Proteomic analysis demonstrates activator- and chromatin-specific recruitment to promoters.
In-depth characterization of RNA polymerase II preinitiation complexes remains an important and challenging goal. We used quantitative mass spectrometry to explore context-dependent Saccharomyces cerevisiae preinitiation complex formation at the HIS4 promoter reconstituted on naked and chromatinized DNA templates. The transcription activators Gal4-VP16 and Gal4-Gcn4 recruited a limited set of chromatin-related coactivator complexes, namely the chromatin remodeler Swi/Snf and histone acetyltransferases SAGA and NuA4, suggesting that transcription stimulation is mediated through these factors. Moreover, the two activators differentially recruited the coactivator complexes, consistent with specific activator-coactivator interactions. Chromatinized templates suppressed recruitment of basal transcription factors, thereby amplifying the effect of activators, compared with naked DNA templates. This system is sensitive, highly reproducible, and easily applicable to mapping the repertoire of proteins found at any promoter.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; Department of Cancer Biology, Harvard Medical School, Boston, Massachusetts 02115.
Timothy W
TW
Sikorski
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Yoo Jin
YJ
Joo
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; Department of Cancer Biology, Harvard Medical School, Boston, Massachusetts 02115; Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Scott B
SB
Ficarro
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; Department of Cancer Biology, Harvard Medical School, Boston, Massachusetts 02115; Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Manor
M
Askenazi
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115. Electronic address: steveb@hms.harvard.edu.
Stephen
S
Buratowski
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; Department of Cancer Biology, Harvard Medical School, Boston, Massachusetts 02115; Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115. Electronic address: jarrod_marto@dfci.harvard.edu.
Jarrod A
JA
Marto
D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D050976:Basic-Leucine Zipper Transcription Factors; D002843:Chromatin; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C501391:GCN4 protein, S cerevisiae; D009687:Nuclear Proteins; C507785:SAGA complex, S cerevisiae; C072981:SWI3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C504740:NuA4 protein, S cerevisiae