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

• Proteomics of DNA-bound E2-liganded ERα defines an initial set of stably bound CoRs
• DNA-dependent protein kinase dynamically “activates” CoR-ERE complexes
• An ERE from the GREB1 gene recruits similar CoR complexes as does consensus EREs
• Histone mark-dependent “readers” produce direct effects on ERα-driven transcription

SummaryChromatin 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.

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