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 High-resolution and High-accuracy Topographic and Transcriptional Maps of the Nucleosome Barrier

2019 ◽  
Author(s):  
Zhijie Chen ◽  
Ronen Gabizon ◽  
Aidan I. Brown ◽  
Antony Lee ◽  
Aixin Song ◽  
...  
Keyword(s):  
High Resolution ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
High Accuracy ◽  
List Type ◽  
Control Of Gene Expression ◽  
Pol Ii ◽  
Time Dynamics ◽  
Crossing Time ◽  
Transcriptional Landscape

AbstractNucleosomes 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.HighlightsA single-molecule unzipping assay mimics DNA unwinding by Pol II and maps the topography of human canonical, H2A.Z and uH2B nucleosome barriers at high resolutionReal-time dynamics and full molecular trajectories of Pol II crossing the nucleosomal barrier reveal the transcriptional landscape of the barrier at high accuracyH2A.Z enhances the width and uH2B the height of the barrierA unified mechanical model links position-dependent dwell times of Pol II on the nucleosome with energetics of the barrier

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 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 Role of polycomb repressive complex 2 in regulation of human transcription factor gene expression

2021 ◽  
Author(s):  
Jay Brown
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Binding Sites ◽  
Regulatory Control ◽  
Polycomb Repressive Complex ◽  
Control Of Gene Expression ◽  
Polycomb Repressive Complex 2

Control of gene expression is now recognized as a central issue in the field of molecular biology. We now know the sequences of many genomes including that of the human genome, and we know the nature of many pathways involved in control of gene expression. It remains difficult, however, to look at the DNA sequences surrounding a particular gene and tell which methods of regulatory control are in use. I have been pursuing the idea that progress might be made by comparing the regulatory regions of paired gene populations in which one population is strongly expressed and the other weakly. Here I report the results obtained with human genes encoding transcription factors (TF). In this population, broadly expressed genes are strongly expressed while tissue targeted TF expression is suppressed in most tissues. The results demonstrated that the promoter region of broadly expressed TF genes is enriched in binding sites for POLR2A, a component of RNA polymerase II while promoters of tissue targeted genes are enriched in EZH2, a subunit of polycomb repressive complex 2 (PRC2). It was rare to observe promoters with binding sites for both POLR2A and EZH2. The findings are interpreted to indicate that strong expression of broadly expressed TF genes is due to the presence of RNA polymerase II at the promoter while weak expression of tissue targeted promoters results from the presence of PRC2. Finally, transcription factor families were compared in the proportion of broadly expressed and tissue targeted genes they contain. The results demonstrated that most families possess both broadly expressed and tissue targeted members. For instance, this was the case with 16 of 20 TF families examined. The results are interpreted to indicate that while individual TFs such as EZH2 may be specific for broadly expressed or tissue targeted genes, this is not a property of most TF families.

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 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.

scholarly journals PTEN modulates gene transcription by redistributing genome-wide RNA polymerase II occupancy

2019 ◽  
Vol 28 (17) ◽  
pp. 2826-2834 ◽  
Author(s):  
Ata Abbas ◽  
Roshan Padmanabhan ◽  
Todd Romigh ◽  
Charis Eng
Keyword(s):  
Gene Regulation ◽  
Transcriptional Regulation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Control Of Gene Expression ◽  
Pol Ii ◽  
Genome Wide ◽  
Pol Ii Pausing

Abstract Control of gene expression is one of the most complex yet continuous physiological processes impacting cellular homeostasis. RNA polymerase II (Pol II) transcription is tightly regulated at promoter-proximal regions by intricate dynamic processes including Pol II pausing, release into elongation and premature termination. Pol II pausing is a phenomenon where Pol II complex pauses within 30–60 nucleotides after initiating the transcription. Negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF) contribute in the establishment of Pol II pausing, and positive transcription elongation factor b releases (P-TEFb) paused complex after phosphorylating DSIF that leads to dissociation of NELF. Pol II pausing is observed in most expressed genes across the metazoan. The precise role of Pol II pausing is not well understood; however, it’s required for integration of signals for gene regulation. In the present study, we investigated the role of phosphatase and tensin homolog (PTEN) in genome-wide transcriptional regulation using PTEN overexpression and PTEN knock-down models. Here we identify that PTEN alters the expression of hundreds of genes, and its restoration establishes genome-wide Pol II promoter-proximal pausing in PTEN null cells. Furthermore, PTEN re-distributes Pol II occupancy across the genome and possibly impacts Pol II pause duration, release and elongation rate in order to enable precise gene regulation at the genome-wide scale. Our observations demonstrate an imperative role of PTEN in global transcriptional regulation that will provide a new direction to understand PTEN-associated pathologies and its management.

scholarly journals In Vivo Requirement of the RNA Polymerase II Elongation Factor Elongin A for Proper Gene Expression and Development

2004 ◽  
Vol 24 (22) ◽  
pp. 9911-9919 ◽  
Author(s):  
Mark Gerber ◽  
Joel C. Eissenberg ◽  
Stephanie Kong ◽  
Kristen Tenney ◽  
Joan Weliky Conaway ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Regulation Of Gene Expression ◽  
Polytene Chromosomes ◽  
Elongation Factor ◽  
Physiological Role ◽  
In Vivo Model ◽  
Pol Ii

ABSTRACT A number of transcription factors that increase the catalytic rate of mRNA synthesis by RNA polymerase II (Pol II) have been purified from higher eukaryotes. Among these are the ELL family, DSIF, and the heterotrimeric elongin complex. Elongin A, the largest subunit of the elongin complex, is the transcriptionally active subunit, while the smaller elongin B and C subunits appear to act as regulatory subunits. While much is known about the in vitro properties of elongin A and other members of this class of elongation factors, the physiological role(s) of these proteins remain largely unclear. To elucidate in vivo functions of elongin A, we have characterized its Drosophila homologue (dEloA). dEloA associates with transcriptionally active puff sites within Drosophila polytene chromosomes and exhibits many of the expected biochemical and cytological properties consistent with a Pol II-associated elongation factor. RNA interference-mediated depletion of dEloA demonstrated that elongin A is an essential factor that is required for proper metamorphosis. Consistent with this observation, dEloA expression peaks during the larval stages of development, suggesting that this factor may be important for proper regulation of developmental events during these stages. The discovery of the role of elongin A in an in vivo model system defines the novel contribution played by RNA polymerase II elongation machinery in regulation of gene expression that is required for proper development.

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