Dynamic assembly and activation of estrogen receptor α enhancers through coregulator switching

Abstract

Although many features of active transcriptional enhancers have been defined by genomic assays, we lack a clear understanding of the order of events leading to enhancer formation and activation as well as the dynamics of coregulator interactions within the enhancer complex. Here, we used selective loss-or gain-of-function mutants of estrogen receptor α (ERα) to define two distinct phases of ligand-dependent enhancer formation. In the first phase (0-20 min), p300 is recruited to ERα by Mediator as well as p300's acetylhistone-binding bromodomain to promote initial enhancer formation, which is not competent for sustained activation. In the second phase (20-45 min), p300 is recruited to ERα by steroid receptor coregulators (SRCs) for enhancer maturation and maintenance. Successful transition between these two phases ("coregulator switching") is required for proper enhancer function. Failure to recruit p300 during either phase leads to abortive enhancer formation and a lack of target gene expression. Our results reveal an ordered and cooperative assembly of ERα enhancers requiring functional interplay among p300, Mediator, and SRCs, which has implications for hormone-dependent gene regulation in breast cancers. More broadly, our results demonstrate the unexpectedly dynamic nature of coregulator interactions within enhancer complexes, which are likely to be a defining feature of all enhancers.

Regulatory Sharing Between Estrogen Receptor α Bound Enhancers

“…CISH-2). These findings are consistent with previous work showing that recruitment of p300 and subsequent histone acetylation appears to be a crucial step in estrogen-dependent gene regulation, as inhibitors of the p300 catalytic domain abrogate estrogen-dependent transcription (Murakami et al, 2017). The fact that permissible chromatin in the absence of estrogens appears important suggests that these sites may be somehow primed for activation; however, it remains unclear what factors are involved in priming these sites.…”

ERα-Mediated Nuclear Sequestration of RSK2 Is Required for ER+ Breast Cancer Tumorigenesis

“…The interaction of RSK2 with ERa, which is disrupted by mutagenesis of RSK2, antiestrogens, or silencing ERa, allows ERK1/2-activated RSK2 to accumulate to high levels in the nucleus. Loss of RSK2 from the nucleus results in reduced ER þ tumor formation, which is due to the inactivation of the proneoplastic transcriptional network comprised of ESR1, EP300, GATA3, and FOXA1 that is critical to maintain ER þ breast cancer (42,46). This conclusion is further supported by the ability of the RSK2 gene signature to stratify breast cancer patients according to their ERa status.…”

Distinct Roles for BET Family Members in Estrogen Receptor α Enhancer Function and Gene Regulation in Breast Cancer Cells

“…Changes in the steady-state levels of mRNAs/eRNAs were analyzed by qRT-PCR, as described previously (5). The resulting cDNA was analyzed by qPCR using primer sets listed in the Supplementary Information.…”

“…ChIP was performed as described previously (5). The ChIPed DNA was dissolved in water and analyzed by qPCR using the primer sets listed in the Supplementary Information.…”

“…3,4). Upon binding to chromatin, ERa recruits additional transcription coregulators (e.g., the steroid receptor coactivator proteins, SRC) and chromatin remodelers to establish fully active transcription enhancer complexes (2,5,6). Active ERa enhancer complexes, in turn, loop to target gene promoters to drive the recruitment of the RNA polymerase II (Pol II) machinery and subsequent transcriptional output (5)(6)(7)(8).…”