scholarly journals Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Emily Hsu ◽  
Mario A. Pennella ◽  
Nathan R. Zemke ◽  
Carol Eng ◽  
Arnold J. Berk
Keyword(s):  
Rna Polymerase Ii ◽  
Posttranslational Modifications ◽  
Lysine Acetylation ◽  
Activation Domain ◽  
Pol Ii ◽  
Protein Activation ◽  
Adenovirus E1a ◽  
Viral Promoters ◽  
H3 Acetylation ◽  
Kinetics Of

ABSTRACTHow histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integratedlacOarray of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCEDespite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res37:1095–1106, 2009,https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.

scholarly journals Transcription and splicing dynamics during early Drosophila development

2020 ◽  
Author(s):  
Pedro Prudêncio ◽  
Kenny Rebelo ◽  
Rosina Savisaar ◽  
Rui Gonçalo Martinho ◽  
Maria Carmo-Fonseca
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Dynamic Features ◽  
Antisense Gene ◽  
Pol Ii ◽  
Human Genes ◽  
Yeast Genes ◽  
Splice Junctions ◽  
Kinetics Of ◽  
Intron Definition

ABSTRACTWidespread co-transcriptional splicing has been demonstrated from yeast to human. However, measuring the kinetics of splicing relative to transcription has been hampered by technical challenges. Here, we took advantage of native elongating transcript sequencing (NET-seq) to identify the position of RNA polymerase II (Pol II) when exons become ligated in the newly synthesized RNA. We analyzed Drosophila melanogaster embryos because the genes transcribed initially during development have few and short introns (like yeast genes), whereas genes transcribed later contain multiple long introns (more similar to human genes). We detected spliced NET-seq reads connected to Pol II molecules that were positioned just a few nucleotides downstream of the 3’ splice site. Although the majority of splice junctions were covered by spliced reads, many introns remained unspliced, resulting in a complex range of heterogeneity in splicing dynamics. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. Both canonical and recursive splicing were associated with a higher Pol II density, suggesting a splicing-coupled mechanism that slows down transcription elongation. We further observed that transcription termination was very efficient for isolated genes but that the presence of an overlapping antisense gene was often associated with transcriptional read-through. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

scholarly journals β-Globin Intergenic Transcription and Histone Acetylation Dependent on an Enhancer

2007 ◽  
Vol 27 (8) ◽  
pp. 2980-2986 ◽  
Author(s):  
AeRi Kim ◽  
Hui Zhao ◽  
Ina Ifrim ◽  
Ann Dean
Keyword(s):  
Histone Acetylation ◽  
Rna Polymerase Ii ◽  
Target Gene ◽  
Globin Gene ◽  
Tata Box ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Transcription Complexes ◽  
And Function ◽  
H3 Acetylation

ABSTRACT Histone acetyltransferases are associated with the elongating RNA polymerase II (Pol II) complex, supporting the idea that histone acetylation and transcription are intertwined mechanistically in gene coding sequences. Here, we studied the establishment and function of histone acetylation and transcription in noncoding sequences by using a model locus linking the β-globin HS2 enhancer and the embryonic ε-globin gene in chromatin. An intact HS2 enhancer that recruits RNA Pol II is required for intergenic transcription and histone H3 acetylation and K4 methylation between the enhancer and target gene. RNA Pol II recruitment to the target gene TATA box is not required for the intergenic transcription or intergenic histone modifications, strongly implying that they are properties conferred by the enhancer. However, Pol II recruitment at HS2, intergenic transcription, and intergenic histone modification are not sufficient for transcription or modification of the target gene: these changes require initiation at the TATA box of the gene. The results suggest that intergenic and genic transcription complexes are independent and possibly differ from one another.

scholarly journals Emerging Views on the CTD Code

2012 ◽  
Vol 2012 ◽  
pp. 1-19 ◽  
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.

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 Herpesvirus infection reduces Pol II occupancy of host promoters but spares viral promoters

2019 ◽  
Author(s):  
Ella N Hartenian ◽  
Britt A Glaunsinger
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Viral Transcript ◽  
Pol Ii ◽  
Murine Gammaherpesvirus ◽  
A Genome ◽  
Viral Promoters ◽  
Gammaherpesvirus 68

AbstractIn mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. Viral genes, despite the fact that they require Pol II for transcription, escape this transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We hypothesize that the ability to escape from transcriptional repression is linked to the localization of viral DNA in replication compartments, providing a means for these viruses to counteract decay-induced viral transcript loss.

Diet-induced epigenetic regulation in vivo of the intestinal fructose transporter Glut5 during development of rat small intestine

2011 ◽  
Vol 435 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Takuji Suzuki ◽  
Veronique Douard ◽  
Kazuki Mochizuki ◽  
Toshinao Goda ◽  
Ronaldo P. Ferraris
Keyword(s):  
Rna Polymerase Ii ◽  
Glucose Transporter ◽  
Histone H3 ◽  
Solute Carrier Family ◽  
Metabolic Complications ◽  
Pol Ii ◽  
Histone H4 Acetylation ◽  
Histone H3 Acetylation ◽  
Solute Carrier ◽  
H3 Acetylation

Metabolic complications arising from excessive fructose consumption are increasing dramatically even in young children, but little is known about ontogenetic mechanisms regulating Glut5 [glucose transporter 5; encoded by the Slc2a5 (solute carrier family 2 member 5) gene]. Glut5 expression is low postnatally and does not increase, unless luminal fructose and systemic glucocorticoids are present, until ≥14 days of age, suggesting substrate-inducible age- and hormone-sensitive regulation. In the present study, we perfused intestines of 10- and 20-day-old rats with either fructose or glucose then analysed the binding of Pol II (RNA polymerase II) and GR (glucocorticoid receptor), as well as acetylation of histones H3 and H4 by chromatin immunoprecipitation. Abundance of Glut5 mRNA increased only with fructose perfusion and age, a pattern that matched that of Pol II binding and histone H3 acetylation to the Glut5 promoter. Although many regions of the Glut5 promoter respond to developmental signals, fewer regions perceive dietary signals. Age- but not fructose-dependent expression of Sglt1 [sodium-dependent glucose co-transporter 1 encoded by the Slc5a1(solute carrier family 5 member 1) gene] also correlated with Pol II binding and histone H3 acetylation. In contrast, G6Pase (glucose-6-phosphatase; encoded by the G6pc gene) expression, which decreases with age and increases with fructose, is associated only with age-dependent changes in histone H4 acetylation. Induction of Glut5 during ontogenetic development appears to be specifically mediated by GR translocation to the nucleus and subsequent binding to the Glut5 promoter, whereas the glucocorticoid-independent regulation of Sglt1 by age was not associated with any GR binding to the Sglt1 promoter.

scholarly journals ArabidopsisHistone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

2018 ◽  
Vol 92 (16) ◽  
Author(s):  
Tami Coursey ◽  
Milica Milutinovic ◽  
Elizabeth Regedanz ◽  
Jelena Brkljacic ◽  
David M. Bisaro
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Posttranslational Modifications ◽  
Chromatin Structure ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
In Planta ◽  
Pol Ii ◽  
Content Type ◽  
Active Gene

ABSTRACTHistone posttranslational modifications (PTMs) impart information that regulates chromatin structure and activity. Their effects are mediated by histone reader proteins that bind specific PTMs to modify chromatin and/or recruit appropriate effectors to alter the chromatin landscape. Despite their crucial juxtaposition between information and functional outcome, relatively few plant histone readers have been identified, and nothing is known about their impact on viral chromatin and pathogenesis. We used the geminivirusCabbage leaf curl virus(CaLCuV) as a model to functionally characterize two recently identified reader proteins, EMSY-LIKE 1 (EML1) and EML3, which contain Tudor-like Agenet domains predictive of histone PTM binding function. Here, we show that mutantArabidopsisplants exhibit contrasting hypersusceptible (eml1) and tolerant (eml3) responses to CaLCuV infection and that EML1 deficiency correlates with RNA polymerase II (Pol II) enrichment on viral chromatin and upregulated viral gene expression. Consistent with reader activity, EML1 and EML3 associate with nucleosomes and with CaLCuV chromatin, suggesting a direct impact on pathogenesis. We also demonstrate that EML1 and EML3 bind peptides containing histone H3 lysine 36 (H3K36), a PTM usually associated with active gene expression. The interaction encompasses multiple H3K36 PTMs, including methylation and acetylation, suggesting nuanced regulation. Furthermore, EML1 and EML3 associate with similar regions of viral chromatin, implying possible competition between the two readers. Regions of EML1 and EML3 association correlate with sites of trimethylated H3K36 (H3K36me3) enrichment, consistent with regulation of geminivirus chromatin by direct EML targeting.IMPORTANCEHistone PTMs convey information that regulates chromatin compaction and DNA accessibility. Histone reader proteins bind specific PTMs and translate their effects by modifying chromatin and/or by recruiting effectors that alter chromatin structure or activity. In this study, CaLCuV was used to characterize the activities of twoArabidopsisAgenet domain histone readers, EML1 and EML3. We show thateml1mutants are hypersusceptible to CaLCuV, whereaseml3plants are more tolerant of infection than wild-type plants. We also demonstrate that EML1 and EML3 associate with histones and viral chromatinin plantaand that both proteins bind peptides containing H3K36, a PTM associated with active gene expression. Consistent with antiviral activity, EML1 suppresses CaLCuV gene expression and reduces Pol II access to viral chromatin. By linking EML1 and EML3 to pathogenesis, these studies have expanded our knowledge of histone reader proteins and uncovered an additional level of viral chromatin regulation.

scholarly journals Cytomegalovirus late transcription factor target sequence diversity orchestrates viral early to late transcription

2021 ◽  
Vol 17 (8) ◽  
pp. e1009796
Author(s):  
Ming Li ◽  
Qiaolin Hu ◽  
Geoffrey Collins ◽  
Mrutyunjaya Parida ◽  
Christopher B. Ball ◽  
...  
Keyword(s):  
Dna Replication ◽  
Rna Polymerase Ii ◽  
Core Promoter ◽  
Target Sequence ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Pol Ii ◽  
Viral Promoters ◽  
Late Transcription ◽  
Target Sequences

Beta- and gammaherpesviruses late transcription factors (LTFs) target viral promoters containing a TATT sequence to drive transcription after viral DNA replication has begun. Human cytomegalovirus (HCMV), a betaherpesvirus, uses the UL87 LTF to bind both TATT and host RNA polymerase II (Pol II), whereas the UL79 LTF has been suggested to drive productive elongation. Here we apply integrated functional genomics (dTag system, PRO-Seq, ChIP-Seq, and promoter function assays) to uncover the contribution of diversity in LTF target sequences in determining degree and scope to which LTFs drive viral transcription. We characterize the DNA sequence patterns in LTF-responsive and -unresponsive promoter populations, determine where and when Pol II initiates transcription, identify sites of LTF binding genome-wide, and quantify change in nascent transcripts from individual promoters in relation to core promoter sequences, LTF loss, stage of infection, and viral DNA replication. We find that HCMV UL79 and UL87 LTFs function concordantly to initiate transcription from over half of all active viral promoters in late infection, while not appreciably affecting host transcription. Both LTFs act on and bind to viral early-late and late kinetic-class promoters. Over one-third of these core promoters lack the TATT and instead have a TATAT, TGTT, or YRYT. The TATT and non-TATT motifs are part of a sequence block with a sequence code that correlates with promoter transcription level. LTF occupancy of a TATATA palindrome shared by back-to-back promoters is linked to bidirectional transcription. We conclude that diversity in LTF target sequences shapes the LTF-transformative program that drives the viral early-to-late transcription switch.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

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.

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