NELF and DSIF cause promoter proximal pausing on thehsp70promoter inDrosophila

Abstract

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 from a Drosophila nuclear extract reduced the level of polymerase that paused in the promoter proximal region of hsp70. Depletion of one NELF subunit in salivary glands using RNA interference also reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborated the cross-linking result and showed 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 were strongly recruited to chromosomal puffs harboring the hsp70 genes. We propose 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.

Imaging Drosophila gene activation and polymerase pausing in vivo

“…Some, but not all, activators have been shown to interact with P-TEFb, so other mechanisms of P-TEFb recruitment need to be considered 26 . The transition into productive elongation seems to correlate with the loss of NELF from the promoter 27 . In contrast, DSIF remains associated with productively elongating Pol II and is thought to have a positive role after escape from the pause 2,28 .…”

“…Using ChIP assays, both DSIF and NELF were found to be located along with paused Pol II in the promoter-proximal regions of uninduced heat shock genes (Fig. 4) 2,27 . The P-TEFb kinase is recruited to active genes where it overcomes the negative effects through its kinase activity, which can phosphorylate DSIF, NELF and Ser 2 residues of the carboxy-terminal domain of the largest subunit of Pol II 2,26 .…”

“…Whereas these melted residues tend to cluster in the region of 20-50 base pairs and highly correlate with paused Pol II, the pattern of reactivity can be influenced by other proteins either protecting or altering the reactivity of T residues. Additional signatures can be detected by ChIP assays and include the presence of NELF, and Ser 5-but not Ser 2-phosphorylated carboxy-terminal domain of the largest subunit (RpII215) of Pol II 20,27 .…”

Nucleosome organization in the Drosophila genome

“…Rather, the positioned +1 nucleosome might be contributing to pol II pausing, which is consistent with other studies [34][35][36][37] . Other factors including NELF are likely to make significant contributions to pausing as well 30,38,39 .…”

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

“…There is ample genetic and biochemical evidence from budding yeast, zebrafish, roundworm, fruitfly, and mammalian systems that Spt5 and Spt4 exert both negative and positive effects on transcription elongation (Hartzog et al 1998;Andrulis et al 2000;Guo et al 2000;Renner et al 2001;Shim et al 2002;Wu et al 2003;Jennings et al 2004). Spt5 is a large polypeptide (z1000-1200 amino acids) composed of multiple domain modules, including an acidic N-terminal domain, central NusG-like NGN (NusG N-terminal homology), and KOW domains (Hartzog et al 1998;Wade et al 1998a;Ponting 2002), and a CTD targeted for threonine phosphorylation Yamada et al 2006).…”