Nucleosome structure of the yeast CHA1 promoter: analysis of activation-dependent chromatin remodeling of an RNA-polymerase-II-transcribed gene in TBP and RNA pol II mutants defective invivo in response to acidic activators

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

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Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

Reproduction ◽

10.1530/rep-09-0578 ◽

2010 ◽

Vol 140 (2) ◽

pp. 273-285 ◽

Cited By ~ 25

Author(s):

Irina Lagutina ◽

Helena Fulka ◽

Tiziana A L Brevini ◽

Stefania Antonini ◽

Dario Brunetti ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Transfer ◽

Blastocyst Development ◽

Cell Stage ◽

Rna Pol Ii ◽

Pol Ii ◽

Pol I ◽

Embryonic Genome ◽

Genome Activity

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine–pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16–25-cell stage for pig–bovine iSCNT embryos and at the four-cell stage for bovine–pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus–cytoplasm compatibility between these animal species.

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Piwi suppresses transcription of Brahma-dependent transposons via Maelstrom in ovarian somatic cells

Science Advances ◽

10.1126/sciadv.aaz7420 ◽

2020 ◽

Vol 6 (50) ◽

pp. eaaz7420

Author(s):

Ryo Onishi ◽

Kaoru Sato ◽

Kensaku Murano ◽

Lumi Negishi ◽

Haruhiko Siomi ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Remodeling ◽

Adenosine Triphosphatase ◽

Somatic Cells ◽

Pol Ii ◽

Heterochromatin Formation ◽

Rna Transcripts ◽

The Core ◽

Chromatin Remodeling Complex

Drosophila Piwi associates with PIWI-interacting RNAs (piRNAs) and represses transposons transcriptionally through heterochromatinization; however, this process is poorly understood. Here, we identify Brahma (Brm), the core adenosine triphosphatase of the SWI/SNF chromatin remodeling complex, as a new Piwi interactor, and show Brm involvement in activating transcription of Piwi-targeted transposons before silencing. Bioinformatic analyses indicated that Piwi, once bound to target RNAs, reduced the occupancies of SWI/SNF and RNA polymerase II (Pol II) on target loci, abrogating transcription. Artificial piRNA-driven targeting of Piwi to RNA transcripts enhanced repression of Brm-dependent reporters compared with Brm-independent reporters. This was dependent on Piwi cofactors, Gtsf1/Asterix (Gtsf1), Panoramix/Silencio (Panx), and Maelstrom (Mael), but not Eggless/dSetdb (Egg)–mediated H3K9me3 deposition. The λN-box B–mediated tethering of Mael to reporters repressed Brm-dependent genes in the absence of Piwi, Panx, and Gtsf1. We propose that Piwi, via Mael, can rapidly suppress transcription of Brm-dependent genes to facilitate heterochromatin formation.

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A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

Journal of Experimental Medicine ◽

10.1084/jem.20112145 ◽

2012 ◽

Vol 209 (11) ◽

pp. 2099-2111 ◽

Cited By ~ 47

Author(s):

Katharina L. Willmann ◽

Sara Milosevic ◽

Siim Pauklin ◽

Kerstin-Maike Schmitz ◽

Gopinath Rangam ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Protein Complexes ◽

Chromatin Modification ◽

Cytosine Deamination ◽

Rna Pol Ii ◽

Pol Ii ◽

Antibody Diversification ◽

Dna Instability ◽

Paf Complex

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

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HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription

Biochemical Journal ◽

10.1042/bj20011191 ◽

2002 ◽

Vol 364 (3) ◽

pp. 649-657 ◽

Cited By ~ 41

Author(s):

Sergei NEKHAI ◽

Meisheng ZHOU ◽

Anne FERNANDEZ ◽

William S. LANE ◽

Ned J.C. LAMB ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Extract ◽

Viral Gene ◽

Transcriptional Elongation ◽

Rna Pol Ii ◽

Hela Nuclear Extract ◽

Pol Ii ◽

Terminal Domain ◽

Hiv 1

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246–256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.

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Negotiating the nucleosome: factors that allow RNA polymerase II to elongate through chromatinThis paper is one of a selection of papers published in this Special Issue, entitled 28th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o07-054 ◽

2007 ◽

Vol 85 (4) ◽

pp. 426-434 ◽

Cited By ~ 9

Author(s):

Jennifer A. Armstrong

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Remodeling ◽

Peer Review Process ◽

Histone Variants ◽

Transcriptional Elongation ◽

Histone Chaperones ◽

Nucleosome Assembly ◽

Pol Ii ◽

Assembly Factors

Initiation by RNA polymerase II (Pol II) involves a host of enzymes, and the process of elongation appears similarly complex. Transcriptional elongation through chromatin requires the coordinated efforts of Pol II and its associated transcription factors: C-terminal domain kinases, elongation complexes, chromatin-modifying enzymes, chromatin remodeling factors, histone chaperones (nucleosome assembly factors), and histone variants. This review examines the following: (i) the consequences of the encounter between elongating Pol II and a nucleosome, and (ii) chromatin remodeling factors and nucleosome assembly factors that have recently been identified as important for the elongation stage of transcription.

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Maize RAMOSA3 accumulates in nuclear condensates enriched in RNA POLYMERASE II isoforms during the establishment of axillary meristem determinacy.

10.1101/2021.04.06.438639 ◽

2021 ◽

Author(s):

Edgar Demesa-Arevalo ◽

Maria Jazmin Abraham-Juarez ◽

Xiaosa Xu ◽

Madelaine Bartlett ◽

David Jackson

Keyword(s):

Rna Polymerase Ii ◽

Chromatin Remodeling ◽

Transcriptional Response ◽

Mrna Processing ◽

Nuclear Speckles ◽

Rna Pol Ii ◽

Pol Ii ◽

Imaging Processing ◽

Whole Mount ◽

Double Immunolabeling

Maize meristem determinacy is regulated by Trehalose-6-Phosphate Phosphatase (TPP) metabolic enzymes RAMOSA3 and TPP4. However, this function is independent of their enzymatic activity, suggesting they have an unpredicted, or moonlighting function. Using whole-mount double immunolabeling and imaging processing, we investigated the co-localization of RA3 nuclear speckles with markers for transcription, chromatin state and splicing. We find evidence for RA3 co-localization with RNA POL II, a transcription marker, and not with markers for promoter chromatin remodeling or mRNA processing, suggesting a function of nuclear RA3 in mediating a transcriptional response during meristem determinacy.

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The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

Molecular Biology of the Cell ◽

10.1091/mbc.10.8.2655 ◽

1999 ◽

Vol 10 (8) ◽

pp. 2655-2668 ◽

Cited By ~ 84

Author(s):

Adayabalam S. Balajee ◽

Amrita Machwe ◽

Alfred May ◽

Matthew D. Gray ◽

Junko Oshima ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cell Lines ◽

Werner Syndrome ◽

Molecular Defect ◽

Rna Pol Ii ◽

Vitro Assay ◽

Pol Ii ◽

Cell Extracts

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

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Emerging Views on the CTD Code

Genetics Research International ◽

10.1155/2012/347214 ◽

2012 ◽

Vol 2012 ◽

pp. 1-19 ◽

Cited By ~ 12

Author(s):

David W. Zhang ◽

Juan B. Rodríguez-Molina ◽

Joshua R. Tietjen ◽

Corey M. Nemec ◽

Aseem Z. Ansari

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Regulation ◽

Chromatin Remodeling ◽

Posttranslational Modifications ◽

Rna Processing ◽

Transcription Initiation ◽

Protein Complexes ◽

Pol Ii

The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the “CTD code” hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a “code.” Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights into transcription regulation that will necessarily emerge upon addressing those challenges.

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Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

Eukaryotic Cell ◽

10.1128/ec.00066-14 ◽

2014 ◽

Vol 13 (7) ◽

pp. 855-865 ◽

Cited By ~ 6

Author(s):

Antônio Augusto Rocha ◽

Nilmar Silvio Moretti ◽

Sergio Schenkman

Keyword(s):

Heat Shock ◽

Trypanosoma Cruzi ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Processing ◽

Transcription Control ◽

Rna Pol Ii ◽

Pol Ii ◽

Content Type ◽

Polycistronic Transcription

ABSTRACT The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans -splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing.

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