Dataset Information

CX-MS of TFIIH and Def1 complex

ABSTRACT: The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans,Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response tomultiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH,the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Nir Kalisman

LAB HEAD: Nir Kalisman

PROVIDER: PXD008223 | Pride | 2018-10-22

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2017_06_18_01_TF2H_DEF1_NoAKTA.raw	Raw
	2017_06_18_02_TF2H_DEF1_NoAKTA.raw	Raw
	2017_06_18_03_TF2H_DEF1_Proteomics.raw	Raw
	2017_06_18_06_TF2H_DEF1_Peptides_A2.raw	Raw
	2017_06_18_09_TF2H_DEF1_Peptides_A3.raw	Raw

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Publications

Def1 interacts with TFIIH and modulates RNA polymerase II transcription.

Damodaren Nivedita N Van Eeuwen Trevor T Zamel Joanna J Lin-Shiao Enrique E Kalisman Nir N Murakami Kenji K

Proceedings of the National Academy of Sciences of the United States of America 20171127 50

The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans, Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response to multiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from ...[more]

PMID: 29180430

Similar Datasets

Project description:Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. In an effort to explore regulatory roles for Elongin A, we performed IP-MS. We constructed a DLD1 cell line expressing Flag-tagged Elongin A at endogenous levels and performed anti-Flag immuno-purification of solubilized chromatin followed by analysis of binding partners by mass spectrometry in triplicates. We identified a large group of Elongin A-associated proteins. We confirmed a subset of possible interacting proteins by Co-IP and western blotting. Consistent with previous studies, we identified RNA Pol II subunits and proteins related to transcription elongation and RNA processing. Among the top hits, we identified nearly all subunits of the PAF1 complex, except for RTF1, which does not stably associate with mammalian PAF1. Concordantly, in our previous PAF1 proteomic study, Elongin A was also among the top hits. We also identified several subunits of the Integrator complex, a multi-subunit complex that has been shown to participate in enhancer RNA (eRNA) processing, a finding consistent with our conclusion that Elongin A localizes to potential enhancers. Collectively, our results suggest that Elongin A stably interacts with RNAPII and the transcription machinery on chromatin. Taken together, our studies suggest that Elongin A associates with the transcription machinery at actively transcribed genomic regions and may be involved in the release of paused RNAPII. However, Elongin A does not appear to be critical for maintaining transcription elongation rates in vivo.