InteractiveFly: GeneBrief

Negative elongation factor E: Biological Overview | References

Transcriptional elongation regulators NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF (FlyBase term: Spt5) from a Drosophila nuclear extract reduces the level of polymerase that pauses in the promoter proximal region of hsp70. Depletion of one Negative elongation factor E (NELF) subunit in salivary glands using RNA interference also reduces the level of paused polymerase. In vivo protein-DNA cross-linking shows that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborates the cross-linking result and shows that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF are strongly recruited to chromosomal puffs harboring the hsp70 genes. It is proposed that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation (Wu, 2003).

It is proposed that promoter proximal pausing occurs when the nascent transcript emerges from the RNA exit channel of the Pol II and is grabbed by the NELF-E subunit. Tethering of the NELF-E to the elongation complex would generate a rigid body that could restrict the movement of the Pol IIa. This model is supported by several observations. The paused polymerase is in the Pol IIa state, and NELF and DSIF only inhibit elongation by Pol IIa. In vitro transcription analysis indicates that the elongation complex is not receptive to inhibition by NELF and DSIF until the nascent transcript is ~30 nucleotides long. This length coincides approximately to the distance polymerase elongates on hsp70 before it pauses. In vitro transcription analyses indicate that DSIF and NELF associate with polymerase shortly after initiation but probably before the polymerase reaches the region of pausing. Finally, NELF-E has an RNA-binding motif that is essential for its inhibitory action (Wu, 2003 and references therein).

Although NELF and DSIF are sufficient to slow the elongation rate of purified Pol IIa, it is suspected that additional proteins are involved in stably pausing Pol II on the hsp70 promoter. In cell-free transcription reactions done with other promoters, the pausing caused by DSIF and NELF appears to be transient -- the polymerase eventually moves forward if given enough time. In contrast, several observations indicate that the Pol II on hsp70 is stably paused. The paused Pol II remains associated with the hsp70 promoter when nuclei are isolated from uninduced cells, and sarkosyl or high salt must accompany addition of nucleotides to cause the Pol II to resume elongation. In a cell-free system, Pol II remains stably paused on the hsp70 promoter for at least 30 min. GAGA factor might be involved in stabilizing the pause because mutations in the GAGA element result in a loss of paused Pol II (Wu, 2003).

Heat shock rapidly induces transcription as a result of the association of HSF with sites located upstream from the TATA element. The data suggest that HSF may activate transcription in part by causing NELF to dissociate from the Pol II. How HSF might cause the release of NELF is unclear. Phosphorylation of Pol IIa is likely to be an important step because the Pol II found in the body of the gene during heat shock is hyperphosphorylated. Phosphorylation of DSIF is another possibility as this has been observed to occur early in elongation in vitro. It is also unclear which kinase might be responsible for phosphorylating the Pol II. P-TEFb (see Cdk9) is a candidate because it associates with the hsp70 gene during heat shock induction, and HSF can be bypassed by directing a Gal4/P-TEFb fusion protein to the hsp70 promoter. No interaction, however, has been detected between P-TEFb and HSF. Recent results show that HSF associates with the mediator. Drosophila mediator contains a kinase that phosphorylates the CTD, and phosphorylation can occur synergistically with the TFIIH kinase. Perhaps HSF recruits the mediator and in turn the mediator releases the paused polymerase by phosphorylating the CTD (Wu, 2003).

The strong immunofluorescence staining observed for DSIF at heat shock loci during heat shock indicates that DSIF is associated with many of the polymerase molecules transcribing the gene. RNA polymerase initiates at a rate of once every few seconds during heat shock resulting in a train of elongation complexes traversing the gene. In the absence of NELF, DSIF might act as a positive elongation factor. Shortly after DSIF was discovered, another investigation identified DSIF as a cofactor required for reconstituting tat-dependent transcription. In this situation, DSIF appears to be stimulating elongation. DSIF has been found in a complex with another positive elongation factor called Tat-SF1. Tat-SF1 was first identified as a stimulatory factor for Tat, but subsequent results indicate that Tat-SF1 may promote elongation on cellular genes. In yeast, DSIF appears to act as either a positive or negative regulator of elongation depending on circumstances. A hypothesis that unites the positive and negative activities of DSIF considers this factor an adaptor that connects other modulators to the elongation complex. In this regard, DSIF has been shown to bind on its own to Pol II, whereas the stable association of NELF with Pol II requires the presence of DSIF (Wu, 2003 and references therein).

NELF and DSIF appear to associate with several hundred interbands in polytene chromosomes. Each interband could contain many genes. The weak staining of interbands by Hoecsht suggests that the DNA in the interbands is in a decondensed state. Residing in these decondensed regions could be genes whose primary control mechanism does not involve a disruption of chromatin structure or even assembly of the initiation complex. Instead, alleviating repression by NELF and DSIF could underlie the mechanism of activation (Wu, 2003).

RNA polymerase II initiates transcription in slp1-repressed cells and pauses downstream from the promoter in a complex that includes the negative elongation factor NELF

The simple combinatorial rules for regulation of the sloppy-paired-1 (slp1) gene by the pair-rule transcription factors during early Drosophila embryogenesis offer a unique opportunity to investigate the molecular mechanisms of developmentally regulated transcription repression. Initial repression of slp1 in response to Runt and Fushi-tarazu (Ftz) does not involve chromatin remodeling, or histone modification. Chromatin immunoprecipitation and in vivo footprinting experiments indicate RNA polymerase II (Pol II) initiates transcription in slp1-repressed cells and pauses downstream from the promoter in a complex that includes the negative elongation factor NELF. The finding that Negative elongation factor E also associates with the promoter regions of wingless (wg) and engrailed (en), two other pivotal targets of the pair-rule transcription factors, strongly suggests that developmentally regulated transcriptional elongation is central to the process of cell fate specification during this critical stage of embryonic development (Wang, 2007).

DNase I hypersensitivity was used to probe the chromatin structure of the slp1 locus. These assays revealed the presence of a DNase I-hypersensitive site near to the 5'-end of the slp1 transcription unit. Chromatin immunoprecipitation (ChIP) experiments with antiserum against histone H3 provide an explanation for this DNase I hypersensitivity. There is significantly reduced association of H3 with the slp1 promoter region compared with both the structural gene as well as sequences upstream of the promoter. These observations strongly suggest that the promoter region is nucleosome free. Importantly, matched collections of wild-type and Runt + Ftz (R+F) embryos show both the same pattern of DNase I hypersensitivity and histone H3 association. These results indicate that the 40-fold decrease in mRNA expression in slp1-repressed embryos is not due to gross changes in the accessibility of the slp1 promoter region (Wang, 2007).

Histone acetylation and deacetylation are important for transcriptional regulation with a general correlation between histone acetylation and active transcription. Indeed, prior work has demonstrated that the Rpd3 histone deacetylase is important for maintaining the Runt-dependent repression of the segment-polarity gene en. ChIP experiments reveal no significant difference in the H3 acetylation pattern of slp1 chromatin from wild-type versus R+F embryos. Although no differences were detected in H3 or Ac-H3 association that correlate with slp1 repression, there are interesting differences in the H3 acetylation levels at different genomic locations. The slp1 structural gene shows stronger Ac-H3 association than the upstream region. This difference is not observed for the association of H3 with these same intervals, suggesting that H3 acetylation marks genomic regions that are permissive for transcription. The relative levels of H3 and Ac-H3 association with Brother (Bro), a gene that is not transcribed in the early embryo (as measured by RT-PCR), provide additional evidence for this trend. Although H3 association with the Bro gene is greater than for any region of the slp1 locus, the level of Ac-H3 association with Bro is lower than for any region of slp1. Based on the observation that differences in H3 and Ac-H3 association can be detected that correlate with transcriptional potential and yet no differences was detected between wild-type and slp1-repressed embryos, it is concluded that H3 acetylation plays a negligible role in the establishment of slp1 repression (Wang, 2007).

The above observations led to a characterization the interactions of the transcriptional machinery with slp1. Association of the TATA-box-binding protein (TBP) is a first step in assembly of the transcriptional machinery on a promoter. As expected, TBP association is detected with a promoter-proximal interval centered 6 base pairs (bp) upstream of the slp1 transcript initiation site in chromatin from wild-type embryos. A weaker signal is detected for an interval within the 5' untranslated region (UTR), centered 124 bp downstream from the start site, whereas all other intervals give background level signals. Very similar levels of TBP association were found in chromatin from R+F embryos. More surprising is the finding that there is almost no difference in the level of Pol II association with the slp1 promoter-proximal interval in chromatin from wild-type and R+F embryos. Pol II is also associated with the slp1 structural gene in wild-type embryos, but at lower levels than at the promoter. In contrast, Pol II association with the slp1 structural gene is markedly reduced in R+F embryos and near to background levels for regions downstream from the 5'-UTR. Based on these results, it is concluded that promoter recruitment of Pol II is not blocked in slp1-repressed embryos. slp1-associated Pol II was further characterized using an antibody that recognizes the Phospho-Ser-5 form of the heptad repeats that comprise the C-terminal domain (CTD) of the largest Pol II subunit. Phospho-Ser-5 modification of the CTD is associated with transcription initiation. This antiserum also gives the strongest signals with the slp1 promoter-proximal interval in wild-type chromatin, and this signal is not reduced in chromatin from R+F embryos. This result indicates that slp1 repression occurs at a step downstream from transcription initiation (Wang, 2007).

The Drosophila hsp70a promoter is an extensively studied example of regulated transcriptional elongation. Pol II initiates transcription at the hsp70a promoter, and then, in the absence of a heat shock, pauses immediately downstream from the promoter. All somatic cells in 3-4-h-AED embryos are capable of activating the hsp70a gene, and as expected, Phospho-Ser-5-modified Pol II is readily detected on the hsp70a promoter in chromatin preparations from non-heat-shocked embryos. The paused Pol II complex on the hsp70a promoter is also readily detected using permanganate footprinting due to the increased sensitivity of thymine residues in single-stranded regions. This same technique was used to carry out footprinting studies on the slp1 promoter region. The results reveal strong hyperreactivity of thymine residues at +15, +28, +30, +38, and +50 downstream from the transcription start site in blastoderm. This interval is similar, though perhaps somewhat larger than the interval detected for hsp70a, within which the most prominent increases in reactivity are at residues +22 and +30. The pattern of reactivity on slp1 is extremely similar in both wild-type and slp1-repressed embryos, indicating that the hyperreactivity is not due to active transcription of the slp1 gene. Importantly, this pattern is not observed in nuclei from Drosophila tissue culture cells. Thus, unlike hsp70a, the footprint on the slp1 5'-UTR is developmentally regulated (Wang, 2007).

The negative elongation factor NELF is thought to play a key role in establishing the paused Pol II complex on the hsp70a promoter. Indeed, NELF association provides a marker for the paused complex as heat-shock-induced transcriptional elongation involves release of NELF (Wu, 2005). In agreement with the results of footprinting studies, ChIP experiments reveal the NELF-D and NELF-E subunits are associated with the slp1 promoter region in chromatin from wild-type embryos, but not in chromatin from Drosophila tissue culture cells. Strong signals are obtained in chromatin from embryos with both the promoter-proximal and 5'-UTR intervals, whereas background level signals are obtained with other intervals of the slp1 locus. It is notable that the promoter-proximal signal is less than or equal to the signal detected for the 5'-UTR interval. This pattern of association contrasts that obtained with TBP, which shows a threefold stronger signal with the promoter-proximal primer pair. These association patterns suggest is that NELF is bound downstream from the slp1 transcription start site, presumably as a component of the paused Pol II complex. Consistent with this interpretation, a similar differential pattern was found of TBP and NELF association with promoter-proximal and 5'-UTR intervals of hsp70a. These results strongly suggest that NELF plays a key role in regulating slp1 elongation in the blastoderm-stage Drosophila embryo (Wang, 2007).

The initial indications that slp1 expression was regulated at a step downstream from transcription initiation came from ChIP experiments on chromatin from a homogeneous population of embryos that uniformly repress slp1. Localized association of NELF in a region downstream from the transcription start site is a hallmark of promoter-proximal pausing. Importantly, this association provides a method for detecting paused Pol II complexes in chromatin from embryos that contain a mixture of cells, some of which are expressing full-length mRNA transcripts. ChIP assays were used to determine whether NELF associates with the promoter regions of wg and en, two pivotal segment-polarity gene targets of the pair-rule transcription factors. The results reveal specific association of NELF with the promoter-proximal and 5'-UTR regions of both genes in 3-4-h-AED embryos. Furthermore, the differential association pattern of TBP and NELF with these two intervals indicates that NELF is localized to a region immediately downstream from the initiation sites for both genes. These findings indicate that regulation of transcriptional elongation is likely to be central in generating the initial patterns of segment-polarity gene expression in the Drosophila embryo (Wang, 2007).

Regulation of transcriptional elongation has been described for several genes in addition to the Drosophila heat-shock genes, including human c-myc, c-myb, c-fos, junB, and p21. A feature shared by these previously characterized examples is rapid induction of gene expression in response to external stimuli. The initial establishment of segment-polarity gene-expression patterns in response to the pair-rule transcription factors occurs within a relatively brief developmental window of ~30 min, spanning the completion of cellularization and the beginning of germ band extension. The temporal advantages offered by regulating these genes at a transcriptional elongation step as compared with chromatin remodeling and/or Pol II initiation complex assembly may be essential for the timely establishment of differing gene expression programs during cell fate specification in the Drosophila blastoderm embryo. The observations that Pol II molecules are enriched at the 5'-ends of a number of genes, coupled with findings that defects in transcriptional elongation factors produce specific developmental defects, strongly suggest that regulation of transcriptional elongation is a hitherto overlooked, but potentially widespread strategy for controlling gene expression during development (Wang, 2007).

Molecular characterization of Drosophila NELF

NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N- and C-terminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to the nucleoside analog DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and ß1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila (Wu, 2005; full text of article).

The amino acid sequences of the various NELF subunits were aligned to learn more about NELF. The sequences of NELF-B and NELF-D orthologs exhibit several regions with >50% identity distributed over their entire lengths. Analysis of subunit interactions has lead to a proposal that NELF-B and NELF-D form a central core that brings together NELF-A and NELF-E. NELF-A associates with NELF-D while NELF-E associates with NELF-B. The high degree of homology between NELF-B and NELF-D orthologs is consistent with constraints that might be dictated by the requirements that these two proteins interact with each other and with other subunits of NELF. The extensive colocalization of NELF-B and NELF-D on polytene chromosomes is also consistent with these two subunits forming the core of the NELF complex (Wu, 2005).

When NELF-A was initially identified through a BLAST search, it was of concern that the genome annotation because dNELF-A was over twice the size of human NELF-A. Immunoblot analysis verifies that dNELF-A is approximately twice the size of hNELF-A. The alignment shows that the size difference is due to a large nonconserved region located approximately between amino acids 300 and 1100. Inspection of the amino acid sequence in this region of Drosophila NELF-A reveals numerous tracts of poly-glutamine, poly-asparagine, poly-threonine and poly-alanine that are absent from hNELF-A. This lack of conservation raises the possibility that this region serves as a linker between two distinct functional domains defined by the regions of homology spanning the first 250 amino acids and the last 100 amino acids of NELF-A. The N-terminal region of homology in hNELF-A has been shown to interact with Pol II and NELF-D. Moreover, this region is required for NELF-mediated repression in vitro. Deletion of amino acids 431-528 of hNELF-A, which corresponds to the last 100 amino acids of dNELF-A, does not impair the ability of the NELF complex to inhibit transcription in vitro. Future analysis using genetic and molecular genetic approaches in Drosophila may help elucidate the function of the large nonconserved region and the C-terminal domain (Wu, 2005 and references therein).

hNELF-E binds RNA, and the region responsible for this activity exhibits 30% identity to amino acids 167-232 in dNELF-E. This RNA recognition motif has been shown to be required for NELF inhibition in human extracts. Two features found in hNELF-E are notably absent in dNELF-E. A 46 amino acid stretch spanning from 196 to 242 in hNELF-E that is composed primarily of alternating arginine and aspartic acid residues is absent -- the function of this region in hNELF-E is not known. Also absent from dNELF-E are serines that correspond to amino acids 181, 185, 187 and 191 in hNELF-E. These serines in hNELF-E are phosphorylated in vitro by P-TEFb, and these modifications reduce the binding between hNELF-E and RNA. It will be interesting to learn if phosphorylation of dNELF-E regulates its RNA binding activity (Wu, 2005 and references therein).

Two results implicate NELF in promoter proximal pausing on the hsp70 heat shock gene. Depleting salivary glands of NELF using RNA interference diminishes the level of promoter proximal pausing occurring on hsp70 in salivary glands. Also, NELF was found to cross-link to the hsp70 promoter region prior to heat shock induction. This study provides additional data strengthening the conclusion that NELF is involved in promoter proximal pausing (Wu, 2005).

Immunodepletion of NELF from nuclear extracts impairs formation of the paused Pol II. Importantly, cell-free transcription reactions with 3' O-methyl GTP indicate that NELF does not contribute to steps in the transcription process encompassing initiation and elongation to +15. It has also been determined that NELF is associated with the ß1-tubulin promoter. Genomic footprinting with permanganate and nuclear run-on analysis indicate that promoter proximal pausing occurs on the ß1-tubulin gene (Wu, 2005).

Based on strong immunofluorescence staining detected for DSIF and the weak staining for NELF at heat shock puffs, it is proposed that NELF dissociates from elongation complexes during activation but DSIF remains associated with the elongation complexes. The observation that the level of NELF cross-linking to the hsp70 promoter region after heat shock is ~5-fold less than before heat shock is consistent with this model. However, a 2-fold decrease in the level DSIF cross-linking to the hsp70 promoter region was also observed after heat shock, in agreement with a report from Saunders (2003). The decrease in DSIF cross-linking near the promoter was unexpected since heat shock puffs on polytene chromosomes stain very strongly with DSIF antibody. However, much of the signal detected on the polytene chromosomes probably originates from DSIF associated with elongation complexes in the body of the gene. In addition, the interaction of DSIF with Pol II in the promoter proximal region might be transient or not occur at all on some elongation complexes in the promoter proximal region. The rate of reinitiation at the hsp70 promoter is exceptionally high, once every few seconds, so a significant portion of the Pol II molecules might escape into the body of the gene before associating with DSIF (Wu, 2005).

The broad distribution of NELF-B and NELF-D on polytene chromosomes could mean that hundreds of genes harbor paused Pol II similar to that found on hsp70. Alternatively, NELF could have additional functions. mRNA capping is coupled to transcription, and recent results lead to the hypothesis that pausing induced by NELF might be important for coupling transcription and mRNA capping (Mandal, 2004). A subset of genes like hsp70 and ß1-tubulin might use cofactors such as GAGA factor to generate a stably paused state (Wu, 2005).

Search PubMed for articles about Drosophila Nelf

Mandal, S.S., Chu, C., Wada, T., Handa, H., Shatkin, A.J., Reinberg, D. (2004). Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II Proc. Natl Acad. Sci. 101: 7572-7577. Medline abstract: 15136722

Saunders, A., Werner, J., Andrulis, E.D., Nakayama, T., Hirose, S., Reinberg, D., Lis, J.T. (2003) Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo Science. 301: 1094-1096. Medline abstract: 12934007

Wang, X., Lee, C., Gilmour, D. S. and Gergen, J. P. (2007). Transcription elongation controls cell fate specification in the Drosophila embryo. Genes Dev. 21(9): 1031-6. Medline abstract: 17473169

Wu, C.-H., et al. (2003). NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17: 1402-1414. Medline abstract: 12782658

Wu, C. H., Lee, C., Fan, R., Smith, M. J., Yamaguchi, Y., Handa, H., Gilmour, D. S. (2005). Molecular characterization of Drosophila NELF. Nucleic Acids Res. 33(4): 1269-79. Medline abstract: 15741180

date revised: 1 December 2007

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