scholarly journals RPRD Proteins Control Transcription in Human Cells

2021 ◽  
Author(s):  
Kinga Winczura ◽  
Hurmuz Ceylan ◽  
Monika Sledziowska ◽  
Matt Jones ◽  
Holly Fagarasan ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Human Cells ◽  
Cellular Stress Response ◽  
Regulation Of Transcription ◽  
Biological Processes ◽  
Substantial Impact ◽  
Pol Ii ◽  
Number Of Factors ◽  
External Signals

The regulation of transcription is an essential process that allows the cell to respond to various internal and external signals. RNA Polymerase II (Pol II) activity is controlled by a number of factors which bind to the C-terminal domain (CTD) of its largest subunit, RPB1, and stimulate or suppress RNA synthesis. Here, we demonstrate that CTD-interacting proteins, RPRD2, RPRD1B and RPRD1A act as negative regulators of transcription and their levels inversely correlate with the accumulation of nascent and newly transcribed RNA in human cells. We show that the RPRD proteins form mutually exclusive complexes with Pol II to coordinate their roles in transcriptional control. Our data indicate that RPRD2 exerts the most substantial impact on transcription and has the potential to alter key biological processes including the cellular stress response and cell growth.

AT7519, a Potent Multi-Targeted CDK Inhibitor, Is Active in CLL Patient Samples Independent of Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3161-3161
Author(s):  
Vicky Lock ◽  
Laurence Cooke ◽  
Murray Yule ◽  
Neil T Thompson ◽  
K. Della Croce ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Rna Polymerase Ii ◽  
Parp Cleavage ◽  
Regulation Of Transcription ◽  
Cytotoxic Effects ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Cyclin Dependent Kinases

Abstract Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II (RNA pol II). The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 on CLL cells isolated from patients. Primary cell samples were isolated from a total of 15 patients with CLL with various stages of disease (8 Stage 0, 0/I or II and 7 Stage IV) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12) as well IgVH mutation and ZAP70 expression. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–700nM. AT7519 appears equally effective at inhibiting the survival of CLL cells harbouring a variety of mutations including those representative of patients that fall within poorer prognosis treatment groups. The amount of AT7519 required to induce cell death in 50% of the CLL cell population increased as exposure time was decreased but significant cell death was obtained at doses approximating to 1uM following 4–6h of treatment. These doses are equivalent to exposures achieved in ongoing AT7519 clinical studies indicating that cytotoxic doses can be achieved in patients on well tolerated schedules. The mechanism of AT7519 cytotoxic effects was investigated by western blotting for a variety of cell cycle and apoptotic markers following incubation with compound. Short term treatments (4–6h) resulted in inhibition of phosphorylation of the transcriptional marker RNA pol II and the downregulation of the anti-apoptotic protein Mcl-1. Additional antiapoptotic proteins including XIAP and Bcl-2 remained unchanged. The reduction in Mcl-1 protein levels was associated with an increase in the apoptotic marker cleaved PARP. No inhibition of cell cycle markers such as phospho-retinoblastoma protein was observed in the same samples suggesting that the cytotoxic effects of AT7519 in CLL patient samples is due to its transcriptional activity alone. Together the data suggest AT7519 offers a promising treatment strategy for patients with advanced B-cell leukemia and lymphoma.

scholarly journals Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output

2021 ◽  
Vol 118 (47) ◽  
pp. e2113757118
Author(s):  
Congyao Xu ◽  
Xiaofeng Fang ◽  
Tiancong Lu ◽  
Caroline Dean
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Antisense Transcript ◽  
Elongation Rate ◽  
Pol Ii ◽  
Alternative Processing ◽  
Floral Repressor ◽  
Related Proteins ◽  
Transcriptional Output

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3′-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins—the spliceosome NineTeen Complex (NTC) and related proteins (NTR)—and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

scholarly journals ELA1 and CUL3 Are Required Along with ELC1 for RNA Polymerase II Polyubiquitylation and Degradation in DNA-Damaged Yeast Cells

2007 ◽  
Vol 27 (8) ◽  
pp. 3211-3216 ◽  
Author(s):  
Balazs Ribar ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
Ring Finger ◽  
Human Cells ◽  
Yeast Cells ◽  
Pol Ii ◽  
Von Hippel Lindau ◽  
Ubiquitin Ligase E3 ◽  
Cullin 3

ABSTRACT Treatment of yeast and human cells with DNA-damaging agents elicits lysine 48-linked polyubiquitylation of Rpb1, the largest subunit of RNA polymerase II (Pol II), which targets Pol II for proteasomal degradation. However, the ubiquitin ligase (E3) responsible for Pol II polyubiquitylation has not been identified in humans or the yeast Saccharomyces cerevisiae . Here we show that elongin A (Ela1) and cullin 3 (Cul3) are required for Pol II polyubiquitylation and degradation in yeast cells, and on the basis of these and other observations, we propose that an E3 comprised of elongin C (Elc1), Ela1, Cul3, and the RING finger protein Roc1 (Rbx1) mediates this process in yeast cells. This study provides, in addition to the identification of the E3 required for Pol II polyubiquitylation and degradation in yeast cells, the first evidence for a specific function in yeast for a member of the elongin C/BC-box protein/cullin family of ligases. Also, these observations raise the distinct possibility that the elongin C-containing ubiquitin ligase, the von Hippel-Lindau tumor suppressor complex, promotes Pol II polyubiquitylation and degradation in human cells.

scholarly journals Multi-step regulation of transcription kinetics explains the non-linear relation between RNA polymerase II density and mRNA expression in Dosage Compensation

2017 ◽  
Author(s):  
Pouria Dasmeh
Keyword(s):  
Rna Polymerase ◽  
Mrna Expression ◽  
Rna Polymerase Ii ◽  
Dosage Compensation ◽  
Mammalian Cells ◽  
Experimental Studies ◽  
Regulation Of Transcription ◽  
Pol Ii ◽  
Non Linear

AbstractIn heterogametic organisms, expression of unequal number of X chromosomes in males and females is balanced by a process called dosage compensation. In Drosophila and mammals, dosage compensation involves nearly two-fold up-regulation of the X chromosome mediated by dosage compensation complex (DCC). Experimental studies on the role of DCC on RNA polymerase II (Pol II) transcription in mammals disclosed a non-linear relationship between Pol II densities at different transcription steps and mRNA expression. An ~20-30% increase in Pol II densities corresponds to a rough 200% increase in mRNA expression and two-fold up-regulation. Here, using a simple kinetic model of Pol II transcription calibrated by in vivo measured rate constants of different transcription steps in mammalian cells, we demonstrate how this non-linearity can be explained by multi-step transcriptional regulation. Moreover, we show how multi-step enhancement of Pol II transcription can increase mRNA production while leaving Pol II densities unaffected. Our theoretical analysis not only recapitulates experimentally observed Pol II densities upon two-fold up-regulation but also points to a limitation of inferences based on Pol II profiles from chromatin immunoprecipitation sequencing (ChIP-seq) or global run-on assays.

scholarly journals The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription in Saccharomyces cerevisiae

Genetics ◽  
2021 ◽  
Author(s):  
Emily Biernat ◽  
Jeena Kinney ◽  
Kyle Dunlap ◽  
Christian Rizza ◽  
Chhabi K Govind
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Biological Processes ◽  
Strongly Correlated ◽  
Coding Sequences ◽  
Pol Ii ◽  
Chromatin Remodeling Complex ◽  
Nucleosomal Dna

Abstract RSC (Remodels the Structure of Chromatin) is a conserved ATP-dependent chromatin remodeling complex that regulates many biological processes, including transcription by RNA polymerase II (Pol II). We report that RSC contributes in generating accessible nucleosomes in transcribed coding sequences (CDSs). RSC MNase ChIP-seq data revealed that RSC-bound nucleosome fragments were very heterogenous (∼80 bp to 180 bp) compared to a sharper profile displayed by the MNase inputs (140 bp to 160 bp), supporting the idea that RSC promotes accessibility of nucleosomal DNA. Notably, RSC binding to + 1 nucleosomes and CDSs, but not with -1 nucleosomes, strongly correlated with Pol II occupancies, suggesting that RSC enrichment s CDSs is linked to transcription. We also observed that Pol II associates with nucleosomes throughout transcribed CDSs, and similar to RSC, Pol II-protected fragments were highly heterogenous, consistent with the idea that Pol II interacts with remodeled nucleosomes in CDSs. This idea is supported by the observation that the genes harboring high-levels of Pol II in their CDSs were the most strongly affected by ablating RSC function. Additionally, rapid nuclear depletion of Sth1 decreases nucleosome accessibility and results in accumulation of Pol II in highly transcribed CDSs. This is consistent with a slower clearance of elongating Pol II in cells with reduced RSC function, and is distinct from the effect of RSC depletion on PIC assembly. Altogether, our data provide evidence in support of the role of RSC in promoting Pol II elongation, in addition to its role in regulating transcription initiation.

scholarly journals Cyclin-Dependent Kinases and CTD Phosphatases in Cell Cycle Transcriptional Control: Conservation across Eukaryotic Kingdoms and Uniqueness to Plants

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 279
Author(s):  
Zhi-Liang Zheng
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Large Scale ◽  
Transcriptional Control ◽  
Cell Cycle Control ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pol Ii ◽  
Precise Control ◽  
Cyclin Dependent Kinases ◽  
Entire Cell

Cell cycle control is vital for cell proliferation in all eukaryotic organisms. The entire cell cycle can be conceptually separated into four distinct phases, Gap 1 (G1), DNA synthesis (S), G2, and mitosis (M), which progress sequentially. The precise control of transcription, in particular, at the G1 to S and G2 to M transitions, is crucial for the synthesis of many phase-specific proteins, to ensure orderly progression throughout the cell cycle. This mini-review highlights highly conserved transcriptional regulators that are shared in budding yeast (Saccharomyces cerevisiae), Arabidopsis thaliana model plant, and humans, which have been separated for more than a billion years of evolution. These include structurally and/or functionally conserved regulators cyclin-dependent kinases (CDKs), RNA polymerase II C-terminal domain (CTD) phosphatases, and the classical versus shortcut models of Pol II transcriptional control. A few of CDKs and CTD phosphatases counteract to control the Pol II CTD Ser phosphorylation codes and are considered critical regulators of Pol II transcriptional process from initiation to elongation and termination. The functions of plant-unique CDKs and CTD phosphatases in relation to cell division are also briefly summarized. Future studies towards testing a cooperative transcriptional mechanism, which is proposed here and involves sequence-specific transcription factors and the shortcut model of Pol II CTD code modulation, across the three eukaryotic kingdoms will reveal how individual organisms achieve the most productive, large-scale transcription of phase-specific genes required for orderly progression throughout the entire cell cycle.

scholarly journals TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei

2011 ◽  
Vol 10 (7) ◽  
pp. 964-976 ◽  
Author(s):  
Tara M. Stanne ◽  
Manish Kushwaha ◽  
Matthew Wand ◽  
Jesse E. Taylor ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase Ii ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Histone Variants ◽  
Unicellular Eukaryote ◽  
Pol Ii ◽  
Content Type ◽  
Pol I ◽  
Switch Regions

ABSTRACTThe unicellular eukaryoteTrypanosoma bruceiis unusual in having very little transcriptional control. The bulk of theT. bruceigenome is constitutively transcribed by RNA polymerase II (Pol II) as extensive polycistronic transcription units. Exceptions to this rule include several RNA Pol I transcription units such as theVSGexpression sites (ESs), which are mono-allelically expressed. TbISWI, a member of the SWI2/SNF2 related chromatin remodeling ATPases, plays a role in repression of Pol I-transcribed ESs in both bloodstream- and procyclic-formT. brucei. We show that TbISWI binds both active and silent ESs but is depleted from the ES promoters themselves. TbISWI knockdown results in an increase inVSGtranscripts from the silentVSGESs. In addition to its role in the repression of the silent ESs, TbISWI also contributes to the downregulation of the Pol I-transcribed procyclin loci, as well as nontranscribedVSGbasic copy arrays and minichromosomes. We also show that TbISWI is enriched at a number of strand switch regions which form the boundaries between Pol II transcription units. These strand switch regions are the presumed sites of Pol II transcription initiation and termination and are enriched in modified histones and histone variants. Our results indicate that TbISWI is a versatile chromatin remodeler that regulates transcription at multiple Pol I loci and is particularly abundant at many Pol II transcription boundaries inT. brucei.

scholarly journals Fission Yeast Cdk7 Controls Gene Expression through both Its CAK and C-Terminal Domain Kinase Activities

2015 ◽  
Vol 35 (9) ◽  
pp. 1480-1490 ◽  
Author(s):  
Maxime Devos ◽  
Elise Mommaerts ◽  
Valerie Migeot ◽  
Harm van Bakel ◽  
Damien Hermand
Keyword(s):  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Ribosome Biogenesis ◽  
Transcriptional Control ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Transcriptome Profiling ◽  
Pol Ii ◽  
Physiological Importance ◽  
Terminal Domain

Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells

Science ◽  
2013 ◽  
Vol 341 (6146) ◽  
pp. 664-667 ◽  
Author(s):  
Ibrahim I. Cisse ◽  
Ignacio Izeddin ◽  
Sebastien Z. Causse ◽  
Lydia Boudarene ◽  
Adrien Senecal ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Eukaryotic Cells ◽  
Average Lifetime ◽  
Pol Ii ◽  
Time Dynamics ◽  
External Signals ◽  
Rate Limiting

Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell’s ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

scholarly journals Dynamics of transcription elongation are finely tuned by dozens of regulatory factors

2021 ◽  
Author(s):  
Mary Couvillion ◽  
Kevin M Harlen ◽  
Kate C Lachance ◽  
Kristine Trotta ◽  
Erin Smtih ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Transcription Elongation ◽  
Antisense Transcription ◽  
Regulation Of Transcription ◽  
Pol Ii ◽  
Density Peaks ◽  
Large Numbers ◽  
Key Points ◽  
Opposing Effects

Understanding the complex network and dynamics that regulate transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of S. cerevisiae lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation dynamics. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Genes that are sensitive to disruptions in transcription elongation tend to couple changes in Pol II pausing and antisense transcription to transcription output. Our findings collectively show that the regulation of transcription elongation by a diverse array of factors affects gene expression levels and co-transcriptional processing by precisely balancing Pol II activity.

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