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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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DSIF does not exert its positive activity on late transcription complexes that are paused downstream. (A) In vitro transcription assays were carried out using P1.0 and pG5MLP as the template, as shown in the lower diagram. After a 40-min pre-incubation step, 60 μM ATP, 10 μM CTP, 1 μM UTP and 5 μCi [α-32P]UTP (800 Ci/mmol) were added to initiate transcription, and reactions were allowed to proceed for 15 to 20 min. In lanes 5 to 7, 7.5 ng of His-DSIF was added after 15 min of initiation/elongation, and reactions were further incubated for 1, 2 and 5 min, respectively. In lanes 8 to 10, a chase experiment was carried out as described for Figure 1C. The incubation time after the first 15-min initiation/elongation is labeled at the top. (B) In vitro transcription assays were carried out as described for panel A, except that 7.5 ng of His-DSIF was added at the indicated time points, as shown in the lower diagram. His-Gal4-VP16 was added together with P1.0 to the reactions in lanes 7 to 12. (C) Schematic representation of the plasmid pG5MLPDG. The template produces transcripts containing two G-free cassettes under the control of the same promoter and GAL4-binding sites as in pG5MLP. The promoter-proximal and -distal G-free cassettes of 84 and 376 bp in length are located 40 and 1522 bp downstream of the transcription start site, respectively. (D) In vitro transcription reaction was carried out using pG5MLPDG as a DNA template with concentrated P1.0. His-DSIF (7.5 ng) and His-Gal4-VP16 (150 ng) were added as indicated. Arrows indicate promoter-proximal (40–124) and -distal (1512–1888) fragments of transcripts. (E) The promoter-proximal and -distal fragments of transcripts in D were quantified using a phosphorimager, and the amounts (in arbitrary units) were illustrated in bars. Quantitative presentation of the ratio between the distal and proximal G-free cassettes was illustrated in the bottom panel.
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Figure 3: DSIF does not exert its positive activity on late transcription complexes that are paused downstream. (A) In vitro transcription assays were carried out using P1.0 and pG5MLP as the template, as shown in the lower diagram. After a 40-min pre-incubation step, 60 μM ATP, 10 μM CTP, 1 μM UTP and 5 μCi [α-32P]UTP (800 Ci/mmol) were added to initiate transcription, and reactions were allowed to proceed for 15 to 20 min. In lanes 5 to 7, 7.5 ng of His-DSIF was added after 15 min of initiation/elongation, and reactions were further incubated for 1, 2 and 5 min, respectively. In lanes 8 to 10, a chase experiment was carried out as described for Figure 1C. The incubation time after the first 15-min initiation/elongation is labeled at the top. (B) In vitro transcription assays were carried out as described for panel A, except that 7.5 ng of His-DSIF was added at the indicated time points, as shown in the lower diagram. His-Gal4-VP16 was added together with P1.0 to the reactions in lanes 7 to 12. (C) Schematic representation of the plasmid pG5MLPDG. The template produces transcripts containing two G-free cassettes under the control of the same promoter and GAL4-binding sites as in pG5MLP. The promoter-proximal and -distal G-free cassettes of 84 and 376 bp in length are located 40 and 1522 bp downstream of the transcription start site, respectively. (D) In vitro transcription reaction was carried out using pG5MLPDG as a DNA template with concentrated P1.0. His-DSIF (7.5 ng) and His-Gal4-VP16 (150 ng) were added as indicated. Arrows indicate promoter-proximal (40–124) and -distal (1512–1888) fragments of transcripts. (E) The promoter-proximal and -distal fragments of transcripts in D were quantified using a phosphorimager, and the amounts (in arbitrary units) were illustrated in bars. Quantitative presentation of the ratio between the distal and proximal G-free cassettes was illustrated in the bottom panel.

Mentions: Concentrated P1.0 fractions were prepared as described previously (33,34). In vitro transcription reactions using the concentrated P1.0 fraction and plasmid DNA templates were carried out as described previously (9,34). Briefly, in reactions using pG5MLP as a template, 12.5 μl reaction mixtures containing 125 ng DNA (32) and the concentrated P1.0 fraction were prepared in the presence or absence of recombinant DSIF and Gal4VP16 in TRX buffer [25 mM Tris–HCl (pH 7.9), 10% (v/v) glycerol, 50 mM KCl, 0.5 mM DTT and 0.5 mM EDTA]. Reactions were incubated for 40 min at 30°C. NTPs and 80 μM 3′-OMe-GTP in TRX buffer were then added, and the mixture was incubated for the indicated times. Where indicated, 1.5 mM each of ATP, UTP and CTP were added and reactions were incubated for an additional period of time. In Figure 3D, pG5MLPDG was used as a template. Transcription reaction was allowed to proceed for 20 min in the presence of 60 μM ATP, 600 μM GTP, 600 μM CTP, 5 μM UTP and 5 μCi of [α-32P]UTP (800 Ci/mmol). G-free RNA fragments derived from transcripts were isolated after RNase T1 treatment, deproteinized, precipitated with ethanol and analyzed using 8% acrylamide denaturing gels, as previously described (4). In Figures 1F and 3E, transcripts were quantified by a phosphorimager (Molecular Dynamics, Storm 860).Figure 1.


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)

DSIF does not exert its positive activity on late transcription complexes that are paused downstream. (A) In vitro transcription assays were carried out using P1.0 and pG5MLP as the template, as shown in the lower diagram. After a 40-min pre-incubation step, 60 μM ATP, 10 μM CTP, 1 μM UTP and 5 μCi [α-32P]UTP (800 Ci/mmol) were added to initiate transcription, and reactions were allowed to proceed for 15 to 20 min. In lanes 5 to 7, 7.5 ng of His-DSIF was added after 15 min of initiation/elongation, and reactions were further incubated for 1, 2 and 5 min, respectively. In lanes 8 to 10, a chase experiment was carried out as described for Figure 1C. The incubation time after the first 15-min initiation/elongation is labeled at the top. (B) In vitro transcription assays were carried out as described for panel A, except that 7.5 ng of His-DSIF was added at the indicated time points, as shown in the lower diagram. His-Gal4-VP16 was added together with P1.0 to the reactions in lanes 7 to 12. (C) Schematic representation of the plasmid pG5MLPDG. The template produces transcripts containing two G-free cassettes under the control of the same promoter and GAL4-binding sites as in pG5MLP. The promoter-proximal and -distal G-free cassettes of 84 and 376 bp in length are located 40 and 1522 bp downstream of the transcription start site, respectively. (D) In vitro transcription reaction was carried out using pG5MLPDG as a DNA template with concentrated P1.0. His-DSIF (7.5 ng) and His-Gal4-VP16 (150 ng) were added as indicated. Arrows indicate promoter-proximal (40–124) and -distal (1512–1888) fragments of transcripts. (E) The promoter-proximal and -distal fragments of transcripts in D were quantified using a phosphorimager, and the amounts (in arbitrary units) were illustrated in bars. Quantitative presentation of the ratio between the distal and proximal G-free cassettes was illustrated in the bottom panel.
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Figure 3: DSIF does not exert its positive activity on late transcription complexes that are paused downstream. (A) In vitro transcription assays were carried out using P1.0 and pG5MLP as the template, as shown in the lower diagram. After a 40-min pre-incubation step, 60 μM ATP, 10 μM CTP, 1 μM UTP and 5 μCi [α-32P]UTP (800 Ci/mmol) were added to initiate transcription, and reactions were allowed to proceed for 15 to 20 min. In lanes 5 to 7, 7.5 ng of His-DSIF was added after 15 min of initiation/elongation, and reactions were further incubated for 1, 2 and 5 min, respectively. In lanes 8 to 10, a chase experiment was carried out as described for Figure 1C. The incubation time after the first 15-min initiation/elongation is labeled at the top. (B) In vitro transcription assays were carried out as described for panel A, except that 7.5 ng of His-DSIF was added at the indicated time points, as shown in the lower diagram. His-Gal4-VP16 was added together with P1.0 to the reactions in lanes 7 to 12. (C) Schematic representation of the plasmid pG5MLPDG. The template produces transcripts containing two G-free cassettes under the control of the same promoter and GAL4-binding sites as in pG5MLP. The promoter-proximal and -distal G-free cassettes of 84 and 376 bp in length are located 40 and 1522 bp downstream of the transcription start site, respectively. (D) In vitro transcription reaction was carried out using pG5MLPDG as a DNA template with concentrated P1.0. His-DSIF (7.5 ng) and His-Gal4-VP16 (150 ng) were added as indicated. Arrows indicate promoter-proximal (40–124) and -distal (1512–1888) fragments of transcripts. (E) The promoter-proximal and -distal fragments of transcripts in D were quantified using a phosphorimager, and the amounts (in arbitrary units) were illustrated in bars. Quantitative presentation of the ratio between the distal and proximal G-free cassettes was illustrated in the bottom panel.
Mentions: Concentrated P1.0 fractions were prepared as described previously (33,34). In vitro transcription reactions using the concentrated P1.0 fraction and plasmid DNA templates were carried out as described previously (9,34). Briefly, in reactions using pG5MLP as a template, 12.5 μl reaction mixtures containing 125 ng DNA (32) and the concentrated P1.0 fraction were prepared in the presence or absence of recombinant DSIF and Gal4VP16 in TRX buffer [25 mM Tris–HCl (pH 7.9), 10% (v/v) glycerol, 50 mM KCl, 0.5 mM DTT and 0.5 mM EDTA]. Reactions were incubated for 40 min at 30°C. NTPs and 80 μM 3′-OMe-GTP in TRX buffer were then added, and the mixture was incubated for the indicated times. Where indicated, 1.5 mM each of ATP, UTP and CTP were added and reactions were incubated for an additional period of time. In Figure 3D, pG5MLPDG was used as a template. Transcription reaction was allowed to proceed for 20 min in the presence of 60 μM ATP, 600 μM GTP, 600 μM CTP, 5 μM UTP and 5 μCi of [α-32P]UTP (800 Ci/mmol). G-free RNA fragments derived from transcripts were isolated after RNase T1 treatment, deproteinized, precipitated with ethanol and analyzed using 8% acrylamide denaturing gels, as previously described (4). In Figures 1F and 3E, transcripts were quantified by a phosphorimager (Molecular Dynamics, Storm 860).Figure 1.

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