scholarly journals Numerous post-translational modifications of RNA polymerase II subunit Rpb4 link transcription to post-transcriptional mechanisms

2019 ◽  
Author(s):  
Stephen Richard ◽  
Lital Gross ◽  
Jonathan Fischer ◽  
Keren Bendalak ◽  
Tamar Ziv ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
The Other ◽  
Mrna Synthesis ◽  
Mutant Type ◽  
Post Translational Modifications ◽  
Pol Ii ◽  
One Stage

AbstractRpb4/7 binds RNA Polymerase II (Pol II) transcripts co-transcriptionally and accompanies them throughout their lives. By virtue of its capacity to interact with key regulators (e.g., Pol II, eIF3, Pat1) both temporarily and spatially, Rpb4/7 regulates the major stages of the mRNA lifecycle. Here we show that Rpb4/7 can undergo over 100 combinations of post-translational modifications (PTMs). Remarkably, the Rpb4/7 PTMs repertoire changes as the mRNA/Rpb4/7 complex progresses from one stage to the next. A mutagenesis approach in residues that undergo PTMs suggests that temporal Rpb4 PTMs regulate its interactions with key regulators of gene expression that control transcriptional and post-transcriptional stages. Moreover, one mutant type specifically affects mRNA synthesis despite its normal association with Pol II, whereas the other affects both mRNA synthesis and decay; both types disrupt the balance between mRNA synthesis and decay (‘mRNA buffering’) and the cell’s capacity to respond to the environment. Taken together, we propose that temporal Rpb4/7 PTMs are involved in cross talks among the various stages of the mRNA lifecycle.

scholarly journals Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7

2021 ◽  
Author(s):  
Adam W Whisnant ◽  
Oliver Mathias Dyck Dionisi ◽  
Arnhild Grothey ◽  
Julia M Rappold ◽  
Ana Luiza Marante ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Early Gene ◽  
Immediate Early ◽  
Pol Ii ◽  
Simplex Virus ◽  
Hsv 1

Transcriptional activity of RNA polymerase II (Pol II) is orchestrated by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes Simplex Virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) of the Pol II CTD by targeting CDK9. The functional implications of this are poorly understood. Here, we report that HSV-1 also induces a global loss of serine 7 phosphorylation (pS7). This effect was dependent on the expression of the two viral immediate-early proteins, ICP22 and ICP27. While lytic HSV-1 infection results in efficient Pol II degradation late in infection, we show that pS2/S7 loss precedes the drop in Pol II level. Interestingly, mutation of the RPB1 polyubiquitination site mutation K1268, which prevents proteasomal RPB1 degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained much higher overall RPB1 protein levels even at late times of infection, indicating that this pathway mediates bulk Pol II protein loss late in infection but is not involved in early CTD dysregulation. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for production of viral proteins, with Ser2 facilitating viral immediate-early gene expression and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments by immunofluorescence. These data expand the known means that HSV-1 employs to create pro-viral transcriptional environments at the expense of host responses.

scholarly journals The Chd1 chromatin remodeler shifts hexasomes unidirectionally

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Robert F Levendosky ◽  
Anton Sabantsev ◽  
Sebastian Deindl ◽  
Gregory D Bowman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Histone Variants ◽  
Chromatin Remodeler ◽  
Post Translational Modifications ◽  
Pol Ii ◽  
Specific Orientation ◽  
Chromatin Reorganization

Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.

scholarly journals High-resolution and high-accuracy topographic and transcriptional maps of the nucleosome barrier

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Zhijie Chen ◽  
Ronen Gabizon ◽  
Aidan I Brown ◽  
Antony Lee ◽  
Aixin Song ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Resolution ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Residence Time ◽  
Base Pair ◽  
High Accuracy ◽  
Control Of Gene Expression ◽  
Pol Ii ◽  
Crossing Time

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.

scholarly journals The yeast exoribonuclease Xrn1 and associated factors modulate RNA polymerase II processivity in 5' and 3' gene regions

2020 ◽  
Vol 295 (33) ◽  
pp. 11435-11454 ◽  
Author(s):  
Jonathan Fischer ◽  
Yun S. Song ◽  
Nir Yosef ◽  
Julia di Iulio ◽  
L. Stirling Churchman ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mrna Decay ◽  
Indirect Evidence ◽  
Yeast Cells ◽  
Mrna Levels ◽  
Mrna Synthesis ◽  
Pol Ii ◽  
Polyadenylation Sites

mRNA levels are determined by the balance between mRNA synthesis and decay. Protein factors that mediate both processes, including the 5'-3' exonuclease Xrn1, are responsible for a cross-talk between the two processes that buffers steady-state mRNA levels. However, the roles of these proteins in transcription remain elusive and controversial. Applying native elongating transcript sequencing (NET-seq) to yeast cells, we show that Xrn1 functions mainly as a transcriptional activator and that its disruption manifests as a reduction of RNA polymerase II (Pol II) occupancy downstream of transcription start sites. By combining our sequencing data and mathematical modeling of transcription, we found that Xrn1 modulates transcription initiation and elongation of its target genes. Furthermore, Pol II occupancy markedly increased near cleavage and polyadenylation sites in xrn1Δ cells, whereas its activity decreased, a characteristic feature of backtracked Pol II. We also provide indirect evidence that Xrn1 is involved in transcription termination downstream of polyadenylation sites. We noted that two additional decay factors, Dhh1 and Lsm1, seem to function similarly to Xrn1 in transcription, perhaps as a complex, and that the decay factors Ccr4 and Rpb4 also perturb transcription in other ways. Interestingly, the decay factors could differentiate between SAGA- and TFIID-dominated promoters. These two classes of genes responded differently to XRN1 deletion in mRNA synthesis and were differentially regulated by mRNA decay pathways, raising the possibility that one distinction between these two gene classes lies in the mechanisms that balance mRNA synthesis with mRNA decay.

scholarly journals Separation of the Saccharomyces cerevisiae Paf1 Complex from RNA Polymerase II Results in Changes in Its Subnuclear Localization

2005 ◽  
Vol 4 (1) ◽  
pp. 209-220 ◽  
Author(s):  
Stephanie E. Porter ◽  
Kristi L. Penheiter ◽  
Judith A. Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
No Reduction ◽  
The Other ◽  
Rrna Processing ◽  
Wild Type ◽  
Pol Ii ◽  
Subnuclear Localization ◽  
Yeast Strains ◽  
Paf1 Complex

ABSTRACT The yeast Paf1 complex (Paf1C), composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, associates with RNA polymerase II (Pol II) at promoters and in the actively transcribed portions of mRNA genes. Loss of Paf1 results in severe phenotypes and significantly reduced levels of the other Paf1C components. In contrast, loss of Rtf1 causes relatively subtle phenotypic changes and no reduction in the other Paf1C factors but disrupts the association of these factors with Pol II and chromatin. To elucidate the fate of the Paf1C when dissociated from Pol II, we examined the localization of the Paf1C components in paf1 and rtf1 mutant yeast strains. We found that although the Paf1C factors remain nuclear in paf1 and rtf1 strains, loss of Paf1 or Rtf1 results in a change in the subnuclear distribution of the remaining factors. In wild-type cells, Paf1C components are present in the nucleoplasm but not the nucleolus. In contrast, in both paf1 and rtf1 strains, the remaining factors are found in the nucleolus as well as the nucleoplasm. Loss of Paf1 affects nucleolar function; we observed that expression of MAK21 and RRP12, important for rRNA processing, is reduced concomitant with an increase in rRNA precursors in a paf1 strain. However, these changes are not the result of relocalization of the Paf1C because loss of Rtf1 does not cause similar changes in rRNA processing. Instead, we speculate that the change in localization may reflect a link between the Paf1C and newly synthesized mRNAs as they exit the nucleus.

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 Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Nicolas Descostes ◽  
Martin Heidemann ◽  
Lionel Spinelli ◽  
Roland Schüller ◽  
Muhammad Ahmad Maqbool ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Consensus Sequence ◽  
Post Translational Modifications ◽  
Pol Ii ◽  
Carboxy Terminal Domain ◽  
Mrna Maturation ◽  
Carboxy Terminal ◽  
Rna Polymerase Ii Ctd

In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability.

scholarly journals HSV-1 ICP22 Is a Selective Viral Repressor of Cellular RNA Polymerase II-Mediated Transcription Elongation

Vaccines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1054
Author(s):  
Nur Firdaus Isa ◽  
Olivier Bensaude ◽  
Nadiah C. Aziz ◽  
Shona Murphy
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Viral Gene ◽  
Elongation Factors ◽  
Pol Ii ◽  
Early Protein ◽  
Hsv 1

The Herpes Simplex Virus (HSV-1) immediate-early protein ICP22 interacts with cellular proteins to inhibit host cell gene expression and promote viral gene expression. ICP22 inhibits phosphorylation of Ser2 of the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) and productive elongation of pol II. Here we show that ICP22 affects elongation of pol II through both the early-elongation checkpoint and the poly(A)-associated elongation checkpoint of a protein-coding gene model. Coimmunoprecipitation assays using tagged ICP22 expressed in human cells and pulldown assays with recombinant ICP22 in vitro coupled with mass spectrometry identify transcription elongation factors, including P-TEFb, additional CTD kinases and the FACT complex as interacting cellular factors. Using a photoreactive amino acid incorporated into ICP22, we found that L191, Y230 and C225 crosslink to both subunits of the FACT complex in cells. Our findings indicate that ICP22 interacts with critical elongation regulators to inhibit transcription elongation of cellular genes, which may be vital for HSV-1 pathogenesis. We also show that the HSV viral activator, VP16, has a region of structural similarity to the ICP22 region that interacts with elongation factors, suggesting a model where VP16 competes with ICP22 to deliver elongation factors to viral genes.

scholarly journals HSV-1 ICP22 is a selective viral repressor of cellular RNA polymerase II-mediated transcription elongation

2021 ◽  
Author(s):  
Nur Firdaus Isa ◽  
Olivier Bensaude ◽  
Nadiah C. Aziz ◽  
Shona Murphy
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Viral Gene ◽  
Elongation Factors ◽  
Pol Ii ◽  
Early Protein ◽  
Hsv 1

The Herpes Simplex Virus (HSV-1) immediate early protein ICP22 interacts with cellular proteins to inhibit host cell gene expression and promote viral gene expression. ICP22 inhibits phosphorylation of Ser2 of the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) and productive elongation of pol II. Here we show that ICP22 affects elongation of pol II through both the early-elongation checkpoint and the poly(A)-associated elongation checkpoint on a protein-coding gene model. Coimmunoprecipitation assays using tagged ICP22 expressed in human cells and pulldown assays with recombinant ICP22 in vitro coupled with mass spectrometry identify transcription elongation factors, including P-TEFb, additional CTD kinases and the FACT complex as interacting cellular factors. Using a photoreactive amino acid incorporated into ICP22, we found that L191, Y230 and C225 crosslink to both subunits of the FACT complex in cells.  Our findings indicate that ICP22 physically interacts with critical elongation regulators to inhibit transcription elongation of cellular genes, which may be vital for HSV-1 pathogenesis. We also show that the HSV viral activator, VP16 has a region of structural similarity to the ICP22 region that interacts with elongation factors, suggesting a model where VP16 competes with ICP22 to deliver elongation factors to viral genes.

scholarly journals Disruption of cardiac Med1 inhibits RNA polymerase II promoter occupancy and promotes chromatin remodeling

2019 ◽  
Vol 316 (2) ◽  
pp. H314-H325 ◽  
Author(s):  
Duane D. Hall ◽  
Kathryn M. Spitler ◽  
Chad E. Grueter
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Chromatin Accessibility ◽  
Gene Promoters ◽  
Specific Expression ◽  
Pol Ii ◽  
Transcriptional Start Sites

The Mediator coactivator complex directs gene-specific expression by binding distal enhancer-bound transcription factors through its Med1 subunit while bridging to RNA polymerase II (Pol II) at gene promoters. In addition, Mediator scaffolds epigenetic modifying enzymes that determine local DNA accessibility. Previously, we found that deletion of Med1 in cardiomyocytes deregulates more than 5,000 genes and promotes acute heart failure. Therefore, we hypothesized that Med1 deficiency disrupts enhancer-promoter coupling. Using chromatin immunoprecipitation-coupled deep sequencing (ChIP-seq; n = 3/ChIP assay), we found that the Pol II pausing index is increased in Med1 knockout versus floxed control mouse hearts primarily due to a decrease in Pol II occupancy at the majority of transcriptional start sites without a corresponding increase in elongating species. Parallel ChIP-seq assays reveal that Med1-dependent gene expression correlates strongly with histone H3 K27 acetylation, which is indicative of open and active chromatin at transcriptional start sites, whereas H3 K27 trimethylated levels, representing condensed and repressed DNA, are broadly increased and inversely correlate with absolute expression levels. Furthermore, Med1 deletion leads to dynamic changes in acetyl-K27 associated superenhancer regions and their enriched transcription factor-binding motifs that are consistent with altered gene expression. Our findings suggest that Med1 is important in establishing enhancer-promoter coupling in the heart and supports the proposed role of Mediator in establishing preinitiation complex formation. We also found that Med1 determines chromatin accessibility within genes and enhancer regions and propose that the composition of transcription factors associated with superenhancer changes to direct gene-specific expression. NEW & NOTEWORTHY Based on our previous findings that transcriptional homeostasis and cardiac function are disturbed by cardiomyocyte deletion of the Mediator coactivator Med1 subunit, we investigated potential underlying changes in RNA polymerase II localization and global chromatin accessibility. Using chromatin immunoprecipitation sequencing, we found that disrupted transcription arises from a deficit in RNA polymerase II recruitment to gene promoters. Furthermore, active versus repressive chromatin marks are redistributed within gene loci and at enhancer regions correlated with gene expression changes.

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