hpi still prevented hypermethylation during late infection at multiple histone lysine residues (Fig 30). With this experimental set-up, the reduction on bacterial progeny is of about 20%. One preliminary experiment indicates that treatment of infected cells with 50 nM doxycycline reduced the progeny by 50% without affecting histone hypermethylation. Quantification of the results is pending and should allow us to rule out the possibility that the reduction in histone hypermethylation with DMKG supplementation during infection is accounted for by a reduction in bacterial growth. At this point it is unclear why DMKG supplementation reduces bacterial proliferation. It could be because of the indirect effect this treatment has on host gene expression,and thus on the environment faced by the bacteria. Alternatively, it could result from an imbalance in the host metabolism that affects the nutrients required for optimal bacterial growth. In an attempt to address this question, we treated DMKG-supplemented cells with low concentration (0.5 M)

Figure 38 .

 38 Figure 38. Levels of TCA cycle intermediates during C. trachomatis infection Figure 38 Levels of TCA metabolites during C. trachomatis infection. HeLa cells were infected with C. trachomatis LGV L2 at MOI 1 or left uninfected for 12, 24, 36 and 48 h. Metabolite levels were analyzed using gas chromatography-mass spectrometry (GC-MS). Histograms represent the log2 values of the ratios of the fold changes between infected and non-infected samples (mean ± SD) Boxed in red are the TCA intermediates capable of inhibiting JmjC histone lysine demethylases via competitive inhibition with -ketoglutarate (boxed in green). Abbreviations of the metabolites: 2OHG: 2-Hydroxyglutarate; aKG: a-ketoglutarate; Cit: Citrate; Fum: Fumarate; GABA: -Aminobutyric acid; Glu: Glutamate; Mal: Malate; Succ: Succinate. (Adapted from Rother et al 2018).

A

  microarray study was carried out to investigate how preventing histone hypermethylation by DMKG supplementation influenced host transcription (Fig 32). Between each of the conditions, the number of elements differentially up-or down-regulated was split fairly evenly (Fig 33A). Interestingly, ~1/3 of DREs normally differentially regulated during infection were no longer so with DMKG supplementation (Fig 33B). Hierarchical clustering was performed on the DREs which were differentially regulated during infection with DMKG supplementation (Fig 33C).

  Le phylum des Chlamydiae est constitué de bactéries intracellulaires obligatoires à Gram négatif. Ses membres se distinguent par un cycle de développement original, se déroulant à l'intérieur d'un hôte eucaryote, pouvant être un protozoaire (amibes par exemple) ou un métazoaire. Le phylum ne compte pour le moment qu'un seul Ordre et qu'une seule Classe, mais plusieurs Familles, telles que les Chlamydiaceae. Cette dernière regroupe des pathogènes d'animaux, dont plusieurs ont un impact en santé vétérinaire et humaine. Deux espèces de Dès 20 hpi, certains RBs changent de programme transcriptionel et initient leur conversion en EB, de manière asynchrone, de sorte que 48 hpi, l'inclusion renferme très majoritairement des EBs. Les EBs sont relargués de la cellule-hôte soit parce que celle-ci est lysée, soit par un processus d'extrusion qui laisse l'inclusion intacte, extracellulaire. Les EB libérés commencent un nouveau cycle dans les cellules épithéliales avoisinantes.

	Chlamydiaceae

sont couramment retrouvées dans des infections humaines : Chlamydia trachomatis et Chlamydia pneumoniae. C. pneumoniae est capable d'infecter des hôtes variés (koala, cheval, reptiles...) et cause des infections de l'appareil respiratoire chez l'Homme. En revanche, C. trachomatis utilise exclusivement l'espère humaine pour se reproduire. Certaines souches (serovars A à C) infectent la conjonctive de l'oeil. En absence de traitement, des infections répétées peuvent conduire à une altération de la cornée. Les séquelles de l'infection donnent lieu à ce qu'on appelle le trachome, première cause de cécité acquise au monde, essentiellement circonscrite à des régions ne bénéficiant pas de condition d'hygiène adéquates. Les autres sérovars sont retrouvés dans toutes les populations et infectent le tractus uro-génital. Les infections sont le plus souvent asymptomatiques, et, non détectées, ne sont pas toujours traitées. A l'origine d'inflammations pelviennes chroniques, de grossesse extra-utérines, elles peuvent être causes d'infertilité. Globalement, l'infection par C. trachomatis représente la première infection sexuellement transmissible d'origine bactérienne, et l'on recense plus de 100 millions de nouveaux cas par an. Le cycle de développement des Chlamydiae est désigné par le terme « biphasique ». En effet, deux formes de la bactérie de morphologie et de métabolisme distincts alternent au cours de ce cycle. La forme infectieuse est le corps élémentaire (EB), qui est non réplicatif et adapté à la survie en milieu extracellulaire. La forme réplicative est le corps réticulé (RB). Elle n'est pas infectieuse, et ne survit que dans l'environnement intracellulaire à laquelle elle est adaptée. Le cycle infectieux est initié par l'adhésion d'un EB à la surface d'une cellule-hôte, et son internalisation au sein d'une vacuole parasitophore appelée inclusion. L'internalisation de l'EB induit une décondensation progressive du chromosome bactérien, et un changement du programme d'expression de ses gènes, qui accompagne la transformation progressive vers la forme RB. La première division bactérienne a lieu environ 6 h après l'initiation de l'infection (6 hpi), suivi d'une croissance exponentielle, jusqu'à atteindre plusieurs centaines de bactéries par inclusion.

Table 2 .

 2 Antibodies for Immunofluorescence

		Target	Species	Supplier	Catalog #
		H3K9me3	Rabbit	Abcam	ab8898
		H4K20me3	Mouse	Abcam	ab78517
		H3K27me3	Mouse	Bp biosciences	25243
		H3K4me3	Rabbit	Diagenode	C15410003
		H3K79me2	Rabbit	Abcam	ab3594
				Cell
	Primary	Phospho-H2AX	Rabbit	Signaling	2577
	Antibodies			Technology
		H2AX	Mouse	Merck Millipore	05-636
		Phospho-ATM Ser 1981	Rabbit	R&D	AF1655
		53BP1	Rabbit	Novus Biologics	NB100-304
		H3phosphoSer10	Rabbit	Abcam	ab5176
		H3phosphoSer28	Rabbit	Abcam	ab5169
		Alexa Fluor TM 488 goat anti-rabbit IgG (H+L)	Goat	Invitrogen	A-11034
	Secondary	Alexa Fluor TM 647 goat anti-rabbit IgG (H+L)	Goat	Invitrogen	A-21244
	Antibodies	Alexa Fluor TM 488 goat anti-mouse IgG (H+L)	Goat	Invitrogen	A-11029
		Alexa Fluor TM 647 goat anti-mouse IgG (H+L)	Goat	Invitrogen	A-21235

Acknowledgements

When I reflect upon these last few years, all I can think is that it has been quite a wild ride. This experience has been one of the most challenging and rewarding of my life, and I am so grateful to everyone that I've surrounded myself with for their support.

Agathe, I have to start this list of thanks with you, as I would not have had the privilege of coming to live and work here if you hadn't taken a chance on me. Thank you for giving me the opportunity to do this thesis work in your lab. I appreciate how available you make yourself to your students, and how easy it is to just pop by your office to go over ideas or troubleshoot. Your guidance gave me a boost on multiple occasions, especially when I got to a place with the work that felt impossible, or like a dead end. You lead with Acknowledgements ............................................................................................................... Table of Contents ..................................................................................................................

Similar to the HKMTs, the specificity of HKDMs is often determined by the complex in which it is associated. For instance, when LSD1 complexes with the Co-REST repressor complex, its demethylates H3K4me1/2. However, its specificity shifts to H3K9me1/2 demethylation when coupled with androgen receptor [START_REF] Klose | Regulation of histone methylation by demethylimination and demethylation[END_REF].

Jumonji (JmjC) domain demethylases

Table 1 Histone Lysine Demethylases and their associated substrates (adapted from [START_REF] Accari | Emerging Roles of JmjC Domain-Containing Proteins[END_REF]. H3K4 methylation is enriched near promoter regions and transcriptional start sites. It is associated with gene activation. H3K4 is methylated via protein complexes that contain a methyltransferase from the SET1 family. The target specificity of these methyltransferases is often determined by other subunits within the multi-protein complex. For example, the SET domain of MLL1 (mixedlineage leukemia 1) alone is weakly capable of monomethylating H3K4. However, its specificity is enhanced when it works within the WRAD complex The WRAD complex is composed of four common subunits: WD repeat containing protein 5 (WDR5), Retinoblastoma Binding Protein 5 (RbBP5), Absent-Small-Homeotic-2-Like protein (ASH2L) and Dumpy-30 protein (DPY30) [START_REF] Patel | On the mechanism of multiple lysine methylation by the human mixed lineage leukemia protein-1 (MLL1) core complex[END_REF].

Monomethylation (me1) of H3K4 is enriched at enhancer regions, and dimethylation (me2) occurs most frequently at the 5' end of transcribed genes. Trimethylation (me3) of H3K4 is associated with promoter regions of actively transcribed genes [START_REF] Jeon | Writing, erasing and reading histone lysine methylations[END_REF]. Methylation of H3K4 is also involved in poised chromatin of embryonic stem cells (ESCs). Poised chromatin is a term used to describe chromatin that is in transition between active and inactive states. It has been well characterized in embryonic stem cells (ESCs) and is important for cell-type differentiation.

H4K3me3 and H3K27me3 have been shown to be important in regulating transcription at these bivalent promoters during development [START_REF] Bae | H3K4me1 Distribution Predicts Transcription State and Poising at Promoters[END_REF].

H3K4 methylation is recognized by chromatin remodelers and transcription factors. For example, CHD1, an ATP-dependent chromatin remodeler, recognizes H3K4 methylation via its chromodomains, and then utilizes ATPase hydrolytic activity to reposition nucleosomes and promote transcriptional elongation (Sims et al, 2019).

H3K4 demethylase activity results in gene repression. There are a number of enzymes capable of demethylating H3K4, among them are FAD-dependent demethylases, LSD1 and LSD2 (KDM1A/B respectively) (Shi et al, 2004;[START_REF] Ciccone | KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints[END_REF]. Several jumonji domaincontaining (JmjC) demethylases have also been implicated in demethylation activity, such as:

S-adenosyl-L-methionine (SAM) metabolism influences histone methylation

Figure 18. Schematic summarizing the metabolites involved in histone methylation and demethylation Figure 18 Schematic summarizing the metabolites involved in histone methylation and demethylation. SAM is required for histone methylation and is synthesized from methionine via the methionine and folate cycles. Once SAM donates its methyl group, it is converted to SAH. Restoration of SAM is also dependent on the methionine and folate cycles. LSD1/2 activity are dependent on FAD, generated in the cytosol, while the JmjC demethylase require -ketoglutarate (-KG) generated by the TCA cycle in the mitochondria. Succinate, fumarate, and 2-HG (2-hydroxyglutarate) are -KG analogs that act as competitive inhibitors for the JmjC demethylases. (Adapted from [START_REF] Reid | The impact of cellular metabolism on chromatin dynamics and epigenetics[END_REF].

MATERIALS AND

METHODS

RESULTS