scholarly journals P-TEFb is degraded by Siah1/2 in quiescent cells

2022 ◽  
Author(s):  
Fang Huang ◽  
Yongmei Feng ◽  
B. Matija Peterlin ◽  
Koh Fujinaga
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Region ◽  
Resting Cells ◽  
Primary Cells ◽  
Proliferating Cells ◽  
E3 Ligases ◽  
Quiescent Cells

P-TEFb, composed of CycT1 and CDK9, regulates the elongation of transcription by RNA polymerase II. In proliferating cells, it is regulated by 7SK snRNA in the 7SK snRNP complex. In resting cells, P-TEFb is absent, because CycT1 is dephosphorylated, released from CDK9 and rapidly degraded. In this study, we identified the mechanism of this degradation. We mapped the ubiquitination and degradation of free CycT1 to its N-terminal region from positions 1 to 280. This region is ubiquitinated at six lysines, where E3 ligases Siah1 and Siah2 bind and degrade these sequences. Importantly, the inhibition of Siah1/2 rescued the expression of free CycT1 in proliferating as well as resting primary cells. We conclude that Siah1/2 are the E3 ligases that bind and degrade the dissociated CycT1 in resting, terminally differentiated, anergic and/or exhausted cells.

scholarly journals In vivo live imaging of RNA polymerase II transcription factories in primary cells

2013 ◽  
Vol 27 (7) ◽  
pp. 767-777 ◽  
Author(s):  
A. Ghamari ◽  
M. P. C. van de Corput ◽  
S. Thongjuea ◽  
W. A. van Cappellen ◽  
W. van IJcken ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Live Imaging ◽  
Primary Cells ◽  
Transcription Factories ◽  
Rna Polymerase Ii Transcription

scholarly journals An N-Terminal Region of Lassa Virus L Protein Plays a Critical Role in Transcription but Not Replication of the Virus Genome

2009 ◽  
Vol 84 (4) ◽  
pp. 1934-1944 ◽  
Author(s):  
Michaela Lelke ◽  
Linda Brunotte ◽  
Carola Busch ◽  
Stephan Günther
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Scale ◽  
Critical Role ◽  
Terminal Region ◽  
Lassa Virus ◽  
Mrna Synthesis ◽  
L Protein ◽  
Mrna Pool ◽  
Capping Enzymes

ABSTRACT The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor α-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

scholarly journals Incorporation of CENP-A/CID into centromeres during early Drosophila embryogenesis does not require RNA polymerase II–mediated transcription

Chromosoma ◽  
2022 ◽  
Author(s):  
Samadri Ghosh ◽  
Christian F. Lehner
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cultured Cells ◽  
Marginal Effect ◽  
Proliferating Cells ◽  
Centromeric Chromatin ◽  
Embryonic Cleavage ◽  
Histone H3 Variant ◽  
Alpha Amanitin

AbstractIn many species, centromere identity is specified epigenetically by special nucleosomes containing a centromere-specific histone H3 variant, designated as CENP-A in humans and CID in Drosophila melanogaster. After partitioning of centromere-specific nucleosomes onto newly replicated sister centromeres, loading of additional CENP-A/CID into centromeric chromatin is required for centromere maintenance in proliferating cells. Analyses with cultured cells have indicated that transcription of centromeric DNA by RNA polymerase II is required for deposition of new CID into centromere chromatin. However, a dependence of centromeric CID loading on transcription is difficult to reconcile with the notion that the initial embryonic stages appear to proceed in the absence of transcription in Drosophila, as also in many other animal species. To address the role of RNA polymerase II–mediated transcription for CID loading in early Drosophila embryos, we have quantified the effects of alpha-amanitin and triptolide on centromeric CID-EGFP levels. Our analyses demonstrate that microinjection of these two potent inhibitors of RNA polymerase II–mediated transcription has at most a marginal effect on centromeric CID deposition during progression through the early embryonic cleavage cycles. Thus, we conclude that at least during early Drosophila embryogenesis, incorporation of CID into centromeres does not depend on RNA polymerase II–mediated transcription.

scholarly journals Roles for Ctk1 and Spt6 in Regulating the Different Methylation States of Histone H3 Lysine 36

2008 ◽  
Vol 28 (16) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael L. Youdell ◽  
Kelby O. Kizer ◽  
Elena Kisseleva-Romanova ◽  
Stephen M. Fuchs ◽  
Eris Duro ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Histone H3 ◽  
Terminal Region ◽  
Histone Deacetylation ◽  
Terminal Domain ◽  
Level Of Activity ◽  
Cryptic Promoters

ABSTRACT Set2 (KMT3)-dependent methylation (me) of histone H3 at lysine 36 (H3K36) promotes deacetylation of transcribed chromatin and represses cryptic promoters within genes. Although Set2 is the only methyltransferase (KMTase) for H3K36 in yeast, it is not known if Set2 is regulated or whether the different methylation states at H3K36 are functionally distinct. Here we show that the N-terminal 261 residues of Set2 (Set21-261), containing the SET KMTase domain, are sufficient for H3K36me2, histone deacetylation, and repression of cryptic promoters at STE11. Set2-catalyzed H3K36me2 does not require either Ctk1-dependent phosphorylation of RNA polymerase II (RNAPII) or the presence of the phospho-C-terminal domain (CTD) interaction (SRI) domain of Set2. This finding is consistent with a known correlation between H3K36me2 and whether a gene is on or off, but not the level of activity of a gene. By contrast, H3K36me3 requires Spt6, proline 38 on histone H3 (H3P38), the CTD of RNAPII, Ctk1, and the C-terminal SRI domain of Set2. We suggest that the C-terminal region of Set2, in conjunction with the phosphorylated CTD of RNAPII, influences the KMTase activity to promote H3K36me3 during transcription elongation.

Effectiveness of Transcriptional CDK Inhibitors in Targeting Non-Proliferating CD34+CD38- AML Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3482-3482
Author(s):  
Monica Pallis ◽  
Francis Burrows ◽  
Abigail Whittall ◽  
Claire Seedhouse ◽  
Nicholas Boddy ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Rna Polymerase Ii ◽  
Rna Synthesis ◽  
Cdk Inhibitors ◽  
Proliferating Cells ◽  
Median Percentage ◽  
Homogeneous Population ◽  
Quiescent Cells ◽  
Species Production

Abstract Abstract 3482 Quiescent cells conserve energy and are characterised by low RNA synthesis. In contrast, cancer cells are thought to be addicted to high RNA synthesis, particularly synthesis of survival molecules. We hypothesised that quiescent cancer cells, already low in RNA, would be sensitive to apoptosis induced by transcriptional cyclin-dependent kinase (CDK) inhibitors that further deplete RNA. We cultured the CD34+CD38- KG1a cell line continuously in the presence of an mTOR inhibitor, which maintained excellent viability and enriched the cells for quiescent stem cell features including low RNA content, low metabolism, low reactive oxygen species production and decreased size. Sensitivity to mitochondrial pore transition was similar in proliferating and quiescence-enriched cells, indicating that the basal mitochondrial apoptotic machinery is neither impaired nor improved. We treated quiescence-enriched cells for 48 hours with the nucleoside analogues ara-C, 5-azacytidine and clofarabine, the topoisomerase targeting agents daunorubicin, etoposide and irinotecan and three multikinase inhibitors with activity against transcriptional CDKs - flavopiridol, roscovotine and TG02. All of the agents used showed increased kill in the unmanipulated compared to the quiescence-enriched cells, emphasising the chemoresistant nature of quiescent cells. To put a value on the difficulty of eradicating the leukamic clone, we used the parameter 2 X IC50 (which theoretically would kill 100% cells in a totally homogeneous population). In quiescence-enriched cells, the percentage of cells killed at 2 X IC50 was roscovotine - 80%, TG02 – 72%, flavopiridol - 62%, 5-azacydidine - 61%, daunorubicin, clofarabine and irinotecan - 60%, etoposide - 58%, and for araC no IC50 for quiescence-enriched cells was reached at 20 times the IC50 of proliferating cells. The data show conclusively that, in this model, transcriptional CDK inhibitors outperformed the agents conventionally used in AML chemotherapy. We also showed that transcriptional CDK inhibitors induced serine 2 dephosphorylation of RNA polymerase II in equal measure in proliferating and quiescence- enriched cells. We have already shown that TGO2 targets primary CD34+CD38- cells in vitro (ASH 2010, abstract 1823). Here we also show that in four primary AML samples sensitive to roscovotine in vitro, the median percentage kill is greater in the CD34+CD38- subset, (which consists largely of quiescent cells and tends to contain the leukaemic stem cells,) than in the bulk cells (40.5% versus 19.5% kill at 2 micromolar roscovotine). We suggest that transcriptional CDK inhibitors may be a useful class of agent for targetting the occult quiescent CD34+CD38- cells thought to contribute to relapse in AML. Disclosures: Burrows: Tragara Pharmaceuticals: Employment.

scholarly journals H2A.Z Is Required for Global Chromatin Integrity and for Recruitment of RNA Polymerase II under Specific Conditions

2001 ◽  
Vol 21 (18) ◽  
pp. 6270-6279 ◽  
Author(s):  
Maryse Adam ◽  
François Robert ◽  
Marc Larochelle ◽  
Luc Gaudreau
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Growth Conditions ◽  
Terminal Region ◽  
Specific Cell ◽  
Evolutionarily Conserved ◽  
Chromatin Integrity ◽  
Specialized Function ◽  
Regulate Gene

ABSTRACT Evolutionarily conserved variant histone H2A.Z has been recently shown to regulate gene transcription in Saccharomyces cerevisiae. Here we show that loss of H2A.Z in this organism negatively affects the induction of GAL genes. Importantly, fusion of the H2A.Z C-terminal region to S phase H2A without its corresponding C-terminal region can mediate the variant histone's specialized function in GAL1-10 gene induction, and it restores the slow-growth phenotype of cells with a deletion of HTZ1. Furthermore, we show that the C-terminal region of H2A.Z can interact with some components of the transcriptional apparatus. In cells lacking H2A.Z, recruitment of RNA polymerase II and TATA-binding protein to the GAL1-10promoters is significantly diminished under inducing conditions. Unexpectedly, we also find that H2A.Z is required to globally maintain chromatin integrity under GAL gene-inducing conditions. We hypothesize that H2A.Z can positively regulate gene transcription, at least in part, by modulating interactions with RNA polymerase II-associated factors at certain genes under specific cell growth conditions.

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