scholarly journals Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription

2021 ◽  
Author(s):  
Fang Wang ◽  
Farrah El-Saafin ◽  
Tao Ye ◽  
Matthieu Stierle ◽  
Luc Negroni ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Embryonic Stem ◽  
Cellular Systems ◽  
Strong Increase ◽  
Modular Organization ◽  
Saga Complex ◽  
Mouse Development ◽  
Pol Ii ◽  
Primary Mouse

AbstractCo-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3−/− embryos show developmental delay already at embryonic day (E) 7.5, while Usp22−/− embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3−/− mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3−/− mESCs, or Atxn7l3−/− MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3−/− cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation.

scholarly journals Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription

2021 ◽  
Author(s):  
Fang Wang ◽  
Farrah El-Saafin ◽  
Tao Ye ◽  
Matthieu Stierle ◽  
Luc Negroni ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Embryonic Stem ◽  
Cellular Systems ◽  
Strong Increase ◽  
Modular Organization ◽  
Saga Complex ◽  
Mouse Development ◽  
Pol Ii ◽  
Primary Mouse

AbstractCo-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3−/− embryos show developmental delay already at embryonic day (E) 7.5, while Usp22−/− embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3−/− mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3−/− mESCs, or Atxn7l3−/− MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3−/− cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation.

scholarly journals SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells

2021 ◽  
Author(s):  
Veronique Fischer ◽  
Elisabeth Scheer ◽  
Elisabeth Lata ◽  
Bastien Morlet ◽  
Damien Plassard ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Gene Promoters ◽  
Saga Complex ◽  
Self Renewal ◽  
Pol Ii ◽  
Histone Fold ◽  
Specific Subset

Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone fold domain (HFD)-containing proteins forming three histone fold (HF) pairs, to which the double HFD-containing SUPT3H adds an HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H; ii) SUPT3H is not essential for mESC survival, iii) SUPT3H is required for mESC growth and self-renewal, and iv) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.

scholarly journals Mediator and RNA polymerase II clusters associate in transcription-dependent condensates

Science ◽  
2018 ◽  
Vol 361 (6400) ◽  
pp. 412-415 ◽  
Author(s):  
Won-Ki Cho ◽  
Jan-Hendrik Spille ◽  
Micca Hecht ◽  
Choongman Lee ◽  
Charles Li ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Spatial Organization ◽  
Embryonic Stem ◽  
Light Sheet ◽  
Gene Control ◽  
Pol Ii ◽  
Stable Clusters ◽  
Large Clusters

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.

scholarly journals A TBP-independent mechanism for RNA Polymerase II transcription

2021 ◽  
Author(s):  
James Z.J. Kwan ◽  
Thomas F. Nguyen ◽  
Marek A. Budzyński ◽  
Jieying Cui ◽  
Rachel M. Price ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Embryonic Stem ◽  
Promoter Regions ◽  
Pol Ii ◽  
Promoter Dna ◽  
Tata Box Binding Protein

AbstractTranscription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the Pre-Initiation Complex onto promoter DNA. Decades of in vitro and yeast research have shown that the TATA-box binding protein (TBP) is essential to Pol II initiation by triggering the binding of other general transcription factors, and ensuring proper Pol II loading. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells (mESCs) has no global effect on ongoing Pol II transcription. Surprisingly, Pol II transcriptional induction through the Heat Shock Response or cellular differentiation also occurs normally in the absence of TBP. In contrast, acute TBP depletion severely impairs initiation by RNA Polymerase III. Lastly, we show that a metazoan-specific paralog of TBP is expressed in mESCs and that it binds to promoter regions of active Pol II genes even in the absence of TBP. Taken together, our findings reveal an unexplored TBP-independent process in mESCs that points to a diversity in Pol II transcription initiation mechanisms.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

scholarly journals RecQL5 Promotes Genome Stabilization through Two Parallel Mechanisms—Interacting with RNA Polymerase II and Acting as a Helicase

2010 ◽  
Vol 30 (10) ◽  
pp. 2460-2472 ◽  
Author(s):  
M. Nurul Islam ◽  
David Fox ◽  
Rong Guo ◽  
Takemi Enomoto ◽  
Weidong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Transcriptional Regulators ◽  
Helicase Activity ◽  
Parallel Mechanisms ◽  
Recq Helicase ◽  
Pol Ii ◽  
Kix Domain ◽  
Genome Stabilization

ABSTRACT The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

scholarly journals Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II

2007 ◽  
Vol 82 (3) ◽  
pp. 1118-1127 ◽  
Author(s):  
Jinhong Chang ◽  
Xingcao Nie ◽  
Ho Eun Chang ◽  
Ziying Han ◽  
John Taylor
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hepatitis Delta Virus ◽  
Cell System ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Delta Antigen ◽  
Nuclear Extracts ◽  
Virus Rna ◽  
Delta Virus

ABSTRACT Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

scholarly journals RNA Polymerase II-Association Factor 1 (Paf1/PD2) Maintains Self-renewal by Its Interaction with Oct3/4 in Mouse Embryonic Stem Cells

Stem Cells ◽  
2009 ◽  
pp. N/A-N/A ◽  
Author(s):  
Moorthy P. Ponnusamy ◽  
Shonali Deb ◽  
Parama Dey ◽  
Subhankar Chakraborty ◽  
Satyanarayana Rachagani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Self Renewal

Genome-wide regulations of the pre-initiation complex formation and elongating RNA polymerase II by an E3 ubiquitin ligase, San1.

2021 ◽  
Author(s):  
Priyanka Barman ◽  
Rwik Sen ◽  
Amala Kaja ◽  
Jannatul Ferdoush ◽  
Shalini Guha ◽  
...  
Keyword(s):  
Quality Control ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
Nuclear Protein ◽  
Protein Quality ◽  
Initiation Complex ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Genome Wide

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in genome-wide association of TBP [that nucleates pre-initiation complex (PIC) formation for transcription initiation] and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences, and hence PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1, but not the incorporation of centromeric histone, Cse4, into the active genes in Δsan1 . Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

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