Browsing by Subject "RNA Polymerase II"
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Item KAP1 Is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs(2020-05-01T05:00:00.000Z) Bacon, Curtis Wayne; Conrad, Nicholas; Kraus, W. Lee; Corey, David; D'Orso, IvánPrecise control of the RNA polymerase II (Pol II) cycle, including pausing and pause release, maintains transcriptional homeostasis and organismal functions. Much previous work to understand individual transcription steps, but insight into how these steps might be integrated is lacking. Here we reveal a mechanism that integrates Pol II cycle transitions. Surprisingly, the transcription factor KAP1/TRIM28 uses a previously uncharacterized chromatin reader cassette to bind hypo-acetylated histone 4 tails at promoters thereby guaranteeing a continuous progression of Pol II entry to, and exit from, the pause state. Upon chromatin docking, KAP1 first associates with Pol II and then recruits a pathway-specific transcription factor (SMAD2) in response to cognate ligands thereby enabling gene-selective CDK9-dependent pause release. This coupling mechanism is exploited by colorectal cancer cells to aberrantly sustain transcriptional programs commonly dysregulated in cancer patients. The discovery of a factor integrating transcription steps expands the functional repertoire by which chromatin readers operate and provides mechanistic understanding of transcription regulation, offering alternative therapeutic opportunities to target transcriptional dysregulation.Item Regulation of Transcription Through RNA Polymerase II Promoter-Proximal Pausing(2018-04-12) Tastemel, Melodi Damla; Kraus, W. Lee; Zhang, Chengcheng "Alec"; Amatruda, James F.; Banaszynski, Laura; Bai, XiaoyingCells need to respond to environmental signals in order to adapt to changing stimuli, maintain cell viability and establish fate decisions. One way the cell accomplishes this adaptation is via altering gene expression of critical genes by influencing transcription of their mRNAs. mRNA Transcription is carried out by the enzyme RNA Polymerase II (Pol II). Regulation of RNA Pol II occurs at every step of transcription, the most well studied one being transcription initiation. However, in many metazoan genes, after transcription initiates, RNA Pol II experiences a pause 20-60 nucleotides downstream of transcription start site. This is mediated by two pausing complexes negative elongation factor (NELF) and DRB-sensitivity inducing factor (DSIF). In order to pursue productive elongation, Pol II needs to be relieved from this promoter-proximal pause by recruitment of P-TEFb. Recent studies have demonstrated that regulation of transcription pausing, and elongation is necessary for cells to respond to external stimuli and for mammalian development, and that dysregulation of this network has been seen in developmental and immunological disorders, heart disease and a variety of cancers. However, what are the molecular and functional roles of Pol II pausing during normal mammalian development are yet to be elucidated. Here, using mouse embryonic stem cell differentiation as my major model system of mammalian development, I asked whether disruptions in RNA Pol II pausing would have an effect on embryonic development. I utilized CRISPR/Cas9 based genome editing to mutate pausing complex subunits nelfe (NELF) and spt5 (DSIF) in mouse embryonic stem cells. Using genomic approaches such as global run-on sequencing, I validated that promoter-proximal pausing is perturbed in these mESCs. By utilizing monolayer and three-dimensional differentiation protocols, I have observed disruption of lineage differentiation in pausing deficient mESCs. Finally, I established CRISPR/Cas9 genome editing protocols to mutate chromatin regulators and pausing factors in order to study their roles in developing organisms.Item Regulatory Mechanisms for the Pol II Associated Histone Methyltransferase Set2(2014-11-20) Wang, Yi; Roth, Michael G.; Kraus, W. Lee; Liu, Yi; Li, BingNucleosomes are building blocks of the eukaryotic chromatin which package genomic DNA with histones. The modification patterns of histones constitute an important signaling pathway for various nuclear processes. H3K36 methylation is catalyzed by the histone methyltranserase Set2 during transcription elongation. This important histone mark is ubiquitously presented in all organisms from yeast to mammals. In this study, we set out to investigate the molecular mechanisms by which the Set2 activity is precisely regulated during dynamic transcription cycle. In the first part of the study we discovered a novel role of the Set2 SRI domain, which is responsible for the binding of Set2 to elongating RNA polymerase II. We show that SRI also binds to DNA which determines the substrate specificity of Set2. In addition, we identified a novel auto-inhibitory role for the middle region of Set2 in regulating catalytic activity of Set2. Remarkably, mutations at this region cause hyperactivities, which in turn lead to synthetic phenotype with an essential histone chaperone FACT. Our data suggests that a temporal control for dynamic chromatin regulation is needed during transcription elongation process. In the second part, we investigated the molecular mechanism by which elongating Pol II regulated the Set2 activity beyond its initial recruitment. Surprisingly, we found the excessive amount of phosphorylated serine residues on Pol II CTD inhibited Set2 activity in vitro. Subsequent biophysical examination revealed that the additional phosphorylated CTD repeats collaterally occupied the surface of SRI where SRI contacts DNA. Finally, we determined that the minimal recognition unit of Set2 on fully phosphorylated CTD tail is three heptad repeats, and demonstrated that this minimal unit is sufficient for the Set2 recruitment without disrupting its catalytic activity. Since Pol II CTD utilizes repeating sequence as a scaffold for multiple factors, our results implicate that an organized spatial arrangement of these factors along CTD is necessary for accommodating their individual functions. In the last part of this work, we examined the state-specific functions of H3K36 methylation. By manipulating the catalytic domain of Set2, we obtained two mutants that can catalyze specific methyl-states of H3K36 both in vitro and in vivo. Genetic studies showed that cells carrying these two mutants displayed distinct phenotypes in several functional pathways, including histone chaperone, CTD proline isomerization and double-strand DNA repair. Our results suggest individual methyl-state of H3K36 plays non-redundant biological roles in cells. In summary, we discovered multiple mechanisms by which Set2 is dynamically regulated by elongating Pol II. Setd2, the human homolog of yeast Set2, has been shown recently to be one of the most important tumor suppressors among chromatin regulators. Given the highly conserved nature of this histone methyltransferase family, we believe that our mechanistic studies here may shed lights on the roles of Setd2 in tumorigenesis.