Dynamics of promoter bivalency and RNAP II pausing in mouse stem and differentiated cells

Mammalian embryonic stem cells display a unique epigenetic and transcriptional state to facilitate pluripotency by maintaining lineage-specification genes in a poised state. Two epigenetic and transcription processes involved in maintaining poised state are bivalent chromatin, characterized by the simultaneous presence of activating and repressive histone methylation marks, and RNA polymerase II (RNAPII) promoter proximal pausing. However, the dynamics of histone modifications and RNAPII at promoters in diverse cellular contexts remains underexplored. We collected genome wide data for bivalent chromatin marks H3K4me3 and H3K27me3, and RNAPII (8WG16) occupancy together with expression profiling in eight different cell types, including ESCs, in mouse. The epigenetic and transcription profiles at promoters grouped in over thirty clusters with distinct functional identities and transcription control. The clustering analysis identified distinct bivalent clusters where genes in one cluster retained bivalency across cell types while in the other were mostly cell type specific, but neither showed a high RNAPII pausing. We noted that RNAPII pausing is more associated with active genes than bivalent genes in a cell type, and was globally reduced in differentiated cell types compared to multipotent

Doctor of Philosophy

dissertationRNA Polymerase III (Pol III) transcribes small noncoding RNAs that are important in protein synthesis. Genome-wide analysis of Pol III and associated transcription factors (TFIIIC, BRF1, and BRF2) localization in various human cell lines was performed using chromatin immunoprecipitation followed by highthroughput sequencing (ChIP-seq). These analyses show that Pol III binds only to a fraction of all its annotated target genes. Comparison of Pol III-bound regions to known chromatin and transcription factor data shows that Pol III localization in a cell is guided by the chromatin landscape setup by Pol II. We believe Pol III binding is opportunistic and relies on a cell's pool of transcription factors and chromatin landscapes to bind its target genes. To understand Pol III localization in a cell line of high interest, human embryonic stem cells (hESCs), we performed Pol III and TFIIIC ChIP-seq analyses in H1 cells. We hoped to understand the role of open chromatin and pluripotency transcription factors in hESC, on Pol III occupancy. We observed that there are significantly more Pol III-bound regions in hESCs when compared to differentiated cells types. We also observe that these Pol III-bound regions correlated positively with active chromatin marks and Pol II. Interestingly, we also observed that pluripotency transcription factors, NANOG (in hESCs) and OCT4 ! iv! (in mESCs), correlated with Pol III-bound regions. We also observed regional H3K27me3 at Pol III-bound regions. We observe Pol II and H3K4me3 peaks situated in between the Pol III and H3K27me3 peaks. We postulate that active chromatin setup by Pol II insulates the Pol III transcription unit from H3K27me3 repression. We also identified several novel Pol III-bound regions in hESCs. This is the first analysis of Pol III in hESCs and our results are consistent with Pol III binding and activity being regulated by active chromatin that is shaped in part by the pluripotency transcription factor network

The Mechanism and Modulation of H-NS Mediated Repression in Escherichia coli.

The histone-like nucleoid structuring protein H-NS acts as a global repressor of genes that are expressed in response to environmental stimuli and stress conditions. Repression by H-NS is presumably mediated by binding of H NS to primary "nucleation sites" close to promoters, and the formation of extended nucleoprotein complex from these nucleation sites to inhibit transcription initiation. Modulation of H-NS mediated repression is a complex process involving specific transcription factors and physiology dependent structural alterations. The E. coli bgl and proU operons are model systems that are repressed by H-NS with exceptional specificity. Both of these systems possess upstream and downstream regulatory elements (URE and DRE) bound by H-NS for efficient repression. The present study demonstrates that repression by H-NS binding upstream and downstream is synergistic in proU (as shown in a parallel study for bgl), and that H-NS when bound within the transcription unit represses transcription initiation at the bgl promoter, as reported before for proU. Repression by binding of H-NS downstream is known to be modulated. Common to both proU and bgl is that an increase in the promoter activity abrogates repression. For bgl it is known, that the H-NS mediated repression of the promoter is counteracted by transcription factors BglJ and LeuO. Further, termination factor Rho and the protease Lon are known to modulate repression by H-NS through the DRE, and as shown here the DnaKJ chaperone system is essential for this repression. In case of proU, the promoter is osmoregulated; the RNA polymerase is poised at the promoter at low osmolarity, while it clears the promoter with better efficiency at high osmolarity. Furthermore, the proU operon is subject to post-transcriptional osmoregulation. The proU mRNA is processed by RNAse III within a stretch of highly conserved sequence, suggesting a common mechanism of regulation among Enterobacteria. In summary, the present study demonstrates that the mechanism of H-NS mediated repression of the bgl and proU operons is very similar. However, its modulation is complex involving numerous additional factors specific to the two systems, and thus is achieved in a context specific manner

Touched by CTCF: Analysis of a multi-functional zinc finger protein

Multicellular organisms contain a complete set of genes in nearly all of their cells. However most cells are very different to each other and are able to form organs with distinct functions. The identity and survival of the cell is regulated by the activity of specific genes in time and space. Specific sets of genes encoding proteins become activated, whereas others are repressed. CTCF is a protein that mediates distinct processes of gene regulation, including transcription and the structural organization of the genome. The aim of this thesis is to investigate the different functions of CTCF by a combined analysis of CTCF-interacting proteins and by deletion of CTCF in vivo and in vitro. Using these approaches we aimed to improve our understanding of the molecular mechanism underlying its functions. Chapter 1 gives an overview of the information required to understand the foundations of studies presented and discussed in this thesis. It gives an introduction to gene regulation and how this process is influenced by chromatin modifications, nuclear organization and compartmentation. A specific nuclear compartment, the nucleolus, and its involvement in ribosomal RNA synthesis, are highlighted. Furthermore the characteristics of CTCF and its homolog CTCFL are described in detail

The Epigenetic Regulation of Cytokine Inducible Mammalian Transcription by the 26S Proteasome

It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes

Mechanistic characterization of ZIC2 function during brain patterning

Zinc finger of the cerebellum (ZIC) proteins constitute a family of transcription factors (TFs) with crucial roles during embryogenesis, particularly during neural development. Defects on the genes encoding these TFs cause a broad range of developmental disorders. In particular, Zic2 defects lead to holoprosencephaly, a congenital brain malformation resulting from the defective cleavage of cerebral hemispheres manifested with variable expressivity and incomplete penetrance. However, the target genes and mechanism of action of ZIC2 during brain development are largely unknown. Consequently, the molecular etiology of ZIC2-associated holoprosencephaly remains poorly characterized. To elucidate the molecular mechanisms by which ZIC2 contributes to proper brain development, I first analyzed how the loss of ZIC2 function affects the differentiation of embryonic stem cells into anterior neural progenitor cells (AntNPCs). Notably, I found that the knockout of Zic2 led to a drastic downregulation of dorsal brain genes, including major roof plate markers such as Lmx1a and Lmx1b. Next, one major objective in this project is to determine if ZIC2 directly activates these dorsal genes during AntNPC differentiation or if, alternatively, it represses ventral regulators which themselves antagonize brain dorsal identity. To achieve this, it is necessary to generate ZIC2 binding profiles genome-wide in AntNPC. Due to the lack of a specific antibody against ZIC2, I used CRISPR-Cas9 technology to generate a mouse embryonic stem cell line (mESC) in which the endogenous Zic2 was tagged with a C-terminal Flag-HA epitope. After demonstrating that Zic2 is expressed in this cell line at the same levels as in WT cells both at mRNA and protein level, I also showed that this cell line can be used to identify ZIC2 binding sites by chromatin immunoprecipitation (ChIP). This Zic2-Flag-HA mESC line will now allow us to perform chromatin immunoprecipitation and sequencing (ChIP-seq) and immunoprecipitation coupled to mass spectrometry (IP-MS) experiments to elucidate ZIC2 genomic binding sites and its possible interacting partners, which should provide major insights into the regulatory networks and mechanisms whereby ZIC2 contributes to brain development and human holoprosencephaly.Máster en Biología Molecular y Biomedicin

Making sense of IL-6 signalling cues in pathophysiology

Unravelling the molecular mechanisms that account for functional pleiotropy is a major challenge for researchers in cytokine biology. Cytokine–receptor cross-reactivity and shared signalling pathways are considered primary drivers of cytokine pleiotropy. However, reports epitomized by studies of Jak-STAT cytokine signalling identify interesting biochemical and epigenetic determinants of transcription factor regulation that affect the delivery of signal-dependent cytokine responses. Here, a regulatory interplay between STAT transcription factors and their convergence to specific genomic enhancers support the fine-tuning of cytokine responses controlling host immunity, functional identity, and tissue homeostasis and repair. In this review, we provide an overview of the signalling networks that shape the way cells sense and interpret cytokine cues. With an emphasis on the biology of interleukin-6, we highlight the importance of these mechanisms to both physiological processes and pathophysiological outcomes

Chromatin binding factor Spn1 contributes to genome instability in Saccharomyces cerevisiae, The

2018 Spring.Includes bibliographical references.Maintaining the genetic information is the most important role of a cell. Alteration to the DNA sequence is generally thought of as harmful, as it is linked with many forms of cancer and hereditary diseases. Contrarily, some level of genome instability (mutations, deletions, amplifications) is beneficial to an organism by allowing for adaptation to stress and survival. Thus, the maintenance of a "healthy level" of genome stability/instability is a highly regulated process. In addition to directly processing the DNA, the cell can regulate genome stability through chromatin architecture. The accessibility of DNA for cellular machinery, damaging agents and spontaneous recombination events is limited by level of chromatin compaction. Remodeling of the chromatin for transcription, repair and replication occurs through the actions of ATP remodelers, histone chaperones, and histone modifiers. These complexes work together to create access for DNA processing and to restore the chromatin to its pre-processed state. As such, many of the chromatin architecture factors have been implicated in genome stability. In this study, we have examined the role of the yeast protein Spn1 in maintaining the genome. Spn1 is an essential and conserved transcription elongation factor and chromatin binding factor. As anticipated, we observed that Spn1 contributes to the maintenance of the genome. Unexpectedly, our data revealed that Spn1 contributes to promoting genome instability. Investigation into a unique growth phenotype in which cells expressing a mutant form of Spn1 displayed resistance to the damaging agent, methyl methanesulfonate revealed Spn1 influences pathway selection during DNA damage tolerance. DNA damage tolerance is utilized during replication and G2 to bypass lesions, which could permanently stall replication machinery. This pathway congruently promotes and prevents genome instability. We theorize that these outcomes are due to the ability of Spn1 to influence chromatin structure throughout the cell cycle