Structure and Function of the Human Transcription Elongation Factor DSIF

Abstract

5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRBsensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160- and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo. 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRBsensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160- and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo. 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole polymerase II DRB sensitivity-inducing factor general transcription factor positive transcription elongation factor b C-terminal domain amino acids hemagglutin polyacrylamide gel electrophoresis glutathioneS-transferase The nucleoside analog DRB1 is a classic inhibitor of transcription elongation by pol II (reviewed in Ref. 1Yamaguchi Y. Wada T. Handa T. Genes Cells. 1998; 3: 9-15Crossref PubMed Scopus (65) Google Scholar). Although it has been used for more than three decades, its mode of action has long been a mystery. DRB is unique in that it shows no effect on transcription reconstituted with purified general transcription factors (GTFs) and pol II, whereas it potently represses transcription in cruder systems or in vivo (2Chodosh L.A. Fire A. Samuels M. Sharp P.A. J. Biol. Chem. 1989; 264: 2250-2257Abstract Full Text PDF PubMed Google Scholar, 3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar, 4Zandomeni R. Mittleman B. Bunick D. Ackerman S. Weinmann R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3167-3170Crossref PubMed Scopus (75) Google Scholar). Therefore, one or more factors apart from GTFs and pol II appear to be involved in DRB-sensitive transcription (1Yamaguchi Y. Wada T. Handa T. Genes Cells. 1998; 3: 9-15Crossref PubMed Scopus (65) Google Scholar). Such putative factors must play general roles in pol II transcription in vivo because DRB affects most of the class II genes (4Zandomeni R. Mittleman B. Bunick D. Ackerman S. Weinmann R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3167-3170Crossref PubMed Scopus (75) Google Scholar, 5Marshall N.F. Price D.H. Mol. Cell. Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (239) Google Scholar, 6Sehgal P.B. Darnell Jr., J.E. Tamm I. Cell. 1976; 9: 473-480Abstract Full Text PDF PubMed Scopus (152) Google Scholar). Recently, we and others have identified two elongation factors essential for DRB-sensitive transcription. One of them, positive transcription elongation factor b (P-TEFb), is a protein kinase that phosphorylates the pol II C-terminal domain (CTD) in a DRB-sensitive fashion (7Mancebo H.S. Lee G. Flygare J. Tomassini J. Luu P. Zhu Y. Peng J. Blau C. Hazuda D. Price D. Flores O. Genes Dev. 1997; 11: 2633-2644Crossref PubMed Scopus (478) Google Scholar, 8Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 9Marshall N.F. Price D.H. J. Biol. Chem. 1995; 270: 12335-12338Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 10Peng J. Zhu Y.T. Milton J. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (447) Google Scholar, 11Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1046) Google Scholar, 12Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (609) Google Scholar). The CTD phosphorylation likely plays a pivotal role in pol II elongation and may be relevant to the P-TEFb function. The other, DSIF, has been purified from HeLa cell nuclear extract, based on its ability to induce DRB-sensitivity in vitro (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). DSIF is composed of 160- and 14-kDa subunits, which are homologs of theSaccharomyces cerevisiae transcription factors Spt5 and Spt4 (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar, 13Hartzog G.A. Basrai M.A. Ricupero-Hovasse S.L. Hieter P. Winston F. Mol. Cell. Biol. 1996; 16: 2848-2856Crossref PubMed Scopus (34) Google Scholar, 14Malone E.A. Fassler J.S. Winston F. Mol. Gen. Genet. 1993; 237: 449-459Crossref PubMed Scopus (54) Google Scholar, 15Swanson M.S. Malone E.A. Winston F. Mol. Cell. Biol. 1991; 11: 3009-3019Crossref PubMed Scopus (117) Google Scholar). DSIF/Spt4-Spt5 genetically and physically interacts with pol II, and thus may directly regulate pol II processivity (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar, 16Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Crossref PubMed Scopus (371) Google Scholar). The function of DSIF in the absence of DRB remains obscure. Small amounts of DSIF, when added back to a DSIF-depleted transcription system, repress transcription only in the presence of DRB, without affecting transcription in its absence (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). Higher doses of DSIF, however, repress transcription even in the absence of DRB (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). In contrast, under limiting concentrations of NTPs, DSIF stimulates transcription elongation in the absence of DRB (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). Genetic analysis in yeast also suggests a stimulatory role for Spt4-Spt5 under low NTP concentrations in vivo (16Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Crossref PubMed Scopus (371) Google Scholar). DSIF p160 has many distinctive structural features (Refs. 1Yamaguchi Y. Wada T. Handa T. Genes Cells. 1998; 3: 9-15Crossref PubMed Scopus (65) Google Scholar and 3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar; see Fig. 1 A). The N-terminal region is highly acidic, and several hexapeptide repeats are found at the C terminus. In the central domain, there are four stretches with significant similarity to NusG, a prokaryotic transcription termination and anti-termination factor (17Greenblatt J. Nodwell J.R. Mason S.W. Nature. 1993; 364: 401-406Crossref PubMed Scopus (206) Google Scholar, 18Mogridge J. Mah T.F. Greenblatt J. Genes Dev. 1995; 9: 2831-2845Crossref PubMed Scopus (85) Google Scholar, 19Sullivan S.L. Gottesman M.E. Cell. 1992; 68: 989-994Abstract Full Text PDF PubMed Scopus (147) Google Scholar). The corresponding region of NusG has recently been found to share homology with a class of proteins involved in translation and is termed the KOW motif, though its function is unknown (20Kyrpides N.C. Woese C.R. Ouzounis C.A. Trends Biochem. Sci. 1996; 21: 425-426Abstract Full Text PDF PubMed Scopus (111) Google Scholar). We have named the four stretches KOW1 to 4, respectively (see Fig.1 A). In this report, we have characterized the structure and function of DSIF p160. We show that p160 is a nuclear protein that forms a stable complex with p14 and interacts with the pol II largest subunit. In addition, we map the regions of p160 that are involved in interactions with p14 and pol II and those that are essential for DSIF activity. Finally, we isolate a p160 mutant that acts in a dominant-negative manner and use this mutant to present evidence that DSIF is a negative regulator of transcription in vivo. For expression of recombinant proteins, the following plasmids were constructed in the expression vector pET-14b (Novagen) by subcloning from parent vectors described elsewhere (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar): pET-p160Δacidic, which lacks amino acids (aa) 1–175 of p160; pET-p160Δp14BD, which lacks aa 176–314 of p160; pET-p160ΔNusG, which lacks aa 314–516 of p160; and pET-p160Δrepeat, which lacks aa 758–936 of p160. For expression of p160ΔKOW1 and ΔKOW2, which lack aa 421–447 and aa 473–499 of p160, respectively, a polymerase chain reaction-based overlap extension technique was employed (21Horton R.M. Ho S.N. Pullen J.K. Hunt H.D. Cai Z. Pease L.R. Methods Enzymol. 1993; 217: 270-279Crossref PubMed Scopus (426) Google Scholar). Forward and reverse mutagenic primers encompassing the deletion points were used and the amplified fragments cloned into pET-14b, generating pET-p160ΔKOW1 and pET-p160ΔKOW2. For expression of GST-p14, a fragment of pBS-DSIFp14 was subcloned into pGEX-5X-3 (Amersham Pharmacia Biotech) to generate pGEX-p14. To construct pCMV-FLAGp160 and pCMV-FLAGp160ΔNusG, fragments of pBS-FLAGp160 and pBS-FLAGp160ΔNusG were inserted into pCAGGS (22Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4561) Google Scholar). To construct pCMV-HAp14, p14 full-length cDNA was inserted into pCHA, a pCAGGS derivative that contains a sequence encoding HA epitope YPYDVPDYA. Histidine-tagged p160 and p14 were expressed inEscherichia coli strain BL21 (DE3) transformed with pET-DSIFp160 or pET-DSIFp14 (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). Cells were harvested and lysed by sonication, the lysates were cleared by centrifugation and filtration, and r-p160 and r-p14 were purified from the supernatants by Ni-affinity column chromatography according to the instructions of the manufacturer (Novagen). To further purify the recombinant proteins, the purified fractions were subjected to SDS-PAGE, and the full-length proteins were recovered from a gel slice. The proteins were then precipitated with acetone, denatured with 6 m guanidine-HCl, and renatured by dialysis against 10 mm HEPES (pH 7.9), 10% glycerol, 50 mm KCl, 0.2 mm EDTA, 0.5 mm dithiothreitol as described (23Wada T. Watanabe H. Usuda Y. Handa H. J. Virol. 1991; 65: 557-564Crossref PubMed Google Scholar). To prepare in vitro translated p160 and its either or was with of which the p160 to the The plasmids were then with either or RNA by translation a in the presence of pCMV-FLAGp160 and were into either or in the were lysed in of mm (pH 7.9), mm and cleared by The lysates were with of for at and then with of protein (Amersham Pharmacia Biotech) for 1 at three with the were with 50 of and by and were with and respectively, and were (Amersham Pharmacia or protein was to of (Amersham Pharmacia The were with in vitro translated p160 or its in of mm (pH 7.9), mm 1 mm for 1 at three with the were with 50 of and by and HeLa cell nuclear and the column fractions and were as described (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). The which a (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google was were as described (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). In Fig. r-p14 or a derivative and were for at were by of NTP and 50 of and of or DRB 50 a were and the by In Fig. HeLa nuclear and mutant were for the and as of a p160 mutant that represses DSIF activity in a dominant-negative manner in and amounts of protein were with HeLa nuclear and for the and transcription were as shown at the The were a The by DRB was and shown HeLa were in essential with 10% and of 10 of of various amounts of pCMV-FLAGp160ΔNusG, and the vector were by a into on were 6 The were harvested and were The were by protein and expressed as In we the of a cDNA encoding the of DSIF (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). The and amino of p160 are shown in Fig. 1 A. was no sequence of the putative translation and the sequence the sequence M. J. Biol. 1989; 108: PubMed Scopus Google there was no in To its the cDNA was and translated in vitro, and the of the was with that of p160 purified from HeLa nuclear and by phosphorylation with kinase II 1 The phosphorylation the of p160 the of the two we that the cDNA full-length p160. have of a of S. cerevisiae K. G. 1996; PubMed Scopus Google Scholar, M. G. H. 1997; PubMed Scopus Google Scholar). The p160 cDNA that we have cloned is to the amino which we and M. G. H. 1997; PubMed Scopus Google are from that by K. G. 1996; PubMed Scopus Google two because of analysis of p160 from several a expressed of a with the cDNA A). p160 putative nuclear 1 we the of p160. vectors p160 and p14 were into HeLa and expression was by is in the G.A. Basrai M.A. Ricupero-Hovasse S.L. Hieter P. Winston F. Mol. Cell. Biol. 1996; 16: 2848-2856Crossref PubMed Scopus (34) Google Scholar). Yamaguchi and H. shown in was also in the of of with the role of p160 as a general transcription elongation Spt5 genetically and physically interacts with Spt4 in yeast (16Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Crossref PubMed Scopus (371) Google Scholar, M.S. Winston F. 1992; PubMed Google Scholar). In addition, p160 was with p14 several of DSIF (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). is likely that p160 with To we expressed and in HeLa either or in and with shown in was with vivo. The interaction was in column or a column was with in and p160 under various with the p160 was and of p160 to the whereas no to the column was and The interaction was by the presence of and 1 m p14 contains a putative G.A. Basrai M.A. Ricupero-Hovasse S.L. Hieter P. Winston F. Mol. Cell. Biol. 1996; 16: 2848-2856Crossref PubMed Scopus (34) Google we the effect of and a of a under conditions, p160 to that the is required for the We the region of p160 involved in forms of p160 were in vitro, and interactions with were The C-terminal of p160 was for the interaction and and of the identified a region of aa for aa of p160 also to p14 the domain is aa We to identify regions for DSIF activity. We constructed a of p160 various structural (see and were expressed in coli and and the of the recombinant proteins was by either or with against the C of p160 amounts of were for DRB sensitivity-inducing activity protein to the of (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). in with transcription to a sensitive to DRB as previously (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). DRB-sensitivity and proteins also the of in the absence of DRB. In contrast, no effect and The in and were The the and the regions of p160, appear to be for DSIF activity. physically interacts with pol II (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar, 16Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Crossref PubMed Scopus (371) Google Scholar). In addition, Spt5 genetically interacts with the two largest of pol II in yeast (16Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Crossref PubMed Scopus (371) Google Scholar). We to which of pol II is involved in interaction with p160. II purified from HeLa cell nuclear was by SDS-PAGE, to and with p160. shown in Fig. a of which with the largest of pol II, was p160 also with protein which was the we that p160 interacts with pol II its largest pol We the region of p160 involved in pol II forms of p160 were in vitro, and with purified pol II and The were and the presence of p160 was shown in the C-terminal deletion of p160 the interaction One role for the KOW is interaction with RNA (20Kyrpides N.C. Woese C.R. Ouzounis C.A. Trends Biochem. Sci. 1996; 21: 425-426Abstract Full Text PDF PubMed Scopus (111) Google Scholar). however, that aa of p160, N-terminal region in to the KOW is for pol II aa of p160, which contains four of the KOW also to pol II of deletion described are in DRB is a it is to the role of DSIF in the absence of DRB. of DSIF to have been to in vitro and there is no evidence its role in vivo. a we to isolate a dominant-negative mutant of p160. We that p160 that complex with but have no DSIF in a dominant-negative We employed one and its dominant-negative effect in amounts of the mutant protein were with HeLa nuclear for and then transcription were in the presence or absence of DRB A). of of the mutant DRB-sensitivity of the nuclear and when the was to DRB was The nuclear used contains of p160 protein (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google Scholar). to of the p160 mutant and a were to most of the is likely that this p160 is by the mutant to DSIF complex with also that the p160 mutant is because of but because a function of p160 the region has been we used to the effect of DSIF on transcription in vivo. plasmids were used in which expression of the is by the the long the and the promoters a but no sequence and are to by of The is by and (23Wada T. Watanabe H. Usuda Y. Handa H. J. Virol. 1991; 65: 557-564Crossref PubMed Google Scholar, C. M. F. Watanabe H. J. Handa H. 1996; PubMed Scopus Google the long is by and K.A. Biochem. PubMed Scopus Google the is by the and M. K.A. G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and the is by and N. Nature. PubMed Scopus Google Scholar). One of plasmids was with amounts of a into HeLa The of added was to 10 with the vector expression to in a of the used A). The were for expression of the with the of the p160 that the and the plasmids were and expressed in of the as by and of the mutant protein were are with that at which the dominant-negative effect is in vitro that DSIF represses transcription from promoters in and of the DSIF activity to transcription. of deletion are in Fig. of p160 to with p14 and pol II were to domains the region and the KOW interaction was shown to be by 1 m or we interaction the this region is interactions may to the stable interaction p160 and interaction for II interaction because p160 the domain to pol II The C-terminal were for DSIF vitro in however, has that deletion of the C-terminal of of M.S. Malone E.A. Winston F. Mol. Cell. Biol. 1991; 11: 3009-3019Crossref PubMed Scopus (117) Google Scholar). is that the C-terminal repeats play roles in vivo. this is in and and has many phosphorylation for several protein DSIF activity may be by phosphorylation in vivo M. G. H. 1997; PubMed Scopus Google Scholar). the most of the was required for DSIF activity. deletion of either the the domain, or one of the two N-terminal KOW p160 function. however, we to the region or the KOW may be involved in interactions with In this we that DSIF acts as a negative at on in vivo. and yeast of the DSIF subunits, have been identified as of of or genes in Ref. F. of a analysis of and transcription factors of S.L. Scholar). the sequence is inserted in the region of a the transcription transcription from the and with transcription of the in the transcription from the of this is that Spt4-Spt5 transcription from the and transcription of the We the that DSIF also as a positive regulator on promoters that we have In Fig. 4, p160 and the mutant only DRB but transcription in the absence of DRB. In are at the We that the DRB sensitivity-inducing activity and the activity described previously (3Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (561) Google are of DSIF, and are to Recently, we found that DSIF and P-TEFb and on pol II In the absence of P-TEFb DSIF represses transcription of the presence or absence of DRB T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. J. 1998; PubMed Scopus Google Scholar). in DRB P-TEFb from the negative effect of DSIF (1Yamaguchi Y. Wada T. Handa T. Genes Cells. 1998; 3: 9-15Crossref PubMed Scopus (65) Google Scholar, F. B. PubMed Google Scholar). The for DRB in the by DSIF may with the of DSIF to In of a to M.S. Malone E.A. Winston F. Mol. Cell. Biol. 1991; 11: 3009-3019Crossref PubMed Scopus (117) Google Scholar). The expression of Spt5 is for its function. from to We in of that and effect of We used a dominant-negative mutant of p160 to the in vivo function of DSIF in this was that the expression was in with the of DSIF activity. that the DSIF represses transcription from the the DSIF only of the the dominant-negative effect be is with the that DSIF affects transcription of the class II as from its in DRB DSIF are also in as cell and in 1Yamaguchi Y. Wada T. Handa T. Genes Cells. 1998; 3: 9-15Crossref PubMed Scopus (65) Google Scholar and F. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). The dominant-negative mutant of p160 be a to DSIF in We M. A. T. J. A. and K. for D. Price for T. for with the and C. for with