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.
Wu
Chwen-Huey
CH
Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Yamaguchi
Yuki
Y
Benjamin
Lawrence R
LR
Horvat-Gordon
Maria
M
Washinsky
Jodi
J
Enerly
Espen
E
Larsson
Jan
J
Lambertsson
Andrew
A
Handa
Hiroshi
H
Gilmour
David
D
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics / Q000502:physiology; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000502:physiology; D017353:Gene Deletion; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins; D012469:Salivary Glands / Q000201:enzymology / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D017931:DNA Primers; C492141:DSIF protein, Drosophila; D029721:Drosophila Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C481011:SUPT4H1 protein, human; C112224:SUPT5H protein, human; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C118437:negative elongation factor; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases