NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila.

• 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

Keyword

Animals

Base Sequence

Cell Nucleus/genetics/physiology

Chromosome Mapping

Cloning; Molecular

DNA Primers

DNA-Directed RNA Polymerases/genetics

Drosophila Proteins/*genetics/metabolism

Drosophila melanogaster/embryology/*genetics

Embryo; Nonmammalian/physiology

Gene Deletion

HSP70 Heat-Shock Proteins/*genetics

Heat-Shock Proteins/genetics

Humans

Nuclear Proteins/*genetics/metabolism

Protein Subunits/genetics

RNA Polymerase II/genetics/metabolism

RNA-Binding Proteins/*genetics/metabolism

Repressor Proteins

Salivary Glands/enzymology/physiology

Transcription Factors/*genetics/metabolism

Publication and Content Type

vet (subject category)

art (subject category)