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Elongin A associates with actively transcribed genes and modulates enhancer RNA levels with limited impact on transcription elongation rate in vivo.

ABSTRACT: Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes to both thousands of Pol II transcribed genes with preference for transcription start site and promoter regions and a large number of active enhancers across the genome. EloA deletion results in accumulation of transcripts from a subset of enhancers and their adjacent genes. Notably, EloA does not substantially enhance the elongation rate of Pol II in vivo. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interfere with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on enhancer RNA synthesis, and interacts with the rRNA producing/processing machinery in the nucleolus. Our work opens new avenues for further investigation of the role of this functionally multifaceted transcription factor in enhancer and ribosomal RNA biology.

SUBMITTER: Ardehali MB

PROVIDER: S-EPMC7948453 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Elongin A displays widespread association with actively transcribed genes and modulates enhancer RNA transcripiton.

Project description:Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes both to thousands of Pol II transcribed genes with a preference for sites of Pol II pausing and as well as a large number of active enhancers across the genome. EloA loss results in accumulation of transcripts at a subset of enhancers and their adjacent genes. However, EloA deletion results in only a modest decrease in the average elongation rate of Pol II globally. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interferes with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on eRNA synthesis and interacts with the rRNA producing/processing machinery in the nucleolus.

2020-12-10 | GSE151582 | GEO

Drosophila Paf1 modulates chromatin structure at actively transcribed genes.

Project description:The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure.

| S-EPMC1317635 | biostudies-literature

Elongin A displays widespread association with actively transcribed genes and modulates enhancer RNA transcripiton.

Project description:Elongin A displays widespread association with actively transcribed genes and modulates enhancer RNA transcripiton.

| PRJNA636401 | ENA

The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes.

Project description:Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of yeast RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process through the distribution of transcribed nucleosome positions. Notably, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer and histone-histone dissociation quantitatively explains our observations and unifies them with results obtained from other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression.

| S-EPMC3279329 | biostudies-literature

Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate.

Project description:The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3'-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5-Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5-Rpb1 complex levels and consequently transcription elongation rate.

| S-EPMC3484106 | biostudies-literature

Zebrafish promoter microarrays identify actively transcribed embryonic genes.

Project description:We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis.

| S-EPMC1779600 | biostudies-literature

The ATP-dependent chromatin remodeler Chd1 is recruited by transcription elongation factors and maintains H3K4me3/H3K36me3 domains at actively transcribed and spliced genes.

Project description:Chd1 (Chromodomain Helicase DNA Binding Protein 1) is a conserved ATP-dependent chromatin remodeler that maintains the nucleosomal structure of chromatin, but the determinants of its specificity and its impact on gene expression are not well defined. To identify the determinants of Chd1 binding specificity in the yeast genome, we investigated Chd1 occupancy in mutants of several candidate factors. We found that several components of the PAF1 transcription elongation complex contribute to Chd1 recruitment to highly transcribed genes and identified Spt4 as a factor that appears to negatively modulate Chd1 binding to chromatin. We discovered that CHD1 loss alters H3K4me3 and H3K36me3 patterns throughout the yeast genome. Interestingly, the aberrant histone H3 methylation patterns were predominantly observed within 1 kb from the transcription start site, where both histone H3 methylation marks co-occur. A reciprocal change between the two marks was obvious in the absence of Chd1, suggesting a role for CHD1 in establishing or maintaining the boundaries of these largely mutually exclusive histone marks. Strikingly, intron-containing genes were most susceptible to CHD1 loss and exhibited a high degree of histone H3 methylation changes. Intron retention was significantly lower in the absence of CHD1, suggesting that CHD1 function as a chromatin remodeler could indirectly affect RNA splicing.

| S-EPMC5499586 | biostudies-literature

Distant positioning of proteasomal proteolysis relative to actively transcribed genes.

Project description:While it is widely acknowledged that the ubiquitin-proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.

| S-EPMC3113580 | biostudies-literature

Interaction between calcium and potassium modulates elongation rate in cotton fiber cells.

Project description:Calcium (Ca2+) is necessary for fiber cell development in cotton (Gossypium hirsutum), both as a cell wall structural component and for environmental signaling responses. It is also known that potassium (K+) plays a critical role in cotton fiber cell elongation. However, it is unclear whether Ca2+ integrates its activities with K+ to regulate fiber elongation. Here, we report the novel discovery that Ca2+ deficiency, when integrated with K+ signaling, promotes fiber elongation. Using inductively coupled plasma-mass spectrometry (ICP-MS), we determined dynamic profiles of the ionome in ovules and fibers at different developmental stages, and found that a high accumulation of macro-elements, but not Ca2+, was associated with longer fibers. Using an in vitro ovule culture system, we found that under Ca2+-deficient conditions, sufficient K+ (52 mM) rapidly induced ovule and fiber browning, while reduced K+ (2 or 27 mM) not only suppressed tissue browning but also altered fiber elongation. Reduced K+ also enhanced reactive oxygen species scavenging ability and maintained abscisic acid and jasmonic acid levels, which in turn compensated for Ca2+ deficiency. Ca2+ deficiency combined with reduced K+ (0 mM Ca2+ and 27 mM K+) produced longer fibers in cultured ovules, due to cell wall loosening by phytosulfokine (PSK), expansin (EXP), and xyloglucan endotransglycosylase/hydrolase (XTH), and an increase of the K+ content of fiber cells. Using transgenic cotton, we showed that the CBL-INTERACTING PROTEIN KINASE 6 (GhCIPK6) gene mediates the uptake of K+ under Ca2+-deficient conditions. This study establishes a new link between Ca2+, K+, and fiber elongation.

| S-EPMC5853336 | biostudies-literature

Actin associates with actively elongating genes

Project description:Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in several biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b (P-TEFb) required for RNA polymerase II (Pol II) pause-release. This interaction is not dependent on kinase activity of Cdk9 and is not involved with releasing P-TEFb from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

2023-09-01 | GSE239771 | GEO

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