Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.
Project description:Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and proposed to regulate transcription initiation. However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate elucidation of the specific role of H3K4me3 in transcriptional regulation. Here, by using mouse embryonic stem cells (mESCs) as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to complete loss of all H3K4 methylation. H3K4me3 turnover occurs more rapidly than H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Surprisingly, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. Notably, we show that H3K4me3 is required for the recruitment of the Integrator Complex Subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation.
Project description:RNA polymerase II promoter-proximal pausing is orchestrated by a ribonucleoprotein complex scaffolded by the noncoding RNA Rn7sk. However, how this interruption of transcription is mechanistically linked to RNA production remains largely unknown. Here, we show that forcing the pause release by germ-line deletion of Rn7sk was embryonic lethal, yet conditional deletion of Rn7sk enhanced stem cell differentiation in skin. To explore the immediate transcriptional mechanisms underpinning enhanced differentiation, we metabolically labelled newly-synthesized RNAs after Rn7sk deletion. Unexpectedly, forced pause release robustly repressed transcription specifically at cell cycle regulators, in the absence of chromatin remodeling at promoters and enhancers. Our results indicate that polymerase pausing affords the core elongation machinery time to properly assemble, and forced elongation triggers splicing defects and nuclear RNA decay. Cell cycle regulators appear highly sensitive to mis-regulation of the elongation machinery due to unique genomic features of high promoter accessibility and low GC–content in the gene body. Transcriptional pausing thus serves as a rate-limiting step in controlling cell division.
Project description:RNA polymerase II promoter-proximal pausing is orchestrated by a ribonucleoprotein complex scaffolded by the noncoding RNA Rn7sk. However, how this interruption of transcription is mechanistically linked to RNA production remains largely unknown. Here, we show that forcing the pause release by germ-line deletion of Rn7sk was embryonic lethal, yet conditional deletion of Rn7sk enhanced stem cell differentiation in skin. To explore the immediate transcriptional mechanisms underpinning enhanced differentiation, we metabolically labelled newly-synthesized RNAs after Rn7sk deletion. Unexpectedly, forced pause release robustly repressed transcription specifically at cell cycle regulators, in the absence of chromatin remodeling at promoters and enhancers. Our results indicate that polymerase pausing affords the core elongation machinery time to properly assemble, and forced elongation triggers splicing defects and nuclear RNA decay. Cell cycle regulators appear highly sensitive to mis-regulation of the elongation machinery due to unique genomic features of high promoter accessibility and low GC–content in the gene body. Transcriptional pausing thus serves as a rate-limiting step in controlling cell division.
Project description:Chickarmane2006 - Stem cell switch irreversible
Kinetic modeling approach of the transcriptional dynamics of the embryonic stem cell switch.
This model is described in the article:
Transcriptional dynamics of the embryonic stem cell switch.
Chickarmane V, Troein C, Nuber UA, Sauro HM, Peterson C
PLoS Computational Biology. 2006; 2(9):e123
Abstract:
Recent ChIP experiments of human and mouse embryonic stem cells have elucidated the architecture of the transcriptional regulatory circuitry responsible for cell determination, which involves the transcription factors OCT4, SOX2, and NANOG. In addition to regulating each other through feedback loops, these genes also regulate downstream target genes involved in the maintenance and differentiation of embryonic stem cells. A search for the OCT4-SOX2-NANOG network motif in other species reveals that it is unique to mammals. With a kinetic modeling approach, we ascribe function to the observed OCT4-SOX2-NANOG network by making plausible assumptions about the interactions between the transcription factors at the gene promoter binding sites and RNA polymerase (RNAP), at each of the three genes as well as at the target genes. We identify a bistable switch in the network, which arises due to several positive feedback loops, and is switched on/off by input environmental signals. The switch stabilizes the expression levels of the three genes, and through their regulatory roles on the downstream target genes, leads to a binary decision: when OCT4, SOX2, and NANOG are expressed and the switch is on, the self-renewal genes are on and the differentiation genes are off. The opposite holds when the switch is off. The model is extremely robust to parameter changes. In addition to providing a self-consistent picture of the transcriptional circuit, the model generates several predictions. Increasing the binding strength of NANOG to OCT4 and SOX2, or increasing its basal transcriptional rate, leads to an irreversible bistable switch: the switch remains on even when the activating signal is removed. Hence, the stem cell can be manipulated to be self-renewing without the requirement of input signals. We also suggest tests that could discriminate between a variety of feedforward regulation architectures of the target genes by OCT4, SOX2, and NANOG.
This model is hosted on BioModels Database
and identified by: MODEL7957942740
.
To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models
.
To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication
for more information.
Project description:Chickarmane2006 - Stem cell switch reversible
Kinetic modeling approach of the transcriptional dynamics of the embryonic stem cell switch.
This model is described in the article:
Transcriptional dynamics of the embryonic stem cell switch.
Chickarmane V, Troein C, Nuber UA, Sauro HM, Peterson C
PLoS Computational Biology. 2006; 2(9):e123
Abstract:
Recent ChIP experiments of human and mouse embryonic stem cells have elucidated the architecture of the transcriptional regulatory circuitry responsible for cell determination, which involves the transcription factors OCT4, SOX2, and NANOG. In addition to regulating each other through feedback loops, these genes also regulate downstream target genes involved in the maintenance and differentiation of embryonic stem cells. A search for the OCT4-SOX2-NANOG network motif in other species reveals that it is unique to mammals. With a kinetic modeling approach, we ascribe function to the observed OCT4-SOX2-NANOG network by making plausible assumptions about the interactions between the transcription factors at the gene promoter binding sites and RNA polymerase (RNAP), at each of the three genes as well as at the target genes. We identify a bistable switch in the network, which arises due to several positive feedback loops, and is switched on/off by input environmental signals. The switch stabilizes the expression levels of the three genes, and through their regulatory roles on the downstream target genes, leads to a binary decision: when OCT4, SOX2, and NANOG are expressed and the switch is on, the self-renewal genes are on and the differentiation genes are off. The opposite holds when the switch is off. The model is extremely robust to parameter changes. In addition to providing a self-consistent picture of the transcriptional circuit, the model generates several predictions. Increasing the binding strength of NANOG to OCT4 and SOX2, or increasing its basal transcriptional rate, leads to an irreversible bistable switch: the switch remains on even when the activating signal is removed. Hence, the stem cell can be manipulated to be self-renewing without the requirement of input signals. We also suggest tests that could discriminate between a variety of feedforward regulation architectures of the target genes by OCT4, SOX2, and NANOG.
This model is hosted on BioModels Database
and identified by: MODEL7957907314
.
To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models
.
To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication
for more information.
Project description:Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.
Project description:Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.
Project description:Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.
Project description:Transcriptional regulation in eukaryotes commonly occurs at promoter-proximal regions wherein transcriptionally engaged RNA Polymerase II (Pol II) pauses before proceeding towards productive elongation. The roles of chromatin in this process remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 regulates transcription elongation by binding to Pol II and anchoring the Negative ELongation Factor NELF, thereby preventing the release of Pol II towards elongation. Absence of SIRT6, or conditions of glucose deprivation, lead to increased levels of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of the CDK9 kinase, phosphorylation of NELF and Pol II, and recruitment of the transcription factors MYC and BRD4, the PAF1 Complex, as well as several positive elongation factors, in turn releasing Pol II into productive elongation. Collectively, we identified SIRT6 as the first pausing-dedicated histone deacetylase, regulating intragenic H3K9ac and H3K56ac as critical chromatin modifications modulating transcriptional pausing and elongation.