Super-resolution microscopy reveals distinct epigenetic states regulated by estrogen receptor activity

Abstract Changes in gene expression regulated by ligand-dependent transcription factors such as estrogen receptor-α (ERα) involves the recruitment of coactivators including p300 that acetylates histone H3 at lysine 27 (H3K27ac). While H3K27ac marks active enhancers, the detailed chromatin architecture of enhancers remains unclear. Using super-resolution microscopy, we reveal distinct structural states of H3K27ac modified chromatin in response to ERα activation. In estradiol (E2)-treated cells, H3K27ac modified chromatin adopts open, elongated structures, while ERα inhibition induces compact, spherical H3K27ac modified chromatin conformations. Using MED1, a core subunit of the Mediator complex that bridges transcription factors with RNA polymerase II (Pol II), we demonstrate that larger H3K27ac structures are preferentially associated with active enhancers, whereas more compact structures show reduced MED1 association, consistent with a less active or inactive state. A constitutively active ERα mutation linked to endocrine therapy resistance in breast cancer maintains open chromatin states independent of ligand, suggesting sustained transcriptional activity. Our findings provide the first direct visualization of H3K27ac associated chromatin structural dynamics, challenging the assumption that H3K27ac modification alone is sufficient to lead to enhancer activation. By demonstrating that H3K27ac architecture is dynamically regulated by ERα, we establish a new paradigm for understanding epigenetic regulation and highlight potential therapeutic targets for endocrine therapy resistant cancers. Competing Interest Statement RJ receives sponsored research support from Lilly and Novartis. RJ is a consultant for Lilly, Novartis, Pfizer, Carrick Therapeutics, and Astra Zeneca. MB receives sponsored research support from Novartis. MB serves on the SAB and holds equity in Kronos Bio and serves on the SAB and holds equity in GV20 Therapeutics. Footnotes ↵10 Lead contact ↵* Co-first authors In this revised submission, we have reorganized Figures 1 to 4 and the supplementary materials, and incorporated new data into the revised Figure 4 in response to the reviewers comments. The manuscript text has been updated accordingly to reflect these changes https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fsecure-web.cisco.com%2F1ZF8q8_aloYFHMxAg2muhvMMMsLwGgaWKbYVgzeqJySMs9GDlw1UoGvRqFDux2or_40HvTMIyO1TWvBi8kqzPwq1Jvn1nt-OsYoLLKwTXXdmv7z8LjXRXQiA6SWXkXEhm6_XNkpFU-F0T5U9upp3axKvMr2bdOTWtmSM5aEb2Fna5K-7mPFCvhxMI2RpPli49vMNVZvN1FZEZ56b-kdUcVSD-RGPimjE4ziHPnp2RjK8L8V-x5jhhKFMSKtl8Wtefw6b3TVdaE3XVe3ih_oCbrF4i9YDi3IVbhx3YSpthPmA4JaWcBumII25j7JQZuBgq%2Fhttps%253A%252F%252Fgithub.com%252Fzang-lab%252F3DSIManalysis&data=05%7C02%7CTara_Akhshi%40dfci.harvard.edu%7Cb944d3f8a29a4e49d71408dd8cd8c33f%7C720edb1f5c4e40438141214a63a7ead5%7C0%7C0%7C638821584792679492%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=LFTCZg7jGBrhoOYZJpZJhOPM3Ju%2BzWupVLyDtgHRA5c%3D&reserved=0