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DSIF contributes to transcriptional activation by DNA-binding activators by preventing pausing during transcription elongation.

Zhu W, Wada T, Okabe S, Taneda T, Yamaguchi Y, Handa H - Nucleic Acids Res. (2007)

Bottom Line: The presence of DSIF reduced pausing, thereby supporting Gal4-VP16-mediated activation.We found that DSIF exerts its positive effects within a short time-frame from initiation to elongation, and that NELF does not affect the positive regulatory function of DSIF.Together, these results provide evidence that higher-level transcription has a stronger requirement for DSIF, and that DSIF contributes to efficient transcriptional activation by preventing RNAPII pausing during transcription elongation.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioscience and Biotechnology and Integrated Research Institute, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.

ABSTRACT
The transcription elongation factor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) regulates RNA polymerase II (RNAPII) processivity by promoting, in concert with negative elongation factor (NELF), promoter-proximal pausing of RNAPII. DSIF is also reportedly involved in transcriptional activation. However, the role of DSIF in transcriptional activation by DNA-binding activators is unclear. Here we show that DSIF acts cooperatively with a DNA-binding activator, Gal4-VP16, to promote transcriptional activation. In the absence of DSIF, Gal4-VP16-activated transcription resulted in frequent pausing of RNAPII during elongation in vitro. The presence of DSIF reduced pausing, thereby supporting Gal4-VP16-mediated activation. We found that DSIF exerts its positive effects within a short time-frame from initiation to elongation, and that NELF does not affect the positive regulatory function of DSIF. Knockdown of the gene encoding the large subunit of DSIF, human Spt5 (hSpt5), in HeLa cells reduced Gal4-VP16-mediated activation of a reporter gene, but had no effect on expression in the absence of activator. Together, these results provide evidence that higher-level transcription has a stronger requirement for DSIF, and that DSIF contributes to efficient transcriptional activation by preventing RNAPII pausing during transcription elongation.

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NELF does not affect the positive activity of DSIF. (A) Silver staining of purified Flag-NELF. (B) In vitro transcription assays were carried out as described for Figure 1E, except that His-DSIF was added to reactions of lanes 2 to 4 and 8 to 10; Flag-NELF was added to the reactions of lanes 3 to 6 and 9 to 12 and DRB was added to the reactions of lanes 7 to 12. Numbers on the left indicate the positions of markers (nucleotides). (C) Western blot analysis of CDK9, RNAPII and CDK8 in the concentrated P1.0 fraction either mock depleted or depleted with anti-CDK9 antibody. (D) In vitro transcription assays were carried out as described in Figure 1E, except that P-TEFb- or mock-immunodepleted concentrated P1.0 fraction was used. P-TEFb was added back prior to incubation as indicated. (E) In vitro transcription assays were carried out using P-TEFb-depleted concentrated P1.0 fraction, His-DSIF and pG5MLP as a template. His-DSIF and Flag-NELF were combined with the mixture during the pre-incubation, while P-TEFb was added 2 min after addition of nucleotides, as shown in the diagram.
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Figure 4: NELF does not affect the positive activity of DSIF. (A) Silver staining of purified Flag-NELF. (B) In vitro transcription assays were carried out as described for Figure 1E, except that His-DSIF was added to reactions of lanes 2 to 4 and 8 to 10; Flag-NELF was added to the reactions of lanes 3 to 6 and 9 to 12 and DRB was added to the reactions of lanes 7 to 12. Numbers on the left indicate the positions of markers (nucleotides). (C) Western blot analysis of CDK9, RNAPII and CDK8 in the concentrated P1.0 fraction either mock depleted or depleted with anti-CDK9 antibody. (D) In vitro transcription assays were carried out as described in Figure 1E, except that P-TEFb- or mock-immunodepleted concentrated P1.0 fraction was used. P-TEFb was added back prior to incubation as indicated. (E) In vitro transcription assays were carried out using P-TEFb-depleted concentrated P1.0 fraction, His-DSIF and pG5MLP as a template. His-DSIF and Flag-NELF were combined with the mixture during the pre-incubation, while P-TEFb was added 2 min after addition of nucleotides, as shown in the diagram.

Mentions: It has been established that DSIF negatively regulates transcription elongation by acting in concert with NELF (7,8). In contrast, how DSIF stimulates transcription elongation is largely unknown except that P-TEFb-mediated phosphorylation of the Spt5 subunit of DSIF is responsible for converting DSIF from a repressor to a positive regulator (9). One possible model is that promoter-proximal pausing leads to processive elongation thereafter, possibly serving as a checkpoint for this subsequent process. We therefore examined the roles of NELF and P-TEFb in the positive activity of DSIF. We purified Flag-epitope-tagged NELF (FLAG-NELF) (Figure 4A) from a Flag-NELF-E-expressing HeLa cell line derivative, and added it to the transcription assay (Figure 4B). There was no appreciable effect of addition of NELF on DSIF-activated transcription, or basal transcription levels in the absence of DSIF (Figure 4B, lanes 3 to 6). In the presence of DRB, an inhibitor of the P-TEFb kinase, the positive activity of DSIF was significantly inhibited (Figure 4B, lanes 2 and 8), suggesting that P-TEFb is critical for the positive regulatory effect of DSIF. Addition of NELF resulted in a much stronger inhibitory effect and the generation of transcripts of less than 150 nt (Figure 4B, lanes 9 and 10). Based on the results of previous studies, this effect is likely due to promoter-proximal pausing induced by NELF and DSIF upon inhibition of P-TEFb activity by DRB (4,36). Consistent with the previously published results (36), NELF was unable to induce such pausing in the absence of DSIF, even in the presence of DRB (Figure 4B, lanes 5, 6, 11 and 12). These results indicated that the positive activity of DSIF is dependent on P-TEFb and independent of NELF.Figure 4.


DSIF contributes to transcriptional activation by DNA-binding activators by preventing pausing during transcription elongation.

Zhu W, Wada T, Okabe S, Taneda T, Yamaguchi Y, Handa H - Nucleic Acids Res. (2007)

NELF does not affect the positive activity of DSIF. (A) Silver staining of purified Flag-NELF. (B) In vitro transcription assays were carried out as described for Figure 1E, except that His-DSIF was added to reactions of lanes 2 to 4 and 8 to 10; Flag-NELF was added to the reactions of lanes 3 to 6 and 9 to 12 and DRB was added to the reactions of lanes 7 to 12. Numbers on the left indicate the positions of markers (nucleotides). (C) Western blot analysis of CDK9, RNAPII and CDK8 in the concentrated P1.0 fraction either mock depleted or depleted with anti-CDK9 antibody. (D) In vitro transcription assays were carried out as described in Figure 1E, except that P-TEFb- or mock-immunodepleted concentrated P1.0 fraction was used. P-TEFb was added back prior to incubation as indicated. (E) In vitro transcription assays were carried out using P-TEFb-depleted concentrated P1.0 fraction, His-DSIF and pG5MLP as a template. His-DSIF and Flag-NELF were combined with the mixture during the pre-incubation, while P-TEFb was added 2 min after addition of nucleotides, as shown in the diagram.
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Figure 4: NELF does not affect the positive activity of DSIF. (A) Silver staining of purified Flag-NELF. (B) In vitro transcription assays were carried out as described for Figure 1E, except that His-DSIF was added to reactions of lanes 2 to 4 and 8 to 10; Flag-NELF was added to the reactions of lanes 3 to 6 and 9 to 12 and DRB was added to the reactions of lanes 7 to 12. Numbers on the left indicate the positions of markers (nucleotides). (C) Western blot analysis of CDK9, RNAPII and CDK8 in the concentrated P1.0 fraction either mock depleted or depleted with anti-CDK9 antibody. (D) In vitro transcription assays were carried out as described in Figure 1E, except that P-TEFb- or mock-immunodepleted concentrated P1.0 fraction was used. P-TEFb was added back prior to incubation as indicated. (E) In vitro transcription assays were carried out using P-TEFb-depleted concentrated P1.0 fraction, His-DSIF and pG5MLP as a template. His-DSIF and Flag-NELF were combined with the mixture during the pre-incubation, while P-TEFb was added 2 min after addition of nucleotides, as shown in the diagram.
Mentions: It has been established that DSIF negatively regulates transcription elongation by acting in concert with NELF (7,8). In contrast, how DSIF stimulates transcription elongation is largely unknown except that P-TEFb-mediated phosphorylation of the Spt5 subunit of DSIF is responsible for converting DSIF from a repressor to a positive regulator (9). One possible model is that promoter-proximal pausing leads to processive elongation thereafter, possibly serving as a checkpoint for this subsequent process. We therefore examined the roles of NELF and P-TEFb in the positive activity of DSIF. We purified Flag-epitope-tagged NELF (FLAG-NELF) (Figure 4A) from a Flag-NELF-E-expressing HeLa cell line derivative, and added it to the transcription assay (Figure 4B). There was no appreciable effect of addition of NELF on DSIF-activated transcription, or basal transcription levels in the absence of DSIF (Figure 4B, lanes 3 to 6). In the presence of DRB, an inhibitor of the P-TEFb kinase, the positive activity of DSIF was significantly inhibited (Figure 4B, lanes 2 and 8), suggesting that P-TEFb is critical for the positive regulatory effect of DSIF. Addition of NELF resulted in a much stronger inhibitory effect and the generation of transcripts of less than 150 nt (Figure 4B, lanes 9 and 10). Based on the results of previous studies, this effect is likely due to promoter-proximal pausing induced by NELF and DSIF upon inhibition of P-TEFb activity by DRB (4,36). Consistent with the previously published results (36), NELF was unable to induce such pausing in the absence of DSIF, even in the presence of DRB (Figure 4B, lanes 5, 6, 11 and 12). These results indicated that the positive activity of DSIF is dependent on P-TEFb and independent of NELF.Figure 4.

Bottom Line: The presence of DSIF reduced pausing, thereby supporting Gal4-VP16-mediated activation.We found that DSIF exerts its positive effects within a short time-frame from initiation to elongation, and that NELF does not affect the positive regulatory function of DSIF.Together, these results provide evidence that higher-level transcription has a stronger requirement for DSIF, and that DSIF contributes to efficient transcriptional activation by preventing RNAPII pausing during transcription elongation.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioscience and Biotechnology and Integrated Research Institute, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.

ABSTRACT
The transcription elongation factor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) regulates RNA polymerase II (RNAPII) processivity by promoting, in concert with negative elongation factor (NELF), promoter-proximal pausing of RNAPII. DSIF is also reportedly involved in transcriptional activation. However, the role of DSIF in transcriptional activation by DNA-binding activators is unclear. Here we show that DSIF acts cooperatively with a DNA-binding activator, Gal4-VP16, to promote transcriptional activation. In the absence of DSIF, Gal4-VP16-activated transcription resulted in frequent pausing of RNAPII during elongation in vitro. The presence of DSIF reduced pausing, thereby supporting Gal4-VP16-mediated activation. We found that DSIF exerts its positive effects within a short time-frame from initiation to elongation, and that NELF does not affect the positive regulatory function of DSIF. Knockdown of the gene encoding the large subunit of DSIF, human Spt5 (hSpt5), in HeLa cells reduced Gal4-VP16-mediated activation of a reporter gene, but had no effect on expression in the absence of activator. Together, these results provide evidence that higher-level transcription has a stronger requirement for DSIF, and that DSIF contributes to efficient transcriptional activation by preventing RNAPII pausing during transcription elongation.

Show MeSH
Related in: MedlinePlus