Summary 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 pulldown 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.

Highlights d Full-length AR follows a unique head-to-head and tail-to-tail dimerization d AR LBD and DBD and NTD all form the dimerization interface d AR interacts with one SRC-3 and one p300 d AR N-terminal domain plays a major role in recruiting SRC-3 and p300

Mining of integrated public transcriptomic and ChIP-Seq (cistromic) datasets can illuminate functions of mammalian cellular signaling pathways not yet explored in the research literature. Here, we designed a web knowledgebase, the Signaling Pathways Project (SPP), which incorporates community classifications of signaling pathway nodes (receptors, enzymes, transcription factors and co-nodes) and their cognate bioactive small molecules. We then mapped over 10,000 public transcriptomic or cistromic experiments to their pathway node or biosample of study. To enable prediction of pathway node-gene target transcriptional regulatory relationships through SPP, we generated consensus ‘omics signatures, or consensomes, which ranked genes based on measures of their significant differential expression or promoter occupancy across transcriptomic or cistromic experiments mapped to a specific node family. Consensomes were validated using alignment with canonical literature knowledge, gene target-level integration of transcriptomic and cistromic data points, and in bench experiments confirming previously uncharacterized node-gene target regulatory relationships. To expose the SPP knowledgebase to researchers, a web browser interface was designed that accommodates numerous routine data mining strategies. SPP is freely accessible at https://www.signalingpathways.org.

Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter in vitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain "poised" for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription.

Integrated mining of public transcriptomic and ChIP-Seq datasets has the potential to illuminate facets of mammalian cellular signaling pathways not yet explored in the research literature.Here, we designed a web knowledgebase, the Signaling Pathways Project (SPP), which incorporates stable community classifications of the four major categories of signaling pathway node (receptors, enzymes, transcription factors and co-nodes) and their cognate bioactive small molecules (BSMs). We then mapped over 10,000 public transcriptomic or cistromic experiments to their relevant signaling pathway node, BSM or biosample of study. To provide for prediction of pathway node-target transcriptional regulatory relationships, we generated consensus 'omics signatures, or consensomes, based on measures of significant differential expression of genomic targets across all underlying transcriptomic experiments. To expose the SPP knowledgebase to researchers, a web browser interface accommodates a variety of routine data mining strategies. Consensomes were validated using alignment with literature-based knowledge, gene target-level integration of transcriptomic and ChIP-Seq data points, and in bench experiments that confirmed previously uncharacterized node-gene target regulatory relationships. SPP is freely accessible at https://beta.signalingpathways.org.Individual dataset pages enable integration of SPP with the research literature via digital object identifier (DOI)-driven links from external sites, as well as for citation of datasets to enhance their FAIR status 3,4 .