scholarly journals Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Calvin Jon A Leonen ◽  
Miho Shimada ◽  
Caroline E Weller ◽  
Tomoyoshi Nakadai ◽  
Peter L Hsu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Strand Break ◽  
Histone H4 ◽  
Post Translational Modification ◽  
Functional Studies ◽  
Chemically Modified ◽  
Active Transcription ◽  
Active Genes

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.

scholarly journals Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

2021 ◽  
Author(s):  
Calvin Jon A Leonen ◽  
Miho Shimada ◽  
Caroline E Weller ◽  
Tomoyoshi Nakadai ◽  
Peter L Hsu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Histone H4 ◽  
Post Translational Modification ◽  
H3k4 Methylation ◽  
Chemically Modified ◽  
Catalytic Module ◽  
Active Genes

Post-translational modification of histone H4 by the small ubiquitin-like modifier (SUMO) protein was associated with gene repression. However, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using H4 chemically modified at Lys12 by SUMO-3 (H4K12su) that was incorporated into mononucleosomes and chromatinized plasmids. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed H4K12su inhibits RNA polymerase II-mediated transcription and H4 tail acetylation. Thus, we uncovered negative crosstalk with acetylation/methylation and the direct inhibition of RNAPII-mediated transcription by H4K12su.

scholarly journals The Recruitment of theSaccharomyces cerevisiaePaf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

2013 ◽  
Vol 33 (16) ◽  
pp. 3259-3273 ◽  
Author(s):  
Manasi K. Mayekar ◽  
Richard G. Gardner ◽  
Karen M. Arndt
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Physical Interaction ◽  
Repeat Domain ◽  
Necessary And Sufficient ◽  
Active Genes ◽  
Interacting Domain

Transcription elongation factors associate with RNA polymerase II and aid its translocation through chromatin. One such factor is the conserved Paf1 complex (Paf1C), which regulates gene expression through several mechanisms, including the stimulation of cotranscriptional histone modifications. Previous studies revealed a prominent role for the Rtf1 subunit in tethering Paf1C to the RNA polymerase II elongation machinery. Here, we investigated the mechanism by which Rtf1 couples Paf1C to active chromatin. We show that a highly conserved domain of Rtf1 is necessary and sufficient for mediating a physical interaction between Rtf1 and the essential transcription elongation factor Spt5. Mutations that alter this Rtf1 domain or delete the Spt5 C-terminal repeat domain (CTR) disrupt the interaction between Rtf1 and Spt5 and release Paf1C from chromatin. When expressed in cells as the only source of Rtf1, the Spt5-interacting domain of Rtf1 can associate independently with active genes in a pattern similar to that of full-length Rtf1 and in a manner dependent on the Spt5 CTR.In vitroexperiments indicate that the interaction between the Rtf1 Spt5-interacting domain and the Spt5 CTR is direct. Collectively, our results provide molecular insight into a key attachment point between Paf1C and the RNA polymerase II elongation machinery.

scholarly journals TFIIH-Associated Cdk7 Kinase Functions in Phosphorylation of C-Terminal Domain Ser7 Residues, Promoter-Proximal Pausing, and Termination by RNA Polymerase II

2009 ◽  
Vol 29 (20) ◽  
pp. 5455-5464 ◽  
Author(s):  
Kira Glover-Cutter ◽  
Stéphane Larochelle ◽  
Benjamin Erickson ◽  
Chao Zhang ◽  
Kevan Shokat ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Histone H4 ◽  
Pol Ii ◽  
Terminal Domain ◽  
Histone H4 Acetylation ◽  
Flanking Regions

ABSTRACT The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7 as/as mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5′ ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase “leaking” into the gene. Consistent with a 5′ pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36—two marks of elongation—within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3′ ends, it was detected within genes and 3′-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.

scholarly journals dCHD3, a Novel ATP-Dependent Chromatin Remodeler Associated with Sites of Active Transcription

2008 ◽  
Vol 28 (8) ◽  
pp. 2745-2757 ◽  
Author(s):  
Magdalena Murawska ◽  
Natascha Kunert ◽  
Joke van Vugt ◽  
Gernot Längst ◽  
Elisabeth Kremmer ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Protein ◽  
Polytene Chromosomes ◽  
Dependent Manner ◽  
Chromatin Remodelers ◽  
Active Transcription ◽  
Nucleosome Binding

ABSTRACT ATP-dependent chromatin remodelers of the CHD family play important roles during differentiation and development. Three CHD proteins, dMi-2, dChd1, and Kismet, have been described for Drosophila melanogaster. Here, we study dCHD3, a novel member of the CHD family. dCHD3 is related in sequence to dMi-2 but lacks several domains implicated in dMi-2 function. We demonstrate that dCHD3 is a nuclear protein and that expression is tightly regulated during fly development. Recombinant dCHD3 remodels mono- and polynucleosomes in an ATP-dependent manner in vitro. Its chromodomains are critical for nucleosome binding and remodeling. Unlike dMi-2, dCHD3 exists as a monomer. Nevertheless, both proteins colocalize with RNA polymerase II to actively transcribed regions on polytene chromosomes, suggesting that both remodelers participate in the process of transcription.

scholarly journals Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

2002 ◽  
Vol 22 (22) ◽  
pp. 8088-8099 ◽  
Author(s):  
Xianming Mo ◽  
William S. Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Genome Stability ◽  
Strand Break ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Rnap Ii

ABSTRACT Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase II (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP II or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominant-negative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined subnuclear structures may also be advantageous in maintenance of genome stability.

scholarly journals Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Xiaowen Zhang ◽  
Huai-Chin Chiang ◽  
Yao Wang ◽  
Chi Zhang ◽  
Sabrina Smith ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Lineage ◽  
Mammary Epithelium ◽  
Strand Break ◽  
Pol Ii ◽  
Genetic Ablation ◽  
Dna Replication Stress ◽  
Pol Ii Pausing

Abstract Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5′ end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development.

scholarly journals Terminal Erythroid Maturation Is Associated with Dynamic Changes in the Abundance of Histone Marks Associated with Active Transcription Elongation and RNA Polymerase II Pausing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 154-154 ◽  
Author(s):  
Zachary C. Murphy ◽  
Tyler A Couch ◽  
Jacquelyn Lillis ◽  
Michael Getman ◽  
Kimberly Lezon-Geyda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Posttranslational Modifications ◽  
Histone H3 ◽  
Histone H4 ◽  
Pol Ii ◽  
Active Transcription ◽  
Pol Ii Pausing ◽  
Erythroid Maturation

Maturation of erythroid progenitors is associated with significant changes in gene expression in the context of a nucleus that dramatically decreases in size in preparation for enucleation, and is regulated by the coordinated action of transcriptional regulators and epigenetic modifiers. In eukaryotes, all DNA is bound by histone proteins into chromatin. Posttranslational modifications of the N-terminal "tails" of these proteins are key regulators of chromatin structure and gene expression. We hypothesized that terminal erythroid maturation is associated with changes in the abundance of specific histone posttranslational modifications. To address this hypothesis, we utilized mass spectrometry to perform an unbiased assessment of the abundance histone post translational modifications in maturing erythroblasts. We cultured peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) down the erythroid lineage using a semi-synchronous culture system (as outlined in Gautier et al. Cell Reports 2016), and sent cells for mass spectrometry on day 7 of erythroid maturation, when the cells are predominately basophilic erythroblasts, and on day 12 of erythroid maturation, when they are predominately poly- and ortho- chromatic erythroblasts. The maturation stage of the cells was confirmed by both cytospins and imaging flow cytometric analyses. Two independent replicates were performed and key results confirmed by western blotting. Terminal erythroid maturation was associated with a dramatic decline in the abundance of multiple histone marks associated with active transcription elongation, including Histone H3 lysine 36 di- and tri-methylation (H3K36me2, H3K36me3), and Histone H3 Lysine 79 di-methylation (H3K79me2). Surprisingly, this was not accompanied by an increase in the abundance of repressive heterochromatin marks (H3K27me3, H3K9me3, and H4K20me3) or a global decline in histone acetylation. Histone H4 lysine 16 acetylation (H4K16Ac), associated with RNA polymerase II pause release (Kapoor-Vazirani MCB 2011) significantly declined, but multiple acetylation marks including H3K36Ac and H3K23Ac increased in abundance. As expected, the abundance histone H4 lysine 20 mono-methylation (H4K20me1), which is implicated both in erythroblast chromatin condensation (Malik Cell Reports 2017) and the regulation of RNA Polymerase II pausing (Kapoor-Vazirani MCB 2011) also significantly increased. Consistent with these data, integration of RNA-seq and ChIP-seq data identified 3,058 genes whose expression decreased from basophilic erythroblast to orthochromatic erythroblasts, which lost enrichment for H3K36me3 (mark of active elongation) without accumulating H3K27me3 (heterochromatin mark). Based on these data, we hypothesized that RNA polymerase II pausing is a critical regulator of gene expression in maturing erythroblasts. RNA Polymerase II (Pol II) pausing is a highly regulated mechanism of transcriptional regulation, whereby transcription is initiated, but pauses 30-60bp downstream of the transcription start site. For paused Pol II to be released into active elongation, pTEFb must hyper-phosphorylate Serine 2 of the Pol II c-terminal domain (CTD). Importantly, pTEFb can be directed to specific loci through interaction with transcription factors, including GATA1 (Elagib Blood 2008; Bottardi NAR 2011). Hexim1 is a key regulator of Pol II pausing that sequesters pTEFb and inhibits its action. Consistent with a central role for Pol II pausing dynamics in the regulation of terminal erythroid maturation, Hexim1 is highly expressed in erythroid cells compared to most other cell types and its expression increases during terminal erythroid maturation. Conversely, the expression of CCNT1 and CKD9, the components of pTEFb, decline during terminal maturation, and the level of elongation competent (Ser2 and Ser2/Ser5 CTD phosphorylated) Pol II also decreases dramatically. To gain insights into the function of Pol II pausing in maturing erythroblasts, we induced Hexim1 expression in HUDEP2 cells (Kurita PLoS One 2013) using hexamethane bisacetamide (HMBA). HMBA treatment increased Hexim1 levels a dose dependent manner and was associated with gene expression and phenotypic changes suggestive of accelerated erythroid maturation. Together, these data suggest that RNA Pol II pausing dynamics are an important regulator of terminal erythroid maturation. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Nuclear Matrix Protein Interacts with the Phosphorylated C-Terminal Domain of RNA Polymerase II

1998 ◽  
Vol 18 (4) ◽  
pp. 2406-2415 ◽  
Author(s):  
Meera Patturajan ◽  
Xiangyun Wei ◽  
Ronald Berezney ◽  
Jeffry L. Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Matrix ◽  
Matrix Protein ◽  
Consensus Sequence ◽  
Nuclear Speckles ◽  
Repeat Domain ◽  
Active Transcription

ABSTRACT Yeast two-hybrid screening has led to the identification of a family of proteins that interact with the repetitive C-terminal repeat domain (CTD) of RNA polymerase II (A. Yuryev et al., Proc. Natl. Acad. Sci. USA 93:6975–6980, 1996). In addition to serine/arginine-rich SR motifs, the SCAFs (SR-like CTD-associated factors) contain discrete CTD-interacting domains. In this paper, we show that the CTD-interacting domain of SCAF8 specifically binds CTD molecules phosphorylated on serines 2 and 5 of the consensus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. In addition, we demonstrate that SCAF8 associates with hyperphosphorylated but not with hypophosphorylated RNA polymerase II in vitro and in vivo. This result suggests that SCAF8 is not present in preinitiation complexes but rather associates with elongating RNA polymerase II. Immunolocalization studies show that SCAF8 is present in granular nuclear foci which correspond to sites of active transcription. We also provide evidence that SCAF8 foci are associated with the nuclear matrix. A fraction of these sites overlap with a subset of larger nuclear speckles containing phosphorylated polymerase II. Taken together, our results indicate a possible role for SCAF8 in linking transcription and pre-mRNA processing.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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