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Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression.

ABSTRACT: The regulation of gene expression catalyzed by RNA polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extraterminal domain (BET) and super elongation complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the proteolysis targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of the SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs.

SUBMITTER: Zheng B

PROVIDER: S-EPMC7849361 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Acute perturbation strategies in interrogating RNA Polymerase II elongation factor function in gene expression

Project description:The regulation of gene expression catalyzed by RNA Polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extra terminal domain (BET) and Super Elongation Complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the Proteolysis-targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: the increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs.

2021-01-15 | GSE145525 | GEO

Acute perturbation strategies in interrogating RNA Polymerase II elongation factor function in gene expression

Project description:Acute perturbation strategies in interrogating RNA Polymerase II elongation factor function in gene expression

| PRJNA607507 | ENA

Elongation factor-specific capture of RNA polymerase II complexes.

Project description:Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often poorly understood. Here, we present an approach for elongation-factor-specific mNET capture (ELCAP) of RNAPII complexes for sequencing and mass spectrometry analysis aimed at investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply ELCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3' end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3' end processing factors but depleted of splicing components. Importantly, the ELCAP sequencing (ELCAP-seq) profiles of SCAF4- and SCAF8-RNAPII complexes nicely reflect their function as mRNA-anti-terminators and their competing functions at the end of genes, where they prevent or promote transcriptional readthrough.

| S-EPMC9795356 | biostudies-literature

Yeast transcription elongation factor Spt5 associates with RNA polymerase I and RNA polymerase II directly.

Project description:Spt5 is a transcription factor conserved in all three domains of life. Spt5 homologues from bacteria and archaea bind the largest subunit of their respective RNA polymerases. Here we demonstrate that Spt5 directly associates with RNA polymerase (Pol) I and RNA Pol II in yeast through its central region containing conserved NusG N-terminal homology and KOW domains. Deletion analysis of SPT5 supports our biochemical data, demonstrating the importance of the KOW domains in Spt5 function. Far Western blot analysis implicates A190 of Pol I as well as Rpb1 of Pol II in binding Spt5. Three additional subunits of Pol I may also participate in this interaction. One of these subunits, A49, has known roles in transcription elongation by Pol I. Interestingly, spt5 truncation mutations suppress the cold-sensitive phenotype of rpa49Δ strain, which lacks the A49 subunit in the Pol I complex. Finally, we observed that Spt5 directly binds to an essential Pol I transcription initiation factor, Rrn3, and to the ribosomal RNA. Based on these data, we propose a model in which Spt5 is recruited to the rDNA early in transcription and propose that it plays an important role in ribosomal RNA synthesis through direct binding to the Pol I complex.

| S-EPMC3099699 | biostudies-literature

NDF is a transcription factor that stimulates elongation by RNA polymerase II.

Project description:RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we found that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than that used by transcription factor TFIIS. Moreover, yeast Pdp3, which is related to NDF, binds to Pol II and stimulates elongation. Thus, NDF is a Pol II binding transcription elongation factor that is localized over gene bodies and is conserved from yeast to humans.

| S-EPMC8973848 | biostudies-literature

Noncanonical tandem SH2 enables interaction of elongation factor Spt6 with RNA polymerase II.

Project description:Src homology 2 (SH2) domains are mostly found in multicellular organisms where they recognize phosphotyrosine-containing signaling proteins. Spt6, a conserved transcription factor and putative histone chaperone, contains a C-terminal SH2 domain conserved from yeast to human. In mammals, this SH2 domain recognizes phosphoserines rather than phosphotyrosines and is essential for the recruitment of Spt6 by elongating RNA polymerase II (RNAPII), enabling Spt6 to participate in the coupling of transcription elongation, chromatin modulation, and mRNA export. We have determined the structure of the entire Spt6 C-terminal region from Antonospora locustae, revealing the presence of two highly conserved tandem SH2 domains rather than a single SH2 domain. Although the first SH2 domain has a canonical organization, the second SH2 domain is highly noncanonical and appears to be unique in the SH2 family. However, both SH2 domains have phosphate-binding determinants. Our biochemical and genetic data demonstrate that the complete tandem, but not the individual SH2 domains, are necessary and sufficient for the interaction of Spt6 with RNAPII and are important for Spt6 function in vivo. Furthermore, our data suggest that binding of RNAPII to the Spt6 tandem SH2 is more extensive than the mere recognition of a doubly phosphorylated C-terminal domain peptide by the tandem SH2. Taken together, our results show that Spt6 interaction with RNAPII via a novel arrangement of canonical and noncanonical SH2 domains is crucial for Spt6 function in vivo.

| S-EPMC2992272 | biostudies-literature

The elongation factor Spt4/5 regulates RNA polymerase II transcription through the nucleosome.

Project description:RNA polymerase II (RNAPII) passes through the nucleosome in a coordinated manner, generating several intermediate nucleosomal states as it breaks and then reforms histone-DNA contacts ahead of and behind it, respectively. Several studies have defined transcription-induced nucleosome intermediates using only RNA Polymerase. However, RNAPII is decorated with elongation factors as it transcribes the genome. One such factor, Spt4/5, becomes an integral component of the elongation complex, making direct contact with the 'jaws' of RNAPII and nucleic acids in the transcription scaffold. We have characterized the effect of incorporating Spt4/5 into the elongation complex on transcription through the 601R nucleosome. Spt4/5 suppressed RNAPII pausing at the major H3/H4-induced arrest point, resulting in downstream re-positioning of RNAPII further into the nucleosome. Using a novel single molecule FRET system, we found that Spt4/5 affected the kinetics of DNA re-wrapping and stabilized a nucleosomal intermediate with partially unwrapped DNA behind RNAPII. Comparison of nucleosomes of different sequence polarities suggest that the strength of the DNA-histone interactions behind RNAPII specifies the Spt4/5 requirement. We propose that Spt4/5 may be important to coordinate the mechanical movement of RNAPII through the nucleosome with co-transcriptional chromatin modifications during transcription, which is affected by the strength of histone-DNA interactions.

| S-EPMC5499766 | biostudies-literature

Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex.

Project description:Negative elongation factor (NELF) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) are involved in pausing RNA Polymerase II (Pol II) in the promoter-proximal region of the hsp70 gene in Drosophila, before heat shock induction. Such blocks in elongation are widespread in the Drosophila genome. However, the mechanism by which DSIF and NELF participate in setting up the paused Pol II remains unclear. We analyzed the interactions among DSIF, NELF, and a reconstituted Drosophila Pol II elongation complex to gain insight into the mechanism of pausing. Our results show that DSIF and NELF require a nascent transcript longer than 18 nt to stably associate with the Pol II elongation complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex. Taken together, these results provide a possible model by which DSIF binds the elongation complex via association with the nascent transcript and subsequently recruits NELF. Although DSIF and NELF were both required for inhibition of transcription, we did not detect a NELF-RNA contact when the nascent transcript was between 22 and 31 nt long, which encompasses the region where promoter-proximal pausing occurs on many genes in Drosophila. This raises the possibility that RNA binding by NELF is not necessary in promoter-proximal pausing.

| S-EPMC2895096 | biostudies-literature

The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes.

Project description:In this article, we provide direct evidence that the evolutionarily conserved transcription elongation factor TFIIS functions during preinitiation complex assembly. First, we identified TFIIS in a mass spectrometric screen of RNA polymerase II (Pol II) preinitiation complexes (PICs). Second, we show that the association of TFIIS with a promoter depends on functional PIC components including Mediator and the SAGA complex. Third, we demonstrate that TFIIS is required for efficient formation of active PICs. Using truncation mutants of TFIIS, we find that the Pol II-binding domain is the minimal domain necessary to stimulate PIC assembly. However, efficient formation of active PICs requires both the Pol II-binding domain and the poorly understood N-terminal domain. Importantly, Domain III, which is required for the elongation function of TFIIS, is dispensable during PIC assembly. The results demonstrate that TFIIS is a PIC component that is required for efficient formation and/or stability of the complex.

| S-EPMC2042163 | biostudies-literature

Negative elongation factor-mediated suppression of RNA polymerase II elongation of Kaposi's sarcoma-associated herpesvirus lytic gene expression.

Project description:Genome-wide chromatin immunoprecipitation assays indicate that the promoter-proximal pausing of RNA polymerase II (RNAPII) is an important postinitiation step for gene regulation. During latent infection, the majority of Kaposi's sarcoma-associated herpesvirus (KSHV) genes is silenced via repressive histone marks on their promoters. Despite the absence of their expression during latency, however, several lytic promoters are enriched with activating histone marks, suggesting that mechanisms other than heterochromatin-mediated suppression contribute to preventing lytic gene expression. Here, we show that the RNAPII-mediated transcription of the KSHV OriLytL, K5, K6, and K7 (OriLytL-K7) lytic genes is paused at the elongation step during latency. Specifically, the RNAPII-mediated transcription is stalled by the host's negative elongation factor (NELF) at the promoter regions of OriLytL-K7 lytic genes during latency, leading to the hyperphosphorylation of the serine 5 residue and the hypophosphorylation of the serine 2 of the C-terminal domain of the RNAPII large subunit, a hallmark of stalled RNAPII. Consequently, depletion of NELF expression induced transition of stalled RNAPII into a productive transcription elongation at the promoter-proximal regions of OriLytL-K7 lytic genes, leading to their RTA-independent expression. Using an RTA-deficient recombinant KSHV, we also showed that expression of the K5, K6, and K7 lytic genes was highly inducible upon external stimuli compared to other lytic genes that lack RNAPII on their promoters during latency. These results indicate that the transcription elongation of KSHV OriLytL-K7 lytic genes is inhibited by NELF during latency, but can also be promptly reactivated in an RTA-independent manner upon external stimuli.

| S-EPMC3446579 | biostudies-literature

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