NELF complex

Abstract: 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.

Abstract: In eukaryotic cells, the regulation of preinitiation complex (PIC) assembly is essential to control gene expression (26), yet recent studies indicate that post-PIC assembly processes are also critical (27) At the human α1-AT locus, transcription initiation is inhibited before induction, although RNA polymerase II (RNAPII) associates with the promoter (29) In contrast, at the human c-fos and c-myc loci and some Drosophila heat shock loci, elongating RNAPII pauses in the promoter-proximal region before induction (12, 23, 24) The latter example is referred to as “promoter-proximal pausing,” although the mechanism and function of this regulatory mode are not well understood (13) At the Drosophila hsp70 locus, promoter-proximal pausing is thought to be mediated by two transcription elongation factors, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) (34) DSIF is a heterodimeric protein complex consisting of the Spt4 and Spt5 subunits (31) Human Spt5 (hSpt5) has a repeat region called the CTR and multiple copies of the KOW motif, which is also found in bacterial elongation factor NusG (39) NELF is a DSIF cofactor that consists of four subunits (38) NELF-A, encoded by Wolf-Hirschhorn syndrome candidate gene 2, has an N-terminal homology to hepatitis delta antigen, which binds to RNAPII and activates elongation (36) NELF-B, encoded by the COBRA1 gene, is known to interact with the BRCA1 protein (41) NELF-C and -D are translational variants of the TH1 gene products (17) NELF-E, also known as RD, has Arg-Asp dipeptide repeats and an RNA recognition motif (38) Biochemical analysis has revealed that DSIF and NELF cooperatively bind to elongating RNAPII and induce transcriptional pausing, possibly through an interaction between NELF-E and nascent RNA (37) Transcriptional pausing is alleviated when positive elongation factor b (P-TEFb) phosphorylates the heptapeptide repeats of the C-terminal domain of RNAPII, as well as the CTR in the hSpt5 subunit of DSIF (32, 35) After pausing is reversed, DSIF instead stimulates elongation by an as-yet-unknown mechanism Interestingly, DSIF and NELF are not evolutionarily conserved to the same extent (17) Although DSIF is highly conserved among eukaryotes and is essential for viability in yeast, some species, including yeast, lack all of the NELF subunits (10, 17) Indeed, promoter-proximal pausing has not been observed in yeast (13) Thus, promoter-proximal pausing may be involved in transcriptional regulation only in some species Only a limited number of studies have been reported on DSIF- and NELF-mediated transcriptional pausing At the Drosophila hsp70 locus, both DSIF and NELF associate with RNAPII paused at positions +20 to +40 before induction (34) After heat shock, NELF dissociates from RNAPII but DSIF translocates downstream with the polymerase Another study showed that human estrogen receptor α recruits the NELF complex to target gene promoters by physically interacting with NELF-B (3) It is suggested that NELF acts as a transcriptional attenuator at these loci and is important for controlling the duration and magnitude of hormonal responses In zebra fish, the mutant called foggy, which carries a point mutation in the Spt5 gene and lacks the repression activity of DSIF, shows specific defects in neuronal differentiation during development, suggesting that DSIF- and NELF-mediated transcriptional pausing may be involved in the expression of only a limited number of genes (9) However, its precise role in gene expression on a genome-wide basis remains unclear The goal of this study was to understand the physiological role of DSIF- and NELF-mediated transcriptional pausing As a model, we used junB, an immediate-early gene (IEG) that is activated transiently and rapidly in response to a wide variety of extracellular stimuli, such as interleukin-6 (IL-6) junB encodes a basic leucine zipper protein, which functions as a component of the AP-1 transcriptional activator Several studies indicate that the regulation of junB gene expression is important for cell growth and differentiation (11, 15, 20) Here we report that DSIF- and NELF-mediated transcriptional pausing has a dual role in the regulation of junB expression in human hepatoma HepG2 cells Our data indicate that pausing contributes to the negative regulation of junB expression not only by inducing transcriptional pausing before induction but also by attenuating the mRNA expression level after induction