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 thehsp70gene inDrosophila. Immunodepletion of DSIF from aDrosophilanuclear extract reduced the level of polymerase that paused in the promoter proximal region ofhsp70. 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 thehsp70genes, 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 thehsp70genes. We propose that NELF and DSIF cause polymerase to pause in the promoter proximal region ofhsp70. 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.