The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila

BACKGROUND: The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. RESULTS AND DISCUSSION: Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. CONCLUSIONS: Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes

Aging alters chromatin accessibility and transcriptional regulation in murine liver tissue

Regulation of gene expression is tightly linked to the organization of the mammalian genome. With age, chromatin alterations occur on all levels of genome organization, accompanied by changes in gene expression profiles. However, little is known about the detailed changes in transcriptional regulation with age. Here, we systematically characterize age-related changes in the local chromatin landscape of murine liver tissue and their link to transcriptional regulation. To the best of our knowledge, this is the first systematic inventory of the connection between aging, chromatin accessibility and transcriptional regulation in vivo, in a whole tissue. We observe that aging of murine liver tissue is accompanied by an increase in chromatin accessibility at promoter regions of protein-coding genes. Yet, although promoter accessibility is a requirement for transcription, the increased accessibility did not result in enhanced transcriptional output. Instead, aging is accompanied by a decrease of promoter-proximal pausing of RNA polymerase II (Pol II). We propose that these changes in transcriptional regulation are due to a reduced stability of the pausing complex and may represent a mechanism to compensate for the age-related increase in chromatin accessibility in order to prevent aberrant transcription

Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters

Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function

SummaryThe functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters

The role of the arginine methyltransferase CARM1 in global transcriptional regulation.

Arginine methylation is a prevalent post-translational modification that is found on many nuclear and cytoplasmic proteins, and has been implicated in the regulation of gene expression. CARM1 is a member of the protein arginine methyltransferase (PRMT) family of proteins, and is a key protein responsible for arginine methylation of a subset of proteins involved in transcription. In this thesis I examine some of the mechanisms through which CARM1 contributes to global transcriptional regulation. Using a ChIP-DSL approach, we show that the p/CIP/CARM1 complex is recruited to 204 proximal promoters following 17β-estradiol (E2) treatment in MCF-7 cells. Many of the target genes have been previously implicated in signaling pathways related to oncogenesis. JAK2, a member of the Jak/Stat signaling cascade, is one of the direct E2-dependent targets of the p/CIP/CARM1 complex. Following E2-treatment, histone modifications at the JAK2 promoter are reflective of a transcriptionally permissive gene, and we observed modest increases in RNA and protein expression. Notably, E2-induced expression of Jak2 was diminished when p/CIP or CARM1 were depleted, suggesting that the p/CIP/CARM1 complex is required for the observed transcriptional response. Collectively, these results suggest that E2-dependent recruitment of the p/CIP/CARM1 complex causes JAK2 to become ‘poised’ for transcription, a finding that may be extendable to other target genes and signalling pathways. Furthermore, bioinformatic examination of p/CIP/CARM1 target promoters suggests that transcription factor crosstalk is the favored mechanism of E2-dependent p/CIP/CARM1 complex recruitment. Using ChIP-Seq, we identified genomic regions to which CARM1 is recruited. Subsequent characterization of binding events suggest a role for CARM1 in transcriptional elongation, and implicate the transcription factor PAX1 as a novel mechanism through which CARM1 can be recruited to the genome. Identification of CARM1-dependent differentially expressed genes revealed that direct recruitment of CARM1 is not essential for the majority of its transcriptional effects in MEFs. However, CARM1 does play a critical role in cellular growth and proliferation, and in the absence of CARM1, the expression of many cell cycle regulators is dramatically affected. Collectively, this work provides insight into some of the mechanisms through which CARM1 modulates transcription, and highlights its importance in diverse cellular processes

H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells

Epigenetic regulation of chromatin plays a critical role in controlling embryonic stem (ES) cell selfrenewal and pluripotency. However, the roles of histone demethylases and activating histone modifications such as trimethylated histone 3 lysine 4 (H3K4me3) in transcriptional events such as RNA polymerase II (RNAPII) elongation and alternative splicing are largely unknown. In this study, we show that KDM5B, which demethylates H3K4me3, plays an integral role in regulating RNAPII occupancy, transcriptional initiation and elongation, and alternative splicing events in ES cells. Depletion of KDM5B leads to altered RNAPII promoter occupancy, and decreased RNAPII initiation and elongation rates at active genes and at genes marked with broad H3K4me3 domains. Moreover, our results demonstrate that spreading of H3K4me3 from promoter to gene body regions, which is mediated by depletion of KDM5B, modulates RNAPII elongation rates and RNA splicing in ES cells. We further show that KDM5B is enriched nearby alternatively spliced exons, and depletion of KDM5B leads to altered levels of H3K4 methylation in alternatively spliced exon regions, which is accompanied by differential expression of these alternatively splice exons. Altogether, our data indicate an epigenetic role for KDM5B in regulating RNAPII elongation and alternative splicing, which may support the diverse mRNA repertoire in ES cells

Mining The Spatial And Temporal Context By Which Transcription Factors Occupy Chromatin

Transcription factors (TFs) occupy chromatin to coordinate widespread regulation of gene expression programs that define cellular identity. These proteins bind diverse DNA sequences genome-wide and in a non-uniform manner across the cell cycle, yet the spatial and temporal determinants of TF chromatin occupancy are largely unknown. First, to define DNA sequence determinants of in vivo TF binding, we developed an approach that exploits natural genetic variation between highly similar erythroid cell lines. From ChIP-seq data we were able to directly identify extensive single nucleotide variants that discriminate these cell lines from each other. By measuring the impact of these variants on TF ChIP-seq binding intensities, we defined at single nucleotide resolution the binding determinants of the GATA1, TAL1, and CTCF factors. We also identified contextual sequences that, in addition to a TF’s core DNA motif, dictate TF specificity and modulate TF binding when mutated. Together, these studies present new approaches and biological insights regarding the DNA sequence requirements for TF binding to chromatin. Next, we tested whether TF chromatin occupancy is dynamic across the cell cycle. In particular, we asked whether the BRD4 TF binds chromatin in erythroid cells during mitosis, and whether it might function as a bookmark of transcriptional programs across mitosis. While we find that BRD4 is preferentially enriched during mitosis at erythroid-specific genes in the G1E-ER4 erythroid cell line, transient removal of BRD4 from chromatin during mitosis does not impair the reactivation of erythroid-specific programs following mitosis. Thus, BRD4 does not function as a bookmark of transcription, and instead we considered that it might passively bind to acetylated histones during mitosis. Given that the histone PTM landscape during mitosis has not been previously characterized, we used histone mass spectrometry and genome-wide location analysis to find extensive preservation of H3K14ac, H3K122ac, and H4K16ac on mitotic chromatin. Furthermore, these marks are predictive of BRD4 and Pol II binding to mitotic chromatin and are preferentially enriched at erythroid-specific genes during mitosis, suggestive of a role in mitotic bookmarking. Together, these studies reveal new insights about the mechanisms by which transcription factors occupy chromatin

Aberrant gene expression induced by a high fat diet is linked to H3K9 acetylation in the promoter-proximal region

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with an estimated global prevalence of 1 in 4 individuals. Aberrant transcriptional control of gene expression is central to the pathophysiology of metabolic diseases. However, the molecular mechanisms leading to gene dysregulation are not well understood. Histone modifications play important roles in the control of transcription. Acetylation of histone 3 at lysine 9 (H3K9ac) is associated with transcriptional activity and is implicated in transcript elongation by controlling RNA polymerase II (RNAPII) pause-release. Hence, changes in this histone modification may shed information on novel pathways linking transcription control and metabolic dysfunction. Here, we carried out genome-wide analysis of H3K9ac in the liver of mice fed a control or a high-fat diet (an animal model of NAFLD), and asked whether this histone mark associates with changes in gene expression. We found that over 70% of RNAPII peaks in promoter-proximal regions overlapped with H3K9ac, consistent with a role of H3K9ac in the regulation of transcription. When comparing high-fat with control diet, approximately 17% of the differentially expressed genes were associated with changes in H3K9ac in their promoters, showing a strong correlation between changes in H3K9ac signal and gene expression. Overall, our data indicate that in response to a high-fat diet, dysregulated gene expression of a subset of genes may be attributable to changes in transcription elongation driven by H3K9ac. Our results point at an added mechanism of gene regulation that may be important in the development of metabolic diseases

The interplay of global chromosomal organisation, promoter-enhancer interactions and transcription

All somatic cells within an organism contain the same genetic material, yet they display pronounced differences in function and morphology. Precise control of gene expression is of fundamental importance to allow cells to properly develop, maintain homeostasis, and respond to external stimuli. The first step in gene expression is transcription, which starts at the core promoter region. While core promoters are crucial for transcriptional initiation, they are insufficient for establishing complex tissue- and condition-specific gene expression patterns in multicellular organisms. Additional transcriptional control elements, such as gene enhancers, are required for this, with many such elements localising considerable distances away from their target promoters. Enhancers commonly convey their regulatory signals to target promoters by forming physical contacts with them through three-dimensional DNA looping, underpinning the importance of chromosomal organisation in transcriptional control. In recent years, the emergence of chromosome conformation capture and related methodologies has dramatically increased our understanding of chromosomal organisation. In particular, high-throughput Hi-C analyses across cell types have led to the identification of spatial genomic structures, including Topologically Associating Domains (TADs). In parallel, high-resolution versions of these technologies (such as 5C, CHiA-PET, HiChIP and Capture Hi-C) have detected multitudes of novel looping interactions, including connections between promoters and enhancers. The interplay between precise regulatory interactions, the higher-order chromosomal organisation, and their joint contribution to transcriptional control is incompletely understood and is the focus of this work. In the first part of this work, I take advantage of high-resolution Promoter Capture Hi-C (PCHi-C) data to investigate the localisation of promoter interactions with respect to TAD boundaries in human primary blood cells and cell-cycle synchronised HeLa cells. I show that the majority of promoter interactions originate at, and are constrained by TAD boundaries. However, a minority of promoter interactions appear to cross TAD boundaries in all analysed cell types. Furthermore, I identify genes with multiple TAD-boundary crossing interactions per promoter and present evidence that these interactions may be supported by transcriptional machinery. These results suggest a role for transcriptional machinery in shaping promoter interactions in a TAD independent manner. In the second part of this work, I investigate promoter interaction rewiring upon perturbations of architectural proteins. For this analysis, I use PCHi-C data from HeLa cells, in which cohesin or CTCF are rapidly depleted using Auxin-induced degradation. I show that promoter interactions that are lost, maintained, or gained upon cohesin depletion possess distinct distance profiles and relate to TAD organisation in markedly different ways. I demonstrate that promoter-interacting regions that are lost upon cohesin depletion associate with architectural proteins, while those that are maintained or gained show characteristics of enhancers. Finally, I show evidence for a functional role of cohesin-mediated interactions in transcriptional regulation. Collectively, this work reveals the interplay between TADs, promoter interactions and transcription, while suggesting that promoter interactions may be supported by TAD independent mechanisms.MRC DTP studentship BBS/E/B/000M0816 and164259