Identification of Long-Range Regulatory Elements in the Human Genome

Genome-wide association studies have shown that the majority of disease-associated genetic variants lie within non-coding regions of the human genome. Subsequently, a challenge following these discoveries is to identify how these variants modulate the risk of disease. Enhancers are non-coding regulatory elements that can be bound by proteins to activate the expression of a gene that may be linearly distant. Experimentally probing all possible enhancer–target gene pairs can be laborious. Hi-C, a technique developed by Job Dekker’s group in 2009, combines high-throughput sequencing with chromosome conformation capture to detect DNA interactions genome-wide and thereby reveals the three-dimensional architecture of chromatin in the nucleus. However, the utility of the datasets produced by this technique for discovering long-range regulatory interactions is largely unexplored. In this thesis, we develop novel approaches to identify DNA-interacting units and their interactions in Hi-C datasets with the goal of uncovering all enhancer–target gene interactions. We began by identifying significantly interacting regions in these datasets, subsequently focusing on candidate enhancer–gene pairs. We found that the identified putative enhancers are enriched for p300 binding activity, while their target promoters are likely to be cell-type-specific. Furthermore, we revealed that enhancers and target genes often interact in many-to-many relationships and the majority of enhancer–target gene interactions are intra-chromosomal and within 1 Mb of each other. Next, we refined our analytical approach to identify physically-interacting DNA regions at ~1 kb resolution and better define the boundaries of likely enhancer elements. By searching for over-represented sequences (motifs) in these putative promoter-interacting enhancers, we were then able to identify bound transcription factors. This newer approach provides the potential to identify protein complexes involved in enhancer–promoter interactions, which can be verified in future experiments. We implemented a high-throughput identification pipeline for promoter-interacting enhancer elements (HIPPIE) using both of the above described approaches. HIPPIE can be run efficiently on typical Linux servers and grid computing environments and is available as open-source software. In summary, our findings demonstrate the potential utility of Hi-C technologies for elucidating the mechanisms by which long-range enhancers regulate gene expression and ultimately result in human disease phenotypes

Transcriptional regulatory logic of the diurnal cycle in the mouse liver.

Many organisms exhibit temporal rhythms in gene expression that propel diurnal cycles in physiology. In the liver of mammals, these rhythms are controlled by transcription-translation feedback loops of the core circadian clock and by feeding-fasting cycles. To better understand the regulatory interplay between the circadian clock and feeding rhythms, we mapped DNase I hypersensitive sites (DHSs) in the mouse liver during a diurnal cycle. The intensity of DNase I cleavages cycled at a substantial fraction of all DHSs, suggesting that DHSs harbor regulatory elements that control rhythmic transcription. Using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq), we found that hypersensitivity cycled in phase with RNA polymerase II (Pol II) loading and H3K27ac histone marks. We then combined the DHSs with temporal Pol II profiles in wild-type (WT) and Bmal1-/- livers to computationally identify transcription factors through which the core clock and feeding-fasting cycles control diurnal rhythms in transcription. While a similar number of mRNAs accumulated rhythmically in Bmal1-/- compared to WT livers, the amplitudes in Bmal1-/- were generally lower. The residual rhythms in Bmal1-/- reflected transcriptional regulators mediating feeding-fasting responses as well as responses to rhythmic systemic signals. Finally, the analysis of DNase I cuts at nucleotide resolution showed dynamically changing footprints consistent with dynamic binding of CLOCK:BMAL1 complexes. Structural modeling suggested that these footprints are driven by a transient heterotetramer binding configuration at peak activity. Together, our temporal DNase I mappings allowed us to decipher the global regulation of diurnal transcription rhythms in the mouse liver

Spatial and Epigenetic Regulation of T-Cell Receptor Beta Gene Assembly

The adaptive immune system endows mammals with a sophisticated mechanism to recognize foreign proteins via surface antigen receptors that are expressed on the surface of all lymphocytes. This defense network is generated by V(D)J recombination, a set of sequentially controlled DNA cleavage and repair events that assembles functional antigen receptor genes from distally located Variable (V), Diversity (D) and Joining (J) gene segments. However, the recombination process must be stringently regulated to prevent formation of chromosomal translocations, which can lead to tumors. The process of V(D)J recombination is controlled at the levels of tissue, stage and allele specificity by a collection of architectural and regulatory elements that are distributed throughout each antigen receptor locus. Our laboratory has characterized several genetic elements that regulate chromatin accessibility and recombination at the T cell receptor beta (Tcrb) locus. These elements include transcriptional promoters and enhancers, which interact with each other in conformational space to form a promoter-enhancer holocomplex, facilitating Dβ to Jβ recombination. Simultaneously, spatial apposition of the Vβ cluster to the DβJβ region (a phenomenon called locus contraction) increases the efficiency of long-range Vβ recombination. Using extensive chromatin profiling of the Tcrb locus, we have discovered that selection of Vβ genes depend upon their association with transcriptionally active chromatin and high quality Recombination Signal Sequences, which serve as substrates for the V(D)J recombinase proteins RAG1/2. We further identify a bi-functional barrier-tethering region upstream of the DβJβ cluster that is essential for stabilizing its long-range interactions with distal Vβ gene segments in progenitor CD4-CD8- double negative (DN) thymocytes. Following Tcrb rearrangement, progenitor thymocytes proliferate and differentiate into CD4+CD8+ Double Positive (DP) cells, where the Vβ genes are epigenetically silenced and the distal ends of Tcrb are spatially segregated (presumably to inhibit further rearrangements). However, we have found that the transcriptionally inactive proximal Vβ genes continue to interact with the DβJβ cluster in a proliferation independent manner. These findings divide the Tcrb locus into two architectural domains, of which only the distal part is spatially segregated in DP cells. The loss of distal Vβ interaction is also observed in DP thymocytes containing a rearranged Tcrb allele, suggesting this conformation is DP-intrinsic. Our results have unraveled new mechanisms that stabilize the long-range Tcrb conformation in DN cells, how the Vβ segments are selected to recombine and how Tcrb topology is retained by DP-intrinsic mechanisms. These studies pave the way for future investigations into the role of boundary elements and tissue specific transcription factors in sculpting AgR gene assembly and regulating genome topology

Genetic and epigenetic regulation of gene expression in pancreatic islets

T2D is a complex disease with evidence of a strong genetic basis. Studies of the human pancreatic islets have provided valuable insight into the islet regulatory landscape and identified enrichment of T2D-associated variants in islet enhancers. The relationship between cis-regulatory variation and changes in gene expression, however, remains unclear. This question is challenging to address in human, as it calls for a systematic analysis of cis-regulatory variation and the impact it has on gene expression in the native genomic context. While a mouse model can be used for this purpose, the regulatory landscape of the mouse pancreatic islets has been poorly characterized. This thesis addresses the questions of genetic and epigenetic regulation of gene expression in pancreatic islets in two parts. First, a genome-wide map of several histone modifications and transcription factor binding sites is created for the mouse pancreatic islet. This enabled identification of the mouse islet regulatory elements and characterization of enhancer clustering, transcription factor occupancy and conservation. A systematic comparison of enhancer clustering between mouse and human islets identified species-common and species-specific subsets of the islet regulatory program, each associated to a distinct biological function of the pancreatic islet. Second, a hybrid mouse was used as a model where naturally occurring genetic variation drives changes in gene expression. High-density mapping of allelic regulatory activity, chromatin accessibility and transcription provided insight into the properties of both genes subject to cis-regulatory variation and the regulatory elements driving the change in expression. As a result, tissue-specific genes associated to clustered enhancers were shown to be most influenced by cis-regulatory variation. Additionally, enhancer clustering emerged as the dominant property of regulatory elements associated to changes in gene expression. Overall, this thesis advanced our knowledge of the mouse islet regulatory landscape and provided novel insight into the properties of functional cis-regulatory variation.Open Acces

Étude de l'influence du variant d'histone H2A.Z sur l'organisation des nucléosomes aux enhancers liés par le récepteur alpha de l'oestrogène

L'identité et la réactivité cellulaires sont établies, maintenues et modulées grâce à l'orchestration de programmes transcriptionnels spécifiques. Les éléments régulateurs, des régions particulières de la chromatine responsables de l'activation ou de la répression des gènes, sont au coeur de cette opération. Ces dernières années, de nombreuses études ont révélé le rôle central des « enhancers » dans ce processus. En effet, des centaines de milliers « enhancers » seraient éparpillés dans le génome humain, majoritairement dans sa portion non-codante, et contrairement au promoteur, leur activation varierait selon le type ou l'état cellulaire ou en réponse à une stimulation physiologique, pathologique ou environnementale. Les « enhancers » sont, en quelque sorte, des carrefours où transitent une multitude de protéines régulées par les signaux intra- et extra-cellulaires et un dialogue s'établit entre ces diverses protéines et la chromatine. L'identification des « enhancers ainsi qu'une compréhension de leur mode de fonctionnement sont donc cruciales, tant au plan fondamental que clinique. La chromatine joue un rôle indéniable dans l'activité des éléments régulateurs, tant par sa composition que par sa structure, en régulant, entre autres, l'accessibilité de l'ADN. En effet, l'ADN des régions régulatrices est bien souvent masqué par un nucléosome occlusif, lequel doit être déplacé ou évincé afin de permettre la liaison des protéines régulatrices, notamment les facteurs de transcription (FTs). Toutefois, la contribution de la composition de la chromatine à ce processus reste incomprise. Le variant d'histone H2A.Z a été identifié comme une composante de la chromatine aux régions accessibles, dont des « enhancers » potentiels. Toutefois son rôle y est inconnu, bien que des études récentes suggèrent qu'il pourrait jouer un rôle important dans la structure de la chromatine à ces régions. Par ailleurs, un lien étroit existe entre H2A.Z et la voie de signalisation des oestrogènes (notamment la 17-[beta]-estradiol (E2)). Ainsi, H2A.Z est essentiel à l'expression de plusieurs gènes cibles de l'E2. Les effets de l'E2 sont en partie exercés par un FT, le récepteur alpha des oestrogènes (ER[alpha]), lequel se lie à l'ADN suite à son activation, et ce majoritairement à des « enhancers », et permet l'établissement d'un programme transcriptionnel spécifique. Cette thèse vise à définir le rôle d'H2A.Z aux « enhancers », et plus particulièrement son influence sur l'organisation des nucléosomes aux « enhancers » liés par ER[alpha]. D'abord, mes travaux effectués à l'échelle du génome ont démontré qu'H2A.Z n'est présent qu'à certains ER[alpha]-« enhancers » actifs. Cette particularité a fait en sorte que nous avons pu comparer directement les « enhancers » actifs occupés par H2A.Z à ceux non-occupés, afin de mettre en évidence sa relation à l'environnement chromatinien. Étonnamment, il est apparu qu'H2A.Z n'introduit pas une organisation unique ou particulière des nucléosomes aux « enhancers ». Par ailleurs, nos résultats montrent qu'H2A.Z joue un rôle crucial dans la régulation de l'activité des « enhancers ». En effet, nous avons observé que suite à leur activation par l'E2, les « enhancers » occupés par H2A.Z recrutent l'ARN polymérase II (ARNPII) et produisent un transcrit. Ils recrutent également RAD21, une composante du complexe cohésine impliqué, entre autres, dans des interactions chromosomiques entre « enhancers » et promoteurs. De façon intéressante, nous avons mis en évidence que ces trois évènements, connus pour leur importance dans l'activité des « enhancers », sont dépendants d'H2A.Z. Ainsi, la présence d'H2A.Z à l' « enhancer » pourrait permettre un environnement chromatinien favorable à trois aspects clés de l'activité des « enhancers » : la présence de l'ARNPII, la transcription et la formation d'une boucle d'interaction, et par la suite, de par la proximité « enhancer »-promoteur ainsi créée, augmenter la concentration d'ARNPII à proximité du promoteur favorisant l'expression du gène cible. Un tel rôle central d'H2A.Z dans l'activité d' « enhancers » spécifiques pourrait participer à un mécanisme épigénétique ciblé de la régulation de l'expression des gènes

Assessment of Histone Tail Modifications and Transcriptional Profiling during Colon Cancer Progression: Effect of Chemoprotective Natural Compounds

During colon cancer development, epigenetic alterations contribute to the dysregulation of major cellular functions and signaling pathways. Recent evidence suggests that nutritionally chemoprotective components that influence cellular dynamics in the colonic epithelium can also directly affect their epigenetic landscape. We hypothesize that the chemoprotective nutritional bioactives found in fish oil and fermentable fiber can act as epigenetic modifiers and mechanistically counteract epigenetic distortions associated with colonic tumorigenesis. Fermentable fiber generates short-chain fatty acids (SCFA), e.g., butyrate, in the lumen of the colon that can serve as a chemoprotective histone deacetylase inhibitor and/or as an acetylation substrate for histone acetylases. In addition, n-3 polyunsaturated fatty acids (n-3 PUFAs) in fish oil can affect the chromatin landscape by acting as ligands for tumor suppressive nuclear receptors. In an effort to gain insight into the extensive dimension of post-translational modifications in histones (including H3K4me3 and H3K9ac) and elucidate the chemoprotective impact of dietary bioactive compounds on transcriptional control in a colon cancer preclinical model, we generated high-resolution genome-wide RNA (RNA-Seq) and “chromatin-state” (H3K4me3-seq and H3K9ac-seq) maps for intestinal (epithelial colonocytes) crypts in rats treated with a colon carcinogen and fed bioactive (i) fish oil (ii) butyrate (in the form of a fermentable fiber a rich source of SCFA), (iii) a combination of fish oil plus butyrate or (iv) control diets. Poor correlation was observed between differentially transcribed (DE) and enriched genes (DERs) at multiple epigenetic levels in fat x fiber dietary combinations and in the presence/absence of carcinogen. The genome-wide carcinogen (AOM) effects were most prevalent at the RNA (116 DE genes) and K4me3 (7678 DERs including 3792 genes) levels. Pathway analysis of the differentially transcribed genes after AOM exposure indicated a strong link to interferon-associated innate immune responses often associated with anti-microbial activity, while K4me3 DERs were strongly linked to colon tumorigenesis. However, these changes in K4me3 enrichment were not reflected at the transcriptional level during the early stages of cancer progression. Therefore, we propose that carcinogen-induced changes in genes with K4me3 DERs are harbingers of future transcriptional events, which drive malignant transformation of the colon cells. We also demonstrated that the combinatorial diet (fish oil + pectin) was synergistically chemoprotective, and uniquely affected epigenetic profiles in the intestinal epithelium, e.g., upregulating lipid catabolism and beta-oxidation associated genes. These genes were linked to activated ligand-dependent nuclear receptors associated with n-3 PUFAs and were also correlated with the inhibition of lipogenesis and a decreased concentration of cholesterol. Interestingly, only a subset of these genes was affected in animals fed fish oil without pectin, and there was a markedly enhanced effect of biological mechanisms associated with n-3 PUFAs in the combinatorial diet. In conclusion, we propose that the chemoprotective effects of the bioactive mediators of the combination fish oil + pectin diet during colon cancer progression are multifaceted and generate specific epigenetic modifications and transcriptional profiles. Our data contribute to the understanding of the regulatory action of chemoprotective bioactive compounds found in fish oil and readily fermentable fiber (n-3 PUFAs and SCFAs) in colonic crypts and provide mechanistic insight into current clinical and epidemiological findings

Conservation and synteny of long non-coding RNAs invertebrate genomes and their identification in novel transcriptomes

Long non-coding RNAs (IncRNAs) are a biological entity defined by what they are not, rather than by what they are. This indicates that our knowledge about them is sensibly limited. The aim of my PhD is to gain insights into the evolution and the functions of IncRNAs through computational approaches and the usage of large scale functional genomics dataset. I developed an annotation pipeline, which can effectively identify IncRNAs in entire transcriptomes. The pipeline is able to accurately annotate the coding genes while predicting a conservative estimate of the IncRNA population. It allowed me to show, for the first time, the presence of lncRNA transcription in a diverse range of organisms. Further, I analysed sequence and positional conservation of lncRNAs, demonstrating the presence of short segments of conserved sequence in IncRNAs and the existence of several syntenically conserved non-coding transcripts over large evolutionary distances. However, I also demonstrate that positional conservation of lncRNAs with a flanking coding gene is generally independent from the conservation of the lncRNA expression with respect to the coding gene. Finally, I have characterised the diversity of lncRNA transcription in specific cells and developmental stages of two teleost fishes. In summary, the work presented in the thesis provides novel findings and contributions in the field of lncRNAomics