<p>In eukaryotic systems, the genetic material of the cell –DNA– is packed into a protein-dense structure called chromatin. Chromatin structure is critical for preservation of the genetic material as well as coordination of vital processes such as DNA replication, transcription and DNA damage repair. The fundamental repeating unit of chromatin is nucleosome which is composed of an octamer of small alkaline proteins called histones and the DNA wrapped around this octamer. The nucleosomes are then packed into higher-order structures leading to formation of 3D chromatin architecture. The chromatin is a dynamic structure; the spacing between nucleosomes, or the folding of the larger chromatin segments is subjected to alterations during embryonic development, tissue specifications or <i>simply during any event that require gene expression changes</i>. Failure in proper regulation of chromatin structure has been associated with embryonic defects and disease such as cancer. </p> <p>This work has focused on a class of ATP-dependent chromatin remodeling complexes known as switch/sucrose-non-fermentable (SWI/SNF) or BRG-associated factors (BAF) complex. This family of complexes act on chromatin and alter its physical structure by mobilizing histones or nucleosome particles through the activity of its ATPase –BRG1 or BRM, enabling more accessible DNA for the other factors such as transcription factors to localize and recruit transcription machinery. In particular, we discovered and biochemically defined a novel version of this family of chromatin complexes that we named as GLTSCR1/1L-BAF (GBAF). GLTSCR1 and GLTSCR1L are two uncharacterized paralogous proteins that have been identified as BRG1-interacting proteins. Biochemically surveying the essence of this interaction, we realized that these proteins incorporates into a previously unknown SWI/SNF family complex that lacks well-characterized SWI/SNF subunits such as ARID1/2, BAF170, BAF47; instead, uniquely comprise GLTSCR1/1L and bromodomain-containing protein BRD9. Focusing on the GLTSCR1 subunit, we observed that its absence is well-tolerated by many different cell types except slight growth retardation by prostate cancer cells. Expanding the cohort of prostate cancer cells, we realized that not the paralogous subunits GLTSCR1 or GLTSCR1L but unique and non-redundant subunit BRD9 is the major GBAF-dependence in prostate cancer cells. We observed that especially the androgen-receptor positive cell lines have severe growth defects upon <i>BRD9 </i>knockdown or inhibition. <i>In vivo, </i>we showed that xenografts with <i>BRD9 </i>knockdown prostate cancer cells (LNCaP) have smaller tumor size. We demonstrated that BRD9 inhibition can block the expression of androgen-receptor targets. Similarly, <i>BRD9 </i>knockdown and treatment with antiandrogen drug (enzalutamide) has overlapping transcriptional effects. Mechanistically, we showed that BRD9 interacts with AR and it colocalizes with AR in subset of AR -binding sites. Surprisingly, we realized that BRD9 depletion has similar transcriptional and phenotypic effects as BET protein inhibitors. BET protein family contains 4 bromodomain containing proteins (BRD2, BRD3, BRD4, BRDT). These proteins were previously shown to be critical for AR-dependent gene expression. We detected interaction between BRD9 and BRD2/4. We demonstrated that BRD4 and BRD9 had shared binding sites on genome, a fraction of which are co-bound by AR. At particular target sites we showed that BRD9 localization is dependent on BET proteins, but not the other way around. Taking together, we provided some evidences that GBAF targeting through BRD9 can be a novel therapeutic approach for prostate cancer. Growing body of reports suggested that current therapy options targeting the androgen receptor is failing due to acquired resistance. Therefore, targeting the AR pathways via its coregulators such as BET proteins or SWI/SNF complexes can serve as potent alternative approaches. Further research is needed to elucidate the roles of GBAF and BET proteins in androgen receptor independent prostate cancer cells, which are still responsive to GBAF or BET manipulations although to a lesser extent.</p>

Les chondrosarcomes sont des tumeurs malignes osseuses, considérés comme radio- et chimio-résistants, du fait de leur environnement hypoxique. Dans ce contexte, cette étude vise à mieux comprendre le rôle de l’hypoxie dans la résistance de ces tumeurs à la chimiothérapie (cisplatine) et à la radiothérapie (rayons X) et à identifier de nouvelles stratégies thérapeutiques permettant de sensibiliser les chondrosarcomes aux traitements, par un ciblage épigénétique de la méthylation de la lysine 27 de l’histone H3 (H3K27).Dans un premier temps, nous avons montré que, contrairement à ce qui est communément admis, l’hypoxie n’a pas d’effet sur la sensibilité au cisplatine ou aux rayons X dans certains chondrosarcomes alors qu’il augmente la résistance au cisplatine et la sensibilité aux rayons X uniquement dans une lignée de chondrosarcome. Dans un second temps, nous avons montré que le 3-deazaneplanocine A (DZNep) induit l’apoptose dans ces tumeurs, par un mécanisme indépendant de la méthylation de H3K27 et de sa méthylase EZH2 et semblerait agir par la voie Rhoβ/EGFR. Cependant, il provoque des effets secondaires sur la fertilité masculine. Par ailleurs, son association avec le cisplatine potentialise ses effets toxiques sur les chondrosarcomes. Le GSK-J4, quant à lui ralentit la croissance cellulaire des chondrosarcomes et son association avec le cisplatine augmente cet effet. Cette étude souligne que les chondrosarcomes possèdent des mécanismes de régulation cellulaires différents, d’où l’importance de mener des études sur plusieurs lignées cellulaires afin de mieux prédire la réponse aux traitements. De plus, ces travaux démontrent les propriétés anti-tumorales du DZNep et du GSK-J4 dans le traitement de ces tumeurs. / Chondrosarcomas are bone malignant tumors, considered as radio- and chemo-resistant, due to their hypoxic environment. In this context, this study aimed to better understand the role of hypoxia in the resistance of these tumors to chemotherapy (cisplatin) and radiotherapy (X-rays) and to identify new therapeutic strategies to re-sensitize chondrosarcomas by epigenetic targeting of H3K27 methylation. First, we showed that, contrary to what is commonly accepted, hypoxia has differential effect on cisplatin or X-ray sensitivity in chondrosarcomas, while it increases cisplatin resistance and X-ray sensitivity only in one cell line. Secondly, 3-deazaneplanocin A (DZNep) induces apoptosis in these tumors by a mechanism independent of H3K27 methylation and its methylase EZH2 and seems to act through the Rhoβ / EGFR pathway. However, it causes side effects on male fertility. In addition, its association with cisplatin potentiates its toxic effects on chondrosarcomas. The GSK-J4, on the other hand, decreases cell growth and its association with cisplatin increases this effect.This study highlights that chondrosarcomas use different cellular regulation mechanisms, showing the importance of conducting studies on several cell lines in order to better predict the response to treatments. In addition, these studies demonstrate the anti-tumoral properties of DZNep and GSK-J4 in the treatment of these tumors.

DNA organization is an intricate and dynamic process. The approximately two meters of DNA in a single cell is wrapped around small proteins called histones. Histones can be compacted into dense coils or loosely distributed along DNA, allowing for cells to control gene expression. This combination of DNA and histones forms chromatin. This work has focused on understanding the role of Polybromo1 (PBRM1), which is a member of a chromatin remodeling complex. PBRM1 is mutated in 3% of all human cancers and is mutated in 40% of renal clear cell carcinomas (ccRCC), the most common type of kidney cancer. Through my work characterizing PBRM1 as a tumor suppressor, we have found PBRM1 acts as a stress sensor. PBRM1 is a member of the Polybromo1 BRG1 associating factors (PBAF) complex which is a subtype of the larger BAF family of chromatin remodelers. Although BAF is essential for cell viability, knockdown of PBRM1 shows minor phenotypic changes in many cell types under standard cell culturing conditions. However, when cells without PBRM1 experience external stress, the reactive oxygen species levels in the cells are elevated and remain high compared to cells with wild type PBRM1. Depending on the cellular environment of the cell, increase in ROS can be growth promoting or growth inhibiting. PBRM1 is a structurally unique protein, containing two bromo-adjacent homologs, a high mobility group and six tandem bromodomains. Due to the multiple reader domains, it is likely PBRM1 acts to target the complex. Taking advantage of a RCCC cell line not expressing PBRM1, we re-expressed full length PBRM1 containing an asparagine to alanine mutation in each bromodomain, disrupting the acetyl-lysine binding. We have found that the bromodomains are cooperative and are facilitating binding of PBAF to chromatin. We found defects in PBRM1’s ability to suppress growth, bind to chromatin, and regulate gene expression when any of the bromodomains were mutated besides the third bromodomain. These results correlated with patient data. Using acetylated histone peptides, we have identified potential combinations of marks that PBRM1 prefers over single marks. Further work needs to be done to characterize how these histone modifications are altered under stress and they contribute to the role of PBRM1 in stress response.

No description available.

Identifying disease risk genes is a central topic of human genetics. Cost-effective exome and whole genome sequencing enabled large-scale discovery of genetic variations. However, the statistical power of finding new risk genes through rare genetic variation is fundamentally limited by sample sizes. As a result, we have an incomplete understanding of genetic architecture and molecular etiology of most of human conditions and diseases. In this thesis, I developed new computational methods that integrate functional genomics data sets, such as epigenomic profiles and single-cell transcriptomics, to improve power for identifying genetic risks and gain more insights on etiology of developmental disorders. The overall hypothesis that disease risk genes contributing to developmental disorders are bottleneck genes under normal development and subject to precise transcriptional regulations to maintain spatiotemporal specific expression during development. In this thesis I describe two major research projects. The first project, Episcore, predicts haploinsufficient genes based on a large integrated epigenomic profiles from multiple tissues and cell lines by supervised machine learning methods. The second one, A-risk, predicts plausibility of being risk genes of autism spectrum disorder based on single-cell RNA-seq data collected in human fetal midbrain and prefrontal cortex. Both methods were shown to be able to improve gene discovery in analysis of de novo mutations in developmental disorders. Overall, my thesis represents an effort to integrate functional genomics data by machine learning to facilitate both discovery and interpretation of genetic studies of human diseases. We believe that such integrative analysis can help us better understand genetic variants and disease etiology.

Histone methyltransferases are key players in eukaryotic gene regulation. The goal of this thesis was to study the role of the histone methyltransferase Mll2 in developing embryos and adult mice. Targeting of mouse ES cells with a multipurpose allele and blastocyst injection had previously generated a mouse line allowing analysis of Mll2 function by knock-out and conditional mutagenesis using Cre/loxP. The first part of the thesis comprised the analysis of the Mll2-/- phenotype, and included the cloning of a targeting construct to generate an ubiquitous, ligand-regulated Cre line. In the second part, we did conditional mutagenesis using the Rosa26-CreER(T2) line obtained from collaborators, and achieved complete knock-out of Mll2 in most tissues of embryos, neonates and adult mice. Mll2 is essential during embryonic development, as mutant embryos were severely growth retarded, had significant increases in apoptosis, and failed in gestation between E 9.5 and E11. Conditional removal of Mll2 protein at gastrulation (E 6.5) produced a similar phenotype at E 11. In contrast, the absence of Mll2 function after E 11 did not result in obvious defects at E16 and indicates an essential role for Mll2 between E6 and E11. Indeed, we identified a loss of expression of 3 important developmental regulators in mutants of this developmental stage: Hoxb1, Mox1 and Six3 are candidate targets for Mll2 regulation that encode homeobox type transcription factors involved in specifying cellular identity. We observed correct establishment of their developmental expression patterns, which than decay in Mll2-/- mutants at E9.5. These data concord with and extend current thoughts about the fly orthologue of Mll2, Trithorax, which suggest that it acts as an epigenetic lock in chromatin to maintain expression of certain transcription factors key to respective cellular identities, after their expression patterns have been established. After birth, Mll2 is dispensable in most tissues, as conditional knock out in neonates and adult mice did not produce any pathological findings except infertility of mutant males and females. Histological analysis of testis revealed progressive loss of spermatogonia, associated with increases in apoptosis but without overt proliferation, meiotic or differentiation defects or loss of the supporting Sertoli cells. Consequently, in addition to its regulation of homeotic genes during development, Mll2 is required for the maintenance of various mitotic cell populations including ES cells, embryonal cells and germ cells.

No description available.

Genomic modifications occur slowly across generations, whereas short-term epigenetic transgenerational inheritance of adaptive phenotypes may be immediately beneficial to large numbers of individuals, acting as a bridge for survival when adverse environments occur. In this study we used dietary exposure to crude oil as an example of an environmental stressor to assess its effects from the molecular to the organismal levels in piscine and avian animal models. In addition, we assessed the role of the parental exposures on their offspring F1 generation. The research developed in this dissertation has contributed to several areas of investigation including molecular biology, animal physiology, and evolutionary biology. The quantitative information from these studies may be utilized to supplement information regarding the proximate and ultimate effects of environmental stressors on fish and bird populations. Furthermore, this information may be used as additional support for understanding the conservation of the responses from the molecular to the whole organismal levels across the vertebrate taxa, as well as their implications for population survival and maintenance. Additionally, the zebrafish (Danio rerio), the Siamese fighting fish (Betta splendens) and the king quail (Coturnix chinensis) have proven to be excellent models to start building a strong basis for understanding the effects of environmental stressors and transgenerational epigenetic phenomena using a multi-level approach. Furthermore, as more raw data and information is discovered, the concatenation of development, organismal variation, epigenetics inheritance, natural selection, speciation and evolution is being slowly decrypted.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL. Further, in the hematopoietic system stem cell aging is characterized by accumulation HSCs with poor self-renewal capacity and myeloid biased differentiation. Despite improved appreciation of the cell intrinsic and cell extrinsic mechanisms driving age-associated HSC functional exhaustion, no interventions have proven effective in delaying HSC aging to date. Here, we show that genetic inactivation of the Phf6 prevents age- associated HSC functional decline. Immunophenotypic and single cell transcriptomics profiling demonstrated markedly decreased accumulation of immunophenotypically-defined HSCs, reduced myeloid bias and decreased upregulation of transcriptional programs associated with stem cell aging in old hematopoietic-specific Phf6 knockout mice. Functionally, Phf6 knockout HSCs from aged mice demonstrated increased hematopoietic reconstitution capacity and preservation of lymphoid differentiation potential. Mechanistically, analysis of long-term HSCs from old Phf6 knockout mice revealed reduced levels of ongoing DNA damage and downregulation of genotoxic stress-induced transcriptional signaturesconducive of HSC aging. These results identify Phf6 as an important epigenetic regulator of HSC aging, whose inactivation counters the functional deterioration of HSC activity induced with age. Microprotein encoding genes are a class of genes which encode poly-peptide gene products comprised by 100 or fewer amino acids. Until recently, many such genes had been considered of low- or no-coding potential given the technical limitations associated with identification of such small proteins. However, recently prominent examples of microprotein encoding genes have been reported with a wide variety of regulatory functions. Therefore, we hypothesized that novel microprotein genes exist within the human genome with oncogenic and tumor suppressive roles. To test this hypothesis, we developed a pipeline for identification of microproteins based on conservation of the open reading frame. Leveraging PLATE-seq to generate a high-dimensional readout in a loss-of-function screen, we then screened for microproteins with potential tumor suppressive or oncogenic function. From this, we identified a brain- specific, 65 amino-acid microprotein encoded in within LINC00617 (TUNAR) which is conserved at the protein level across vertebrates. We experimentally validated the protein-level expression of the TUNAR microprotein. In vitro and in vivo knockout and overexpression experiments demonstrate a role for TUNAR as a tumor suppressor in glioma. Specifically, we show that loss of Tunar in the mouse brain results in lower expression of Fermt1 and genes in the integrin signaling pathway. Consistently, overexpression of TUNAR in human glioblastoma multiforme cell lines significantly impeded cellular migration suggesting a role of Tunar in glioma cell dissemination. Finally, human glioma sequencing and copy number data were mined to determine the prognostic significance of the loss of TUNAR in human gliomas. These analyses demonstrated that copy number loss of TUNAR is associated with poor outcomes in lower grade gliomas and that TUNAR expression and glioma grade are strongly, negatively correlated suggesting that TUNAR likely has tumor suppressive effects in human glioma.

FOXL2 constitue un gène majeur de la différenciation et de la fonction ovarienne. Chez l’Homme, l’haploinsuffisance de ce gène induit des malformations palpébrales qui peuvent être associées ou non à une insuffisance ovarienne prématurée (BPES de type I ou II). Dans certains cas, des anomalies de l’expression de FOXL2 sont liées à des délétions de régions situées très en amont du gène, témoignant de la présence d’activateurs distaux. L’existence de cette régulation à longue distance a aussi été mise en évidence dans l’espèce caprine par notre équipe. La mutation naturelle PIS (Polled Intersex Syndrome) induit à l’état homozygote l’absence d’expression du gène FOXL2 dans les gonades XX, conduisant au développement de testicules à la place d’ovaires. Ainsi, au cours de mon doctorat j’ai été amenée à travailler sur deux aspects différents : (i) l’analyse des cibles/voies activées par FOXL2 dans la gonade, et (ii) l’étude de la régulation à distance de l’expression de FOXL2.En ce qui concerne le premier point, l’analyse du rôle d’un gène candidat, Dmxl2, chez la souris a nécessité la mise en place d’une invalidation conditionnelle de ce gène (Knock-out ou KO) dans les gonades (le KO total étant létal à la naissance). Chez les mâles, une diminution de 60% de la production de spermatozoïdes a été observée à la puberté pendant la première vague de spermatogénèse.En ce qui concerne le second point, l’étude d’une région régulatrice potentielle de l’expression de FOXL2 a permis de définir des éléments très conservés présentant un profil épigénétique caractéristique de régions de type « enhancers ». J’ai ensuite établi un modèle in vitro « d’édition de l’épigénome » du locus FOXL2, en utilisant la technologie CRISPR/dCas9-p300 pour modifier la marque épigénétique H3K27ac et activer l’expression de ce gène. A long terme, ces travaux pourraient aboutir à la création de « médicaments épigénétiques » pour soutenir l’expression de FOXL2 et rétablir la fertilité des patientes atteintes d’une mutation de ce gène. / FOXL2 is a major gene for ovarian differentiation and functions. In humans, FOXL2 haploinsufficiency induces eyelid malformations with or without premature ovarian failure (BPES type I or II). In some cases, abnormalities of FOXL2 expression are related to deletions of regions located far upstream of this gene, indicating the presence of distal activators. The existence of this long-range regulation has also been demonstrated in the goat species by our laboratory. The natural mutation PIS (Polled Intersex Syndrome) when homozygous induces the silencing of FOXL2 expression in XX gonads, leading to the development of testes instead of ovaries. Thus, during my PhD, I worked on two different aspects: (i) the analysis of FOXL2-activated targets/pathways in the gonad, and (ii) the study of the long-range regulation of FOXL2 expression.Regarding the first point, gene function analysis of the candidate gene Dmx12 required the establishment of a conditional knock-out in the mouse gonad (Dmxl2 total KO is lethal at birth). In males, a 60% decrease in sperm production was observed at puberty during the first wave of spermatogenesis.Regarding the second point, the study of a putative regulatory region of FOXL2 expression allowed to define highly conserved elements harbouring typical enhancer epigenetic profile. Then, I established an in vitro model of FOXL2 locus “epigenome editing”, using the CRISPR/dCas9-p300 technology to modify the epigenetic mark H3K27ac. In the long term, this work may lead to the development of "epigenetic drugs" to support the expression of FOXL2 and restore the fertility of patients with a mutation of this gene.