We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.

Previous characterization of the Saccharomyces cerevisiae Spt4, Spt5, and Spt6 proteins suggested that these proteins act as transcription factors that modify chromatin structure. In this work, we report new genetic and biochemical studies of Spt4, Spt5, and Spt6 that reveal a role for these factors in transcription elongation. We have isolated conditional mutations in SPT5 that can be suppressed in an allele-specific manner by mutations in the two largest subunits of RNA polymerase II (Pol II). Strikingly, one of these RNA Pol II mutants is defective for transcription elongation and the others cause phenotypes consistent with an elongation defect. In addition, we show that spt4, spt5, and spt6 mutants themselves have phenotypes suggesting defects in transcription elongation in vivo. Consistent with these findings, we show that Spt5 is physically associated with RNA Pol II in vivo, and have identified a region of sequence similarity between Spt5 and NusG, an Escherichia coli transcription elongation factor that binds directly to RNA polymerase. Finally, we show that Spt4 and Spt5 are tightly associated in a complex that does not contain Spt6. These results, taken together with the biochemical identification of a human Spt4-Spt5 complex as a transcription elongation factor (Wada et al. 1998), provide strong evidence that these factors are important for transcription elongation in vivo.

contributed equally to this work Recently, a positive and a negative elongation factor, implicated in 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, has been identified. P-TEFb is a positive transcription elongation factor and the DRB-sensitive kinase that phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). PIT-ALRE, a member of the Cdc2 family of protein kinases, is the catalytic subunit of P-TEFb. DSIF is a human homolog of the yeast Spt4-Spt5 complex and renders elongation of transcription sensitive to DRB. DRB sensitivity-inducing factor (DSIF) binds to RNA Pol II and may directly regulate elongation. Here we show a functional interaction between P-TEFb and DSIF. The reduction of P-TEFb activity induced by either DRB, antibody against PITALRE, or immunodepletion resulted in a negative effect of DSIF on transcription. DSIF acts at an early phase of elongation, and the prior action of P-TEFb makes transcription resistant to DSIF. The state of phosphorylation of CTD determines the DSIF-RNA Pol II interaction, and may provide a direct link between P-TEFb and DSIF. Taken together, this study reveals a molecular basis for DRB action and suggests that P-TEFb stimulates elongation by alleviating the negative action of DSIF.

The elongation step of RNA polymerase II (RNAPII) transcription is emerging as a critical control point for the expression of various genes and for diverse biological processes. Examples include neuronal fate determination during embryonic development (6, 44), gene expression of human immunodeficiency virus (5,11,13,19,43), replication and transcription of hepatitis delta virus (38), and transcriptional regulation of heat shock genes (1,10,18). In all these cases, the involvement of three transcription elongation factors, namely, DRB (5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF), NELF (negative elongation factor), and positive transcription elongation factor b (P-TEFb), has been demonstrated or implicated.Shortly after the initiation of transcription, RNAPII comes under the negative and positive control of DSIF, NELF, and P-TEFb. DSIF and NELF cause transcriptional pausing through physical association with RNAPII. DSIF binds to RNAPII directly and stably (33, 36). However, this appears to have little effect on the catalytic activity of RNAPII (37). A previous study has pointed out that NELF does not bind substantially to DSIF or RNAPII alone but does bind to the complex of DSIF and RNAPII (40). This association is the likely trigger of transcriptional pausing. Conversely, P-TEFb allows RNAPII to enter the productive elongation phase by preventing the action of DSIF and NELF (27, 37). P-TEFb is the protein kinase whose primary target is thought to be the C-terminal domain (CTD) of RNAPII (26). Most, but not all, evidence suggests that P-TEFb-dependent phosphorylation of the CTD facilitates the release of DSIF and NELF from RNA-PII, thereby reversing the inhibition (3, 24, 37). In theory, such regulation at the elongation step allows for rapid change in mRNA levels and for highly sophisticated control over gene expression when combined with regulation at the (pre)initiation step.The structures and functions of DSIF and P-TEFb have been extensively characterized. Human DSIF is a heterodimer composed of p14 (14 kDa) and p160 (160 kDa), whose Saccharomyces cerevisiae counterparts are Spt4 and Spt5 (7,33). In addition to its role in transcriptional pausing, DSIF has a potential to activate RNAPII elongation. The activation mechanism is not well understood: interaction partners of DSIF other than NELF may be involved (13,14,20,23,28). Spt5 has a highly acidic N-terminal region, multiple copies of the KOW motifs, and a repetitive C-terminal region analogous to the RNAPII CTD (9,25,36). RNAPII interacts with Spt5 through a region encompassing the KOW motifs. KOW motifs are also found in the bacterial transcription elongation factor NusG, which binds to prokaryotic RNA polymerase and controls termination and antitermination (15,17,29). In addition, the extreme C terminus of Spt5 is specifically involved in the transcriptional repression pathway (6). Human P-TEFb is a heterodimer composed of Cdk9 (41 kDa) and one of multiple cyclin subunits T1, T2a, T2b, and K (50 to 90 kDa) (26). The k...

Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.Transcription elongation by RNA polymerase II (RNAPII) is controlled by a number of trans-acting factors called transcription elongation factors as well as by cis-acting elements (2, 26). Transcription elongation factors such as transcription factor IIF (TFIIF), elongin, and TFIIS interact with elongating RNAPII to prevent its pausing or to reactivate it from an arrested configuration. cis-acting elements are mainly located on nascent transcripts. Some RNA elements cause RNAPII to pause or arrest without the aid of protein factors by forming structures that destabilize RNAPII-DNA-RNA complexes (19,26). Other types of RNA elements include the one called TAR, which exists at the 5Ј end of human immunodeficiency virus (HIV) transcripts and serves as a binding site for the viral activator Tat and cellular cofactors (9); together these strongly stimulate RNAPII elongation. The functions of many other RNA elements are largely unknown.The recent discovery of a new class of positive and negative elongation factors, including DRB sensitivity-inducing factor (DSIF), negative elongation factor (NELF), and positive transcription elongation factor b (P-TEFb), has shed new light on the control of RNAPII elongation (21,27,(31)(32)(33)35). Biochemical studies have established that DSIF and NELF cooperatively repress RNAPII elongation, whereas P-TEFb alleviates the repression in a manner sensitive to the kinase inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) (28,29,31). DSIF and NELF coimmunoprecipitate with the unphosphorylated form of RNAPII (IIa), but not with the hyperphosphorylated form (IIo) (28, 31). In addition, P-TEFb strongly phosphorylates the C-terminal domain (CTD) of RNAPII and a subunit of DSIF in a DRB-sensitive manner (8,12,35). From these data, we have proposed that transcription r...

Studying the sensitivity of transcription to the nucleotide analog 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole has led to the discovery of a number of proteins involved in the regulation of transcription elongation by RNA polymerase II. We have developed a highly purified elongation control system composed of three purified proteins added back to isolated RNA polymerase II elongation complexes. Two of the proteins, 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), act as negative transcription elongation factors by increasing the time the polymerase spent at pause sites. P-TEFb reverses the negative effect of DSIF and NELF through a mechanism dependent on its kinase activity. TFIIF is a general initiation factor that positively affects elongation by decreasing pausing. We show that TFIIF functionally competes with DSIF and NELF, and this competition is dependent on the relative concentrations of TFIIF and NELF.The balance of activity between both positive and negative factors achieves accurate control of many cellular processes. Accumulating evidence indicates that such a process regulates the control of transcription elongation (1). It has been proposed that shortly after initiation, negative transcription elongation factors act upon RNA polymerase II to cause production of short transcripts (2). With the action of P-TEFb the polymerase enters productive elongation and transcription is no longer influenced by the negative factors (3, 4). After this transition, the polymerase is acted upon by general elongation factors such as S-II, TFIIF, ELL, and elongin to generate long transcripts (5-7).The key step in the elongation control process, the transition from abortive elongation to productive elongation, requires the positive elongation factor P-TEFb (1, 3, 4). P-TEFb was originally purified from Drosophila nuclear extracts, as a factor required for reconstitution of 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) 1 sensitivity in in vitro transcription assays (3). Active human P-TEFb consists of a heterodimer of cyclin-dependent kinase 9 (Cdk9) and either cyclin T1, cyclin T2, or cyclin K (8, 9). The elongation properties of P-TEFb are dependent on its kinase activity, and both the kinase and elongation activities are sensitive to the nucleotide analog DRB (3, 4, 8, 10 -12), a kinase inhibitor known for its ability to inhibit transcription elongation (13). P-TEFb is also strongly inhibited by flavopiridol, a drug currently in clinical trials as an anti-cancer treatment that might also be useful as an anti-HIV therapy (14). Depletion of P-TEFb from HeLa nuclear extract (HNE) greatly reduces the ability of RNA polymerase II to produce full-length transcripts and eliminates the DRB sensitivity of that extract (8). The addition of purified P-TEFb to HNE depleted of Cdk9 restores the ability of RNA polymerase II to generate full-length transcripts and restores DRB sensitivity (8, 15).Another factor required for DRB sensitivity, DRB sensitivity...

Human plasma membrane-associated sialidase (Neu3) is unique in specifically hydrolyzing gangliosides, thought to participate in cell differentiation and transmembrane signaling, thereby playing crucial roles in the regulation of cell surface functions. We have discovered levels of mRNA for this sialidase to be increased in restricted cases of human colon cancer by 3-to 100-fold compared with adjacent nontumor mucosa (n ‫؍‬ 32), associated with significant elevation in sialidase activity in tumors (n ‫؍‬ 50). In situ hybridization showed the sialidase expression in epithelial elements of adenocarcinomas. In cultured human colon cancer cells, the sialidase level was downregulated in the process of differentiation and apoptosis induced by sodium butyrate, whereas lysosomal sialidase (Neu1) was upregulated. Transfection of the sialidase gene into colon cancer cells inhibited apoptosis and was accompanied by increased Bcl-2 and decreased caspase expression. Colon cancer exhibited a marked accumulation of lactosylceramide, a possible sialidase product, and addition of the glycolipid to the culture reduced apoptotic cells during sodium butyrate treatment. These results indicate that high expression of the sialidase in cancer cells leads to protection against programmed cell death, probably modulation of gangliosides. This finding provides a possible sialidase target for diagnosis and therapy of colon cancer.