Structure and Function of the Human Transcription Elongation Factor DSIF

Abstract

5,6-Dichloro-1-␤-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 DRB sensitivity-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.The nucleoside analog DRB 1 is a classic inhibitor of transcription elongation by pol II (reviewed in Ref. 1). 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 (2-4). Therefore, one or more factors apart from GTFs and pol II appear to be involved in DRB-sensitive transcription (1). Such putative factors must play general roles in pol II transcription in vivo because DRB affects most of the class II genes (4 -6).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 (7-12). 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 (3). DSIF is composed of 160-and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4 (3, 13-15). DSIF/Spt4-Spt5 genetically and physically interacts with pol II, and thus may directly regulate pol II processivity (3, 16).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 (3). Higher doses of DSIF, however, repress transcription even in the absence of DRB (3). In contrast, under limiting concentrations of NTPs, DSIF stimulates transcription elongation in the absence of DRB (3). Genetic a...

Interactions between Fission Yeast Cdk9, Its Cyclin Partner Pch1, and mRNA Capping Enzyme Pct1 Suggest an Elongation Checkpoint for mRNA Quality Control

“…13 and citations therein). Expression of a dominant negative version of Spt5 in human cells results in stimulation of transcription from the HIV-LTR and other promoters, implying that wild-type human Spt5 is a negative regulator of transcription (10). Depletion of Cdk9 or cyclin T in Caenorhabditis elegans embryos results in a general shutoff of pol II transcription (16).…”

“…Spt5 and Tat cooperate to regulate HIV transcription elongation. Spt5 and its binding partner Spt4 comprise the transcription elongation factor DSIF (DRB sensitivity-inducing factor) (9,10). DSIF binds to pol II and, in conjunction with NELF, represses elongation at promoter-proximal positions (Ref.…”

NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly

“…Although few targets of NELF have been defined, NELF is known to influence expression of several genes that are regulated by Pol II stalling, including Drosophila Hsp70, mammalian junB, and HIV RNA (Fujinaga et al 1998;Wu et al 2003;Aida et al 2006;Zhang et al 2007). NELF requires DSIF to interact with Pol II, and the two factors together inhibit transcription elongation in vitro by increasing the duration of intrinsic pauses (Yamaguchi et al 1999;Renner et al 2001;Palangat et al 2005;Cheng and Price 2007). Importantly, NELF is present at the uninduced Hsp70 promoter, but dissociates upon gene activation, and depletion of NELF reduces Pol II stalling at Hsp70 both in vitro and in vivo (Wu et al 2003(Wu et al , 2005.…”

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

“…Wada et al (1998a) estimated by gel filtration a native size of >300 kDa and suggested that the Spt5-Spt4 complex is a multimer of the 165-kDa Spt5 protein. Deletion analysis delineated a central Spt4-binding domain on human Spt5 from amino acids 176-313 (Yamaguchi et al 1999;Ivanov et al 2000), but the methods employed (GST pull-downs, co-immunoprecipitations) did not reveal the stoichiometry or size of the complex.…”

“…www.rnajournal.org 1243 (Yamaguchi et al 1999;Ivanov et al 2000). The apparent resistance of this segment of the S. pombe Spt5-Spt4 complex to trypsin cleavage suggests that it comprises a folded domain.…”