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PMID: 19279664 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S.

Spt6 enhances the elongation rate of RNA polymerase II in vivo.

The EMBO journal ·Vol. 28 ·No. 8 ·2009-04-22 ·Pages 1067-77

Ardehali MB, Yao J, Adelman K, Fuda NJ, Petesch SJ, Webb WW, Lis JT

Abstract

Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.

MeSH Terms
Animals Animals, Genetically Modified Cell Line Chromosomal Proteins, Non-Histone/genetics,metabolism Drosophila Proteins/genetics,metabolism Drosophila melanogaster/genetics,physiology Fluorescence Recovery After Photobleaching HSP70 Heat-Shock Proteins/genetics,metabolism Heat-Shock Proteins/genetics,metabolism Histones/metabolism Peptide Elongation Factors/genetics,metabolism RNA Interference RNA Polymerase II/genetics,metabolism Recombinant Fusion Proteins/genetics,metabolism Transcription, Genetic Transcriptional Elongation Factors/genetics,metabolism
Chemicals
Chromosomal Proteins, Non-Histone Drosophila Proteins HSP70 Heat-Shock Proteins Heat-Shock Proteins Histones Hsp83 protein, Drosophila Peptide Elongation Factors Recombinant Fusion Proteins Spt6 protein, Drosophila Transcriptional Elongation Factors atms protein, Drosophila transcription factor S-II SPT5 transcriptional elongation factor RNA Polymerase II
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Ardehali M Behfar
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Yao Jie
Adelman Karen
Fuda Nicholas J
Petesch Steven J
Webb Watt W
Lis John T
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
1460-2075
DOI
10.1038/emboj.2009.56
Published
2009-04-22
Epub
2009-00-12
Pages
1067-77
Language
English
Region
England
NLM ID
8208664
PMCID
PMC2683705
Subset
IM
Grants
NIGMS NIH HHS · R01 GM025232 · United States
NIGMS NIH HHS · R01 GM025232-33 · United States
NIGMS NIH HHS · R37 GM025232 · United States
NIGMS NIH HHS · GM25232 · United States
External Links
PubMed source DOI: 10.1038/emboj.2009.56 PMC Full Text View journal details
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