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Repression of RNA polymerase II elongation in vivo is critically dependent on the C-terminus of Spt5.

Chen H, Contreras X, Yamaguchi Y, Handa H, Peterlin BM, Guo S - PLoS ONE (2009)

Bottom Line: Second, NSpt5 de-represses the transcription of hsp70-4 in zebrafish embryos and HIVLTR in cultured human cells, which are repressed at the RNAPII elongation step under non-inducible conditions.Third, NSpt5 directly associates with hsp70-4 chromatin in vivo and increases the occupancy of RNAPII, positive transcription elongation factor b (P-TEFb), histone H3 Lys 4 trimethylation (H3K4Me3), and surprisingly, the negative elongation factor A (NELF-A) at the locus, indicating a direct action of NSpt5 on the elongation repressed locus.Together, these results reveal a dominant activity of NSpt5 to de-repress RNAPII elongation, and suggest that the C-terminus of Spt5 is critical for repressing RNAPII elongation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biopharmaceutical Sciences, and Programs in Biological Sciences and Human Genetics, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
The stalling of RNA polymerase II (RNAPII) at the promoters of many genes, including developmental regulators, stress-responsive genes, and HIVLTR, suggests transcription elongation as a critical regulatory step in addition to initiation. Spt5, the large subunit of the DRB sensitivity-inducing factor (DSIF), represses or activates RNAPII elongation in vitro. How RNAPII elongation is repressed in vivo is not well understood. Here we report that CTR1 and CTR2CT, the two repeat-containing regions constituting the C-terminus of Spt5, play a redundant role in repressing RNAPII elongation in vivo. First, mis-expression of Spt5 lacking CTR1 or CTR2CT is inconsequential, but mis-expression of Spt5 lacking the entire C-terminus (termed NSpt5) dominantly impairs embryogenesis in zebrafish. Second, NSpt5 de-represses the transcription of hsp70-4 in zebrafish embryos and HIVLTR in cultured human cells, which are repressed at the RNAPII elongation step under non-inducible conditions. Third, NSpt5 directly associates with hsp70-4 chromatin in vivo and increases the occupancy of RNAPII, positive transcription elongation factor b (P-TEFb), histone H3 Lys 4 trimethylation (H3K4Me3), and surprisingly, the negative elongation factor A (NELF-A) at the locus, indicating a direct action of NSpt5 on the elongation repressed locus. Together, these results reveal a dominant activity of NSpt5 to de-repress RNAPII elongation, and suggest that the C-terminus of Spt5 is critical for repressing RNAPII elongation in vivo.

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ChIP at the hsp70-4 chromatin in Nspt5 expressing zebrafish embryos.(A to D) Charts show the percent of input material immunoprecipitated in different regions of hsp70-4 chromatin. The relative enrichment of ChIP and qRT-PCR values obtained with CDK9 antibody over the IgG control (A), H3K4Me3 antibody over the IgG control (B), or H3K79Me2 antibody over the IgG control (C), NELF-A antibody over the rabbit serum control (D). Error bars represent S.E.M. of duplicate measurements from two independent experiments.
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pone-0006918-g006: ChIP at the hsp70-4 chromatin in Nspt5 expressing zebrafish embryos.(A to D) Charts show the percent of input material immunoprecipitated in different regions of hsp70-4 chromatin. The relative enrichment of ChIP and qRT-PCR values obtained with CDK9 antibody over the IgG control (A), H3K4Me3 antibody over the IgG control (B), or H3K79Me2 antibody over the IgG control (C), NELF-A antibody over the rabbit serum control (D). Error bars represent S.E.M. of duplicate measurements from two independent experiments.

Mentions: We next determined the in vivo chromatin occupancy of other proteins involved in regulating transcription elongation at the hsp70-4 locus in vivo. Cdk9 is the kinase subunit of P-TEFb. In the presence of NSpt5, the occupancy of Cdk9 is significantly increased at the 5′ end, middle, and 3′ end (Figure 6A). Inhibition of Cdk9 activity, either through a specific morpholino antisense oligonucleotide or using the kinase inhibitor flavoperidol (FP), significantly suppressed NSpt5-induced dorsalization phenotype and the de-repression of hsp70-4 (Figure S4 and S5). Several lines of evidence suggest that such a suppression of NSpt5 effects is not due to a general developmental delay but rather, is likely due to a direct requirement of CDK9 in the manifestation of NSpt5's dominant negative effect: First, we checked later stages of Nspt5-expressing and Cdk9-impaired embryos, and still did not see a dorsalization phenotype, suggesting that it is not due to a simple developmental delay. Second, at the molecular level, NSpt5-mediated, increased expression of hsp70-4, which is a marker gene directly regulated by Spt5, is significantly impaired by Cdk9 knockdown. Third, Cdk9, being a subunit of P-TEFb, is known to phosphorylate RNAPII CTD, Spt5 C-terminal domains, and NELF, which together form protein complexes, suggesting a possible direct requirement of RNAPII or NELF phosphorylation for the manisfestation of NSpt5's effect. Taken together, we suggest that Cdk9 activity and hence the phosphorylation of RNAPII or NELF is likely to be directly required for NSpt5's dominant negative effect in vivo. Consistent with the increased occupancy of Cdk9, we also observed increased occupancy of H3k4Me3 (Figure 6B), and a slight increase in occupancy of H3K79Me2 (Figure 6C), both of which represent histone marks for active transcription [45].


Repression of RNA polymerase II elongation in vivo is critically dependent on the C-terminus of Spt5.

Chen H, Contreras X, Yamaguchi Y, Handa H, Peterlin BM, Guo S - PLoS ONE (2009)

ChIP at the hsp70-4 chromatin in Nspt5 expressing zebrafish embryos.(A to D) Charts show the percent of input material immunoprecipitated in different regions of hsp70-4 chromatin. The relative enrichment of ChIP and qRT-PCR values obtained with CDK9 antibody over the IgG control (A), H3K4Me3 antibody over the IgG control (B), or H3K79Me2 antibody over the IgG control (C), NELF-A antibody over the rabbit serum control (D). Error bars represent S.E.M. of duplicate measurements from two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2735033&req=5

pone-0006918-g006: ChIP at the hsp70-4 chromatin in Nspt5 expressing zebrafish embryos.(A to D) Charts show the percent of input material immunoprecipitated in different regions of hsp70-4 chromatin. The relative enrichment of ChIP and qRT-PCR values obtained with CDK9 antibody over the IgG control (A), H3K4Me3 antibody over the IgG control (B), or H3K79Me2 antibody over the IgG control (C), NELF-A antibody over the rabbit serum control (D). Error bars represent S.E.M. of duplicate measurements from two independent experiments.
Mentions: We next determined the in vivo chromatin occupancy of other proteins involved in regulating transcription elongation at the hsp70-4 locus in vivo. Cdk9 is the kinase subunit of P-TEFb. In the presence of NSpt5, the occupancy of Cdk9 is significantly increased at the 5′ end, middle, and 3′ end (Figure 6A). Inhibition of Cdk9 activity, either through a specific morpholino antisense oligonucleotide or using the kinase inhibitor flavoperidol (FP), significantly suppressed NSpt5-induced dorsalization phenotype and the de-repression of hsp70-4 (Figure S4 and S5). Several lines of evidence suggest that such a suppression of NSpt5 effects is not due to a general developmental delay but rather, is likely due to a direct requirement of CDK9 in the manifestation of NSpt5's dominant negative effect: First, we checked later stages of Nspt5-expressing and Cdk9-impaired embryos, and still did not see a dorsalization phenotype, suggesting that it is not due to a simple developmental delay. Second, at the molecular level, NSpt5-mediated, increased expression of hsp70-4, which is a marker gene directly regulated by Spt5, is significantly impaired by Cdk9 knockdown. Third, Cdk9, being a subunit of P-TEFb, is known to phosphorylate RNAPII CTD, Spt5 C-terminal domains, and NELF, which together form protein complexes, suggesting a possible direct requirement of RNAPII or NELF phosphorylation for the manisfestation of NSpt5's effect. Taken together, we suggest that Cdk9 activity and hence the phosphorylation of RNAPII or NELF is likely to be directly required for NSpt5's dominant negative effect in vivo. Consistent with the increased occupancy of Cdk9, we also observed increased occupancy of H3k4Me3 (Figure 6B), and a slight increase in occupancy of H3K79Me2 (Figure 6C), both of which represent histone marks for active transcription [45].

Bottom Line: Second, NSpt5 de-represses the transcription of hsp70-4 in zebrafish embryos and HIVLTR in cultured human cells, which are repressed at the RNAPII elongation step under non-inducible conditions.Third, NSpt5 directly associates with hsp70-4 chromatin in vivo and increases the occupancy of RNAPII, positive transcription elongation factor b (P-TEFb), histone H3 Lys 4 trimethylation (H3K4Me3), and surprisingly, the negative elongation factor A (NELF-A) at the locus, indicating a direct action of NSpt5 on the elongation repressed locus.Together, these results reveal a dominant activity of NSpt5 to de-repress RNAPII elongation, and suggest that the C-terminus of Spt5 is critical for repressing RNAPII elongation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biopharmaceutical Sciences, and Programs in Biological Sciences and Human Genetics, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
The stalling of RNA polymerase II (RNAPII) at the promoters of many genes, including developmental regulators, stress-responsive genes, and HIVLTR, suggests transcription elongation as a critical regulatory step in addition to initiation. Spt5, the large subunit of the DRB sensitivity-inducing factor (DSIF), represses or activates RNAPII elongation in vitro. How RNAPII elongation is repressed in vivo is not well understood. Here we report that CTR1 and CTR2CT, the two repeat-containing regions constituting the C-terminus of Spt5, play a redundant role in repressing RNAPII elongation in vivo. First, mis-expression of Spt5 lacking CTR1 or CTR2CT is inconsequential, but mis-expression of Spt5 lacking the entire C-terminus (termed NSpt5) dominantly impairs embryogenesis in zebrafish. Second, NSpt5 de-represses the transcription of hsp70-4 in zebrafish embryos and HIVLTR in cultured human cells, which are repressed at the RNAPII elongation step under non-inducible conditions. Third, NSpt5 directly associates with hsp70-4 chromatin in vivo and increases the occupancy of RNAPII, positive transcription elongation factor b (P-TEFb), histone H3 Lys 4 trimethylation (H3K4Me3), and surprisingly, the negative elongation factor A (NELF-A) at the locus, indicating a direct action of NSpt5 on the elongation repressed locus. Together, these results reveal a dominant activity of NSpt5 to de-repress RNAPII elongation, and suggest that the C-terminus of Spt5 is critical for repressing RNAPII elongation in vivo.

Show MeSH