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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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NSpt5 directly interacts with the hsp70-4 chromatin in vivo.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 Flag antibody over the IgG control. Error bars represent S.E.M. of duplicate measurements from two independent experiments.
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pone-0006918-g005: NSpt5 directly interacts with the hsp70-4 chromatin in vivo.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 Flag antibody over the IgG control. Error bars represent S.E.M. of duplicate measurements from two independent experiments.

Mentions: To determine whether NSpt5 directly or indirectly causes the enhanced RNAPII occupancy on the hsp70-4 chromatin in vivo, we examined the chromatin occupancy of NSpt5, in comparison with Spt5. ChIP was carried out using the anti-FLAG antibody in F-spt5- or FNspt5- expressing embryos. F-NSpt5 was found to associate with the hsp70-4 chromatin (Figure 5). However, F-Spt5 did not exhibit any detectable association with the hsp70-4 chromatin (Figure 5). This observation suggests that NSpt5 directly interacts with the hsp70-4 chromatin to cause the increased RNAPII occupancy at the locus. The fact that the exogenously provided Spt5 has no association with the hsp70-4 chromatin suggests that NSpt5 has better access to the hsp70-4 chromatin than the full length Spt5.


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)

NSpt5 directly interacts with the hsp70-4 chromatin in vivo.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 Flag antibody over the IgG control. Error bars represent S.E.M. of duplicate measurements from two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2735033&req=5

pone-0006918-g005: NSpt5 directly interacts with the hsp70-4 chromatin in vivo.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 Flag antibody over the IgG control. Error bars represent S.E.M. of duplicate measurements from two independent experiments.
Mentions: To determine whether NSpt5 directly or indirectly causes the enhanced RNAPII occupancy on the hsp70-4 chromatin in vivo, we examined the chromatin occupancy of NSpt5, in comparison with Spt5. ChIP was carried out using the anti-FLAG antibody in F-spt5- or FNspt5- expressing embryos. F-NSpt5 was found to associate with the hsp70-4 chromatin (Figure 5). However, F-Spt5 did not exhibit any detectable association with the hsp70-4 chromatin (Figure 5). This observation suggests that NSpt5 directly interacts with the hsp70-4 chromatin to cause the increased RNAPII occupancy at the locus. The fact that the exogenously provided Spt5 has no association with the hsp70-4 chromatin suggests that NSpt5 has better access to the hsp70-4 chromatin than the full length Spt5.

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