The Role of Topologically Associating Domains for Developmental Gene Regulation - ⁠ A Systematic Functional Analysis at the Sox9 and Shh Loci

Precise spatiotemporal gene expression during embryonic developmental is controlled by cis- regulatory elements (CREs) such as enhancers and promoters. Their physical chromatin proximity is correlated with active transcription and thought to be restricted to topologically associated domains (TADs) that help establish interactions between CREs and limit inappropriate contacts. Accordingly, TADs frequently overlap with gene regulatory landscapes, in which are contained diverse enhancers that transmit their activity across the domain towards their target promoter. Large structural variants reorganizing TADs were shown to cause gene misexpression and disease thereby linking gene regulation to chromatin structure. Recently, several studies revealed controversial results questioning the importance of TADs for transcriptional control. Acute depletion of CTCF and other architectural proteins in vitro led to loss of TAD structures with surprisingly modest effects on gene expression. However, the cytotoxicity of such depletion assays hindered analysis of more complex gene regulatory scenarios and their effect during development. This study specifically addresses the connection between TADs and developmental gene regulation through two projects using the murine limb as a model system. First, we took advantage of the Sox9/Kcnj2-locus that is subdivided into two adjacent TADs with distinct expression patterns of Sox9 and Kcnj2. The systematic deletion of individual CTCF binding sites at the TAD boundary and within the TAD resulted in gradual fusion of the neighboring domains without major effects on gene expression. TAD rearrangement by TAD-spanning inversions and repositioning of the boundary, however, redirected the regulatory activity and resulted in pathogenic gene misexpression. Thus, TAD structures may not be essential for developmental gene regulation, yet CTCF-dependent rearrangement of TADs can lead to the redirection of enhancer–promoter contacts and gene misexpression. In the second project, we studied how enhancer position relative to its TAD influences the function of an individual enhancer at the Shh-locus. Therefore, we repositioned the Shh-limb enhancer ZRS to five alternative locations inside and outside of its TAD. As expected, the enhancer lost all function in the positions outside of the Shh-TAD. Interestingly, the new positions inside the TAD also displayed decreased enhancer activity, albeit to varying degrees. Further analysis suggests that CTCF likely functions in some positions as a facilitator of enhancer-promoter contacts, while insulating short-range contacts in others. Ultimately, the ZRS is only able to ectopically activate some genes if repositioned to novel TADs, displaying strong enhancer-promoter selectivity. In summary, the results demonstrate that TADs provide robustness and precision to gene regulation, guiding enhancer-promoter interaction without being essential. The findings in this work build a basis for future studies aiming to understand enhancer-promoter interaction and can help in contextualizing potential disease-causing mutations disrupting TADs

The Role of ZMYND8 in Immunoglobulin Class Switch Recombination

Class switch Recombination (CSR) also known as Immunoglobulin (Ig) Class switching is a genomic recombination/deletion reaction that diversifies the effector component of the antibody response but preserves antigen specificity. CSR is initiated by the enzyme activation induced cytidine deaminase (AID), which produces nucleotide mismatches in actively transcribed immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3’ Regulatory Region (3’RR), a prototypical super-enhancer located at the 3’ of the Igh locus, is essential for acceptor switch region transcription, but the mechanism by which it regulates this process is not well defined. After targeting by AID, nearby mismatches in the donor switch region are processed into DNA double strand breaks (DSBs), translocated to DSBs in the acceptor switch region, and ligated through the DNA Damage Repair (DDR) pathway, non-homologous end-joining (NHEJ). Critical components of CSR are 53BP1 and its effector RIF1 because they inhibit end resection to promote NHEJ and oppose competing pathways in DDR. However, the mechanism by which RIF1 effects end-protection in CSR and binds to 53BP1 is still unknown In these studies, I identified a novel component of the RIF1 interactome, ZMYND8, a chromatin reader and transcriptional repressor that binds to RIF1 and facilitates effective CSR. Unexpectedly, ZMYND8 promotes CSR independently of RIF1. In B cells, ZMYND8 binds active promoters and super-enhancers, including the Igh enhancer the 3’RR. ZMYND8 controls 3’RR activity by regulating polymerase loading. In its absence there is increased 3’ RR polymerase loading and decreased acceptor region transcription and CSR. Thus, ZMYND8 controls CSR by regulating the activity of the 3’ Igh super enhancer

The odyssey of MYC transcript from the nucleus to the cytoplasm : the molecular mechanism of the gene gating in human cancer cells

Pathological expression of the MYC oncogene is a common denominator in a wide range of cancers and is linked with abnormal cell proliferation. To achieve this status, the MYC gene benefits from being embedded in a region rich in enhancers and super-enhancers that are often absent in the normal cell counterparts. How those regions regulate MYC transcription and expression is, however, not well understood, although likely players include enhancer-binding factors, the 3D nuclear architecture and local eRNAs and ncRNAs. In this thesis, two new models governing MYC expression have been identified. The first describes a posttranscriptional mechanism that is based on the gene gating concept proposed in 1985, while the second is based on the ability of the non-coding eRNA, CCAT1 to promote MYC transcription, which paradoxically antagonizes the gating of MYC. In Paper I, a model of gene gating mechanism of MYC in human cancer cells is proposed. Briefly, the Nucleopore Complex (NPC) member ELYS (or AHCTF1) recruits MYC and its distal Oncogenic Super Enhancer (OSE) to the nuclear pore in a b-catenin -dependent manner. This principle increases MYC expression post-transcriptionally by facilitating the nuclear export of its derived mRNAs and thus enabling the escape of MYC transcripts from the faster degradation rate in the nucleus compared to the cytoplasm. In Paper II, a CTCF binding site within the non-coding gene, CCAT1, positioned within the OSE, was mutated using CRISPR technique. Expanded clones carrying the mutated CTCF binding site revealed that this site is essential for the canonical WNT-mediated gating of MYC. Normally ascribed an insulator function, this non-canonical feature of CTCF was essential for the recruitment of ELYS/AHCTF1 to the OSE, thereby effectuating its anchoring to the nuclear pore. In addition, this report shows that CTCF is essential for the WNT-mediated activation of the CCAT1 gene. In Paper III, the role of the OSE transcript CCAT1 in the gating mechanism was further analyzed. siRNA-mediated knockdown of CCAT1 eRNA expression revealed its dual function. While it promotes MYC transcription in the nuclear interior, it impedes the nuclear export of its derived mRNAs. We speculate that the CCAT1 eRNA likely indirectly alleviates transcriptional pausing of MYC transcription. Conversely, transcriptional pausing is proposed to promote the migration of the MYC gene to the nuclear pores to provide a key switch in the nuclear export pathways of MYC mRNAs. In summary, this work has identified two new models of MYC expression regulation in cancer cells, thereby providing opportunities for designing new pharmaceutical strategies targeting pathological expression of this central oncogene during cancer evolution

High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods generally provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at limited numbers of loci. Due to technical limitations, generation of reproducible high-resolution interaction profiles has not been achieved at genome-wide scale. Here, to overcome this barrier, we systematically test each step of 3C and report two improvements over current methods. We show that up to 30% of reporter events generated using the popular in situ 3C method arise from ligations between two individual nuclei, but this noise can be almost entirely eliminated by isolating intact nuclei after ligation. Using Nuclear-Titrated Capture-C, we generate reproducible high-resolution genome-wide 3C interaction profiles by targeting 8055 gene promoters in erythroid cells. By pairing high-resolution 3C interaction calls with nascent gene expression we interrogate the role of promoter hubs and super-enhancers in gene regulation

Single molecule characterization of the roles of long non-coding RNAs in eukaryotic transcription regulation

Récemment, des analyses dans divers organismes eucaryotes ont révélé que l'ensemble du génome est transcrit et produit en plus des ARNs messagers, une grande variété d’ARNs non codants de différentes longueurs. Les ARNs non codants de plus de 200 nucleotides, classés comme longs ARNs non codants (LARNnc), représentent la classe la plus abondante de transcripts non codants. Les études des fonctions des LARNnc suggèrent que beaucoup d'entre eux seraient impliqués dans la régulation de la transcription. L'objectif de ma thèse de doctorat était d'élucider les mécanismes de la régulation transcriptionnelle médiée par des LARNnc dans différents systèmes eucaryotes. Dans mon premier projet, j'ai étudié le rôle d'un long ARN non codant antisens dans la régulation transcriptionnelle du gène PHO84, codant un transporteur de phosphate à haute affinité, chez S. cerevisiae. Des études antérieures ont montré que la suppression d’une proteine de l’exosome Rrp6 entraîne une augmentation de l'expression antisens et la répression de PHO84. Il a été suggéré que la perte de Rrp6 entraîne une stabilisation antisens au locus PHO84, entraînant le recrutement de l'histone de-acétylase Hda1 et la répression de PHO84. Cependant, le mécanisme par lequel Rrp6p régule la transcription de PHO84 n’était pas connu. En combinant des méthodes à l’échelle de cellule unique, des approches biochimiques et génétiques, nous avons montré que les niveaux d'ARN antisens sont régulés principalement lors de l'élongation par le complexe Nrd1-Nab3-Sen1, qui nécessite Rrp6 pour un recrutement efficace à l`extrémité 3`de PHO84. De plus, nous révélons l'expression anticorrelé du sens et de l'antisens, En résumé, nos données suggèrent que la transcription antisens régule le seuil d'activation du promoteur PHO84. Dans mon second projet, j'ai étudié les rôles des ARNs dérivés des amplificateurs (ARNa) dans la regulation de la transcription. En utilisant les cellules de cancer du sein MCF7 comme système modèle, nous avons cherché à déterminer comment les ARNa induits par l'oestrogène (E2) participent à la régulation de la transcription médiée par le recepteur d’oestrogène (ERα) au niveau de l'allèle unique. À l'aide de l’hybridation fluorescente à l’échelle de molécule unique (smFISH), nous avons révélé qu`après induction d'E2, les ARNa sont induits avec une cinétique similaire à celle des ARNm cibles, sont localisés exclusivement dans le noyau, principalement associés à la chromatine, et sont moins abondants que les ARNm. De manière surprenante, nous avons constaté que les ARNa sont rarement co-transcrits avec leurs loci cibles, indiquant que la transcription active des gènes ne nécessite pas la synthèse continue ou l'accumulation d'ARNa sur l'amplificateur. En outre, en utilisant des mesures de la distance à sous-diffraction, nous avons démontré que la cotranscription des ARNa et des ARNm se produit rarement dans une boucle amplificateurpromoteur. De plus, nous avons révélé que la transcription basale d'ARNa n'exige pas ERα ou l'histone méthyltransférase MLL1 qui active l'amplificateur par la mono-méthylation H3K4. Dans l'ensemble, nos résultats ont montré que les ARNa peuvent jouer un rôle lors de l'activation du promoteur, mais ne sont pas nécessaires pour maintenir la transcription de l'ARNm ou pour stabiliser les interactions amplificateur-promoteur.Transcription is the initial step in gene expression and is subject to extensive regulation. Recently, analyses in diverse eukaryotes have revealed that in addition to protein coding genes, transcription occurs throughout the noncoding genome, producing non-coding RNAs of various lengths. Non-coding RNAs longer than 200 nucleotides, classified as long non-coding RNAs (lncRNAs), represent the most abundant class of non-coding transcripts, whose functions however are poorly understood. Recent studies suggest that many lncRNAs might have roles in transcription regulation. The goal of my PhD thesis was to elucidate the mechanisms of lncRNA mediated transcription regulation in different eukaryotic systems. For my first project, I investigated the role of an antisense long noncoding RNA in transcription regulation of the high-affinity phosphate transporter gene PHO84 in the unicellular eukaryote S. cerevisiae. Previous studies showed that deletion of the nuclear exosome component Rrp6 results in increased antisense expression and repression of PHO84. It was suggested that the loss of Rrp6 results in antisense stabilization at the PHO84 locus, leading to recruitment of the histone de-acetylase Hda1 and repression of PHO84. However, most of the mechanistic details of how Rrp6p functions in regulating PHO84 transcription were not understood. Combining single cell methods with biochemical and genetic approaches, we showed that antisense RNA levels are regulated primarily during transcriptional elongation by the Nrd1-Nab3-Sen1 complex, which requires Rrp6 for efficient recruitment to the 3’end of PHO84. Furthermore, we reveal anti-correlated expression of sense and antisense, which have distinct modes of transcription. In summary, our data suggest a model whereby antisense transcriptional read-through into the PHO84 promoter regulates the activation threshold of the gene. For my second project, I investigated the roles of enhancer derived RNAs (eRNAs). eRNAs are lncRNAs transcribed from enhancers that have been suggested to regulate transcription through different mechanisms, including enhancer-promoter looping, RNA polymerase elongation, and chromatin remodeling. However, no coherent model of eRNA function has yet emerged. Using MCF7 breast cancer cells as a model system, we sought to determine how estrogen (E2) induced eRNAs participate in estrogen receptor alpha (ERα) mediated transcription regulation at the single allele level. Using single molecule fluorescent in situ hybridization (smFISH), we revealed that upon E2 induction eRNAs are induced with similar kinetics as target mRNAs, but are localized exclusively in the nucleus, mostly chromatin associated, and are less abundant than mRNAs. Surprisingly, we found that eRNAs are rarely co-transcribed with their target loci, indicating that active gene transcription does not require the continuous synthesis or accumulation of eRNAs at the enhancer. Furthermore, using sub-diffraction-limit distance measurements, we demonstrated that co-transcription of eRNAs and mRNAs rarely occurs within a closed enhancer-promoter loop. Moreover, we revealed that basal eRNA transcription does not require ERα or the histone methyltransferase MLL1, which activates the enhancer through H3K4 mono-methylation. Altogether, our findings showed that eRNAs may play a role during promoter activation, but are not required to sustain mRNA transcription or stabilize enhancer-promoter looping interactions

Evidence for DNA-mediated nuclear compartmentalization distinct from phase separation.

RNA Polymerase II (Pol II) and transcription factors form concentrated hubs in cells via multivalent protein-protein interactions, often mediated by proteins with intrinsically disordered regions. During Herpes Simplex Virus infection, viral replication compartments (RCs) efficiently enrich host Pol II into membraneless domains, reminiscent of liquid-liquid phase separation. Despite sharing several properties with phase-separated condensates, we show that RCs operate via a distinct mechanism wherein unrestricted nonspecific protein-DNA interactions efficiently outcompete host chromatin, profoundly influencing the way DNA-binding proteins explore RCs. We find that the viral genome remains largely nucleosome-free, and this increase in accessibility allows Pol II and other DNA-binding proteins to repeatedly visit nearby DNA binding sites. This anisotropic behavior creates local accumulations of protein factors despite their unrestricted diffusion across RC boundaries. Our results reveal underappreciated consequences of nonspecific DNA binding in shaping gene activity, and suggest additional roles for chromatin in modulating nuclear function and organization