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At least 19 recordsLinked to original sources
PubMed2024

Pause Patrol: Negative Elongation Factor's Role in Promoter-Proximal Pausing and Beyond.

RNA polymerase (Pol) II is highly regulated to ensure appropriate gene expression. Early transcription elongation is associated with transient pausing of RNA Pol II in the promoter-proximal region. In multicellular organisms, this pausing is stabilized by the association of transcription elongation factors DRB-sensitivity inducing factor (DSIF) and Negative Elongation Factor (NELF). DSIF is a broadly conserved transcription elongation factor whereas NELF is mostly restricted to the metazoan lineage. Mounting evidence suggests that NELF association with RNA Pol II serves as checkpoint for either release into rapid and productive transcription elongation or premature termination at promoter-proximal pause sites. Here we summarize NELF's roles in promoter-proximal pausing, transcription termination, DNA repair, and signaling based on decades of cell biological, biochemical, and structural work and describe areas for future research.

Promoter Regions, Genetic↗
PubMed1999

HIV-1 Tat: coping with negative elongation factors.

The intrinsic processivity of RNA polymerase II complexes arises from a complex interplay between the recently identified positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, DSIF (5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole [DRB]-sensitivity-inducing factor) and the negative elongation factor complex (NELF). Elements in nascent HIV-1 RNA function in concert with these factors and the HIV-1 Tat protein to ensure that viral transcription is induced strongly in activated T cells. Studies in the past year have elucidated key aspects of the Tat trans-activation mechanism that help to define this important paradigm for RNA-mediated control of transcription elongation.

Animals↗
PubMed2005

A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS.

Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-beta-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF/NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF/NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF/NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires > or = 18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of back-tracked nascent RNA, TFIIS inhibition may help DSIF/NELF negatively regulate productive transcription.

Animals↗
PubMed2002

Evidence that negative elongation factor represses transcription elongation through binding to a DRB sensitivity-inducing factor/RNA polymerase II complex and RNA.

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.

Chromatography, Affinity↗
PubMed2004

Attenuation of estrogen receptor alpha-mediated transcription through estrogen-stimulated recruitment of a negative elongation factor.

Estrogen receptor alpha (ERalpha) signaling is paramount for normal mammary gland development and function and the repression of breast cancer. ERalpha function in gene regulation is mediated by a number of coactivators and corepressors, most of which are known to modify chromatin structure and/or influence the assembly of the regulatory complexes at the level of transcription initiation. Here we describe a novel mechanism of attenuating the ERalpha activity. We show that cofactor of BRCA1 (COBRA1), an integral subunit of the human negative elongation factor (NELF), directly binds to ERalpha and represses ERalpha-mediated transcription. Reduction of the endogenous NELF proteins in breast cancer cells using small interfering RNA results in elevated ERalpha-mediated transcription and enhanced cell proliferation. Chromatin immunoprecipitation reveals that recruitment of COBRA1 and the other NELF subunits to endogenous ERalpha-responsive promoters is greatly stimulated upon estrogen treatment. Interestingly, COBRA1 does not affect the estrogen-dependent assembly of transcription regulatory complexes at the ERalpha-regulated promoters. Rather, it causes RNA polymerase II (RNAPII) to pause at the promoter-proximal region, which is consistent with its in vitro biochemical activity. Therefore, our in vivo work defines the first corepressor of nuclear receptors that modulates ERalpha-dependent gene expression by stalling RNAPII. We suggest that this new level of regulation may be important to control the duration and magnitude of a rapid and reversible hormonal response.

Blotting, Northern↗
PubMed1998

Interplay between positive and negative elongation factors: drawing a new view of DRB.

DRB is a classic inhibitor of transcription by RNA polymerase II (pol II). Although it has been demonstrated that DRB inhibits the elongation step of transcription, its mode of action has been elusive. DRB also markedly inhibits human immunodeficiency virus (HIV) transcription, by targeting the elongation which is enhanced by the HIV-encoded transactivator Tat. Two factors essential for DRB action have recently been identified. These factors, positive transcription elongation factor b (P-TEFb) and DRB sensitivity-inducing factor (DSIF), positively and negatively regulate pol II elongation, and are likely to be relevant to the function of Tat. In this review, we summarize the recent findings on these factors, and discuss a possible model for the molecular mechanism of DRB action.

Dichlororibofuranosylbenzimidazole↗
PubMed2006

Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription.

The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has alpha-helical and beta-strand elements adopting a betaalphabetabetaalphabeta fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with K(d) values in the low-micromolar range.

Amino Acid Motifs↗
PubMed2006

Transcriptional pausing caused by NELF plays a dual role in regulating immediate-early expression of the junB gene.

Human 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) negatively regulate transcription elongation by RNA polymerase II (RNAPII) in vitro. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF- and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position +50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF- and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.

Cells, Cultured↗
PubMed1999

NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation.

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.

Amino Acid Sequence↗
PubMed2001

A highly purified RNA polymerase II elongation control system.

Studying the sensitivity of transcription to the nucleotide analog 5,6-dichloro-1-beta-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-beta-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.

Nuclear Proteins↗
PubMed2003

Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex.

The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, A to E. NELF-A has sequence similarity to hepatitis delta antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding activity is critical for NELF function. To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we identified the B, C, and D proteins of human NELF. NELF-B is identical to COBRA1, recently reported to associate with the product of breast cancer susceptibility gene BRCA1. NELF-C and NELF-D are highly related or identical to the protein called TH1, of unknown function. NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression of these four proteins in insect cells resulted in the reconstitution of functionally active NELF. Detailed analyses using mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding and for transcriptional pausing. This study defines several important protein-protein interactions and opens the way for understanding the mechanism of DSIF- and NELF-induced transcriptional pausing.

Amino Acid Sequence↗
PubMed2004

Modulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5.

BACKGROUND: Several cellular positive and negative elongation factors are involved in regulating RNA polymerase II processivity during transcription elongation in human cells. In recruiting several of these regulatory factors to the 5' long terminal repeat (LTR) promoter during transcription elongation, HIV-1 modulates replication of its genome in a process mediated by the virus-encoded transactivator Tat. One particular cellular regulatory factor, DSIF subunit human SPT5 (hSpt5), has been implicated in both positively and negatively regulating transcriptional elongation but its role in Tat transactivation in vivo and in HIV-1 replication has not been completely elucidated. RESULTS: To understand the in vivo function of hSpt5 and define its role in Tat transactivation and HIV-1 replication, we used RNA interference (RNAi) to specifically knockdown hSpt5 expression by degrading hSpt5 mRNA. Short-interfering RNA (siRNA) designed to target hSpt5 for RNAi successfully resulted in knockdown of both hSpt5 mRNA and protein levels, and did not significantly affect cell viability. In contrast to hSpt5 knockdown, siRNA-mediated silencing of human mRNA capping enzyme, a functionally important hSpt5-interacting cellular protein, was lethal and showed a significant increase in cell death over the course of the knockdown experiment. In addition, hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV-1 replication, indicating that hSpt5 was required for mediating Tat transactivation and HIV-1 replication. CONCLUSIONS: The findings presented here showed that hSpt5 is a bona fide positive regulator of Tat transactivation and HIV-1 replication in vivo. These results also suggest that hSpt5 function in transcription regulation and mRNA capping is essential for a subset of cellular and viral genes and may not be required for global gene expression.

Gene Expression Regulation, Viral↗
PubMed2001

Stimulation of RNA polymerase II elongation by hepatitis delta antigen.

Transcription elongation by RNA polymerase II (RNAPII) is negatively regulated by the human factors DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF). A 66-kilodalton subunit of NELF (NELF-A) shows limited sequence similarity to hepatitis delta antigen (HDAg), the viral protein required for replication of hepatitis delta virus (HDV). The host RNAPII has been implicated in HDV replication, but the detailed mechanism and the role of HDAg in this process are not understood. We show that HDAg binds RNAPII directly and stimulates transcription by displacing NELF and promoting RNAPII elongation. These results suggest that HDAg may regulate RNAPII elongation during both cellular messenger RNA synthesis and HDV RNA replication.

Amino Acid Sequence↗
PubMed1998

The vaccinia virus A18R DNA helicase is a postreplicative negative transcription elongation factor.

Loss of vaccinia virus A18R gene function results in an aberrant transcription profile termed promiscuous transcription, defined as transcription within regions of the genome which are normally transcriptionally silent late during infection. Promiscuous transcription results in an increase in the intracellular concentration of double-stranded RNA, which in turn results in activation of the cellular 2-5A pathway and subsequent RNase L-catalyzed degradation of viral and cellular RNAs. One of three hypotheses could account for promiscuous transcription: (i) reactivation of early promoters late during infection, (ii) random transcription initiation, (iii) readthrough transcription from upstream promoters. Transcriptional analysis of several viral genes, presented here, argues strongly against the first two hypotheses. We have tested the readthrough hypothesis by conducting a detailed transcriptional analysis of a region of the vaccinia virus genome which contains three early genes (M1L, M2L, and K1L) positioned directly downstream of the intermediate gene, K2L. The results show that mutation of the A18R gene results in increased readthrough transcription of the M1L gene originating from the K2L intermediate promoter. A18R mutant infection of RNase L knockout mouse fibroblast (KO3) cells does not result in 2-5A pathway activation, yet the virus mutant is defective in late viral gene expression and remains temperature sensitive. These results demonstrate that the A18R gene product is a negative transcription elongation factor for postreplicative viral genes.

Adenosine Triphosphatases↗
PubMed2000

FACT relieves DSIF/NELF-mediated inhibition of transcriptional elongation and reveals functional differences between P-TEFb and TFIIH.

We report that the chromatin-specific transcription elongation factor FACT functions in conjunction with the RNA polymerase II CTD kinase P-TEFb to alleviate transcription inhibition by DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor). We find that the kinase activity of TFIIH is dispensable for this activity, demonstrating that TFIIH-mediated CTD phosphorylation is not involved in the regulation of FACT and DSIF/NELF activities. Thus, we propose a novel transcriptional regulatory network in which DSIF/NELF inhibition of transcription is prevented by P-TEFb in cooperation with FACT. This study uncovers a novel role for FACT in the regulation of transcription on naked DNA that is independent of its activities on chromatin templates. In addition, this study reveals functional differences between P-TEFb and TFIIH in the regulation of transcription.

DNA-Binding Proteins↗
PubMed2004

Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II.

Capping of the 5' ends of nascent RNA polymerase II transcripts is the first pre-mRNA processing event in all eukaryotic cells. Capping enzyme (CE) is recruited to transcription complexes soon after initiation by the phosphorylation of Ser-5 of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here, we analyze the role of CE in promoter clearance and its functional interactions with different factors that are involved in promoter clearance. FCP1-mediated dephosphorylation of the carboxyl-terminal domain results in a drastic decrease in cotranscriptional capping efficiency but is reversed by the presence of DRB sensitivity-inducing factor (DSIF). These results suggest involvement of DSIF in CE recruitment. Importantly, CE relieves transcriptional repression by the negative elongation factor, indicating a critical role of CE in the elongation checkpoint control mechanism during promoter clearance. This functional interaction between CE and the negative elongation factor documents a dynamic role of CE in promoter clearance beyond its catalytic activities.

Nuclear Proteins↗
PubMed2000

DSIF and NELF interact with RNA polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated phosphorylation of RNA polymerase II and DSIF during transcription elongation.

Control of transcription elongation requires a complex interplay between the recently discovered positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing factors (DSIF) and the negative elongation factor (NELF). Activation of HIV-1 gene expression is regulated by a nascent RNA structure, termed TAR RNA, in concert with HIV-1 Tat protein and these positive and negative elongation factors. We have used a stepwise RNA pol II walking approach and Western blotting to determine the dynamics of interactions between HIV-1 Tat, DSIF/NELF, and the transcription complexes actively engaged in elongation. In addition, we developed an in vitro kinase assay to determine the phosphorylation status of proteins during elongation stages. Our results demonstrate that DSIF/NELF associates with RNA pol II complexes during early transcription elongation and travels with elongation complexes as the nascent RNA is synthesized. Our results also show that HIV-1 Tat protein stimulated DSIF and RNA pol II phosphorylation by P-TEFb during elongation. These findings reveal a molecular mechanism for the negative and positive regulation of transcriptional elongation at the HIV-1 promoter.

Cell Nucleus↗
PubMed2001

The regulation of elongation by eukaryotic RNA polymerase II: a recent view.

The elongation of mRNA by RNA polymerase II (RNAPII) is controlled by the diverse actions of cis- and trans-acting elements that respond to a variety of biological signals. Elongation regulation, which determines the processivity of RNAPII, occurs during the steps of transcription initiation and elongation through both negative and positive influences. It has been generally accepted that negative elongation factors repress the elongation reaction. It is also generally accepted that positive elongation factors act by allowing the stalled RNAPII to escape from an inactive state, thus facilitating the production of full-length transcripts. However, recent data suggests that these opposing regulatory mechanisms can be achieved through a coupling pattern, rather than through independent activity. In addition, biochemical experiments using 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) have identified several factors involved in DRB-sensitive transcription, and elucidated the mechanism of interplay between the positive and negative elongation factors. Here, we will review the major findings from studies on RNAPII elongation control. We will further discuss the properties of newly identified factors that have resulted from studies on the action of DRB on elongation.

RNA Polymerase III↗