Proteomic Analysis of Coregulators Bound to ERα on DNA and Nucleosomes Reveals Coregulator Dynamics

Mol Cell. 2013 Jul 25;51(2):185-99. doi: 10.1016/j.molcel.2013.06.007. Epub 2013 Jul 11.

Abstract

Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pull-down assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17β-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an "activated" state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a "natural" ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism*
  • Chromatin Immunoprecipitation
  • DNA / genetics
  • DNA / metabolism*
  • DNA-Activated Protein Kinase / genetics
  • DNA-Activated Protein Kinase / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • E1A-Associated p300 Protein / genetics
  • E1A-Associated p300 Protein / metabolism
  • Estrogen Receptor alpha / genetics
  • Estrogen Receptor alpha / metabolism*
  • Estrogens / pharmacology
  • Female
  • Forkhead Box Protein O1
  • Forkhead Transcription Factors / genetics
  • Forkhead Transcription Factors / metabolism
  • Gene Expression Regulation, Neoplastic*
  • HeLa Cells
  • Humans
  • MCF-7 Cells
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Nuclear Receptor Coactivator 3 / genetics
  • Nuclear Receptor Coactivator 3 / metabolism
  • Nucleosomes / genetics
  • Nucleosomes / metabolism*
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Promoter Regions, Genetic
  • Proteomics*
  • Response Elements / genetics*
  • Sialoglycoproteins / genetics
  • Sialoglycoproteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trans-Activators
  • Transcription, Genetic
  • Transcriptional Activation

Substances

  • DNA-Binding Proteins
  • Estrogen Receptor alpha
  • Estrogens
  • FOXO1 protein, human
  • Forkhead Box Protein O1
  • Forkhead Transcription Factors
  • Multiprotein Complexes
  • Nuclear Proteins
  • Nucleosomes
  • Peptide Fragments
  • Sialoglycoproteins
  • Trans-Activators
  • bone sialoprotein (35-62), human
  • estrogen receptor alpha, human
  • DNA
  • E1A-Associated p300 Protein
  • EP300 protein, human
  • NCOA3 protein, human
  • Nuclear Receptor Coactivator 3
  • DNA-Activated Protein Kinase
  • PRKDC protein, human