ARTICLE https://doi.org/10.1038/s41467-019-14098-x OPEN Epigenetic reprogramming at estrogen-receptor binding sites alters 3D chromatin landscape in endocrine-resistant breast cancer Joanna Achinger-Kawecka 1,2, Fatima Valdes-Mora 1,2, Phuc-Loi Luu1,2, Katherine A. Giles1, C. Elizabeth Caldon3, Wenjia Qu1, Shalima Nair1, Sebastian Soto1, Warwick J. Locke1, Nicole S. Yeo-Teh1, Cathryn M. Gould1, Qian Du1, Grady C. Smith1, Irene R. Ramos4, Kristine F. Fernandez3, Dave S. Hoon 4, Julia M.W. Gee5, Clare Stirzaker1,2 & Susan J. Clark 1,2* 1234567890():,; Endocrine therapy resistance frequently develops in estrogen receptor positive (ER+) breast cancer, but the underlying molecular mechanisms are largely unknown. Here, we show that 3-dimensional (3D) chromatin interactions both within and between topologically associating domains (TADs) frequently change in ER+ endocrine-resistant breast cancer cells and that the differential interactions are enriched for resistance-associated genetic variants at CTCF- bound anchors. Ectopic chromatin interactions are preferentially enriched at active enhancers and promoters and ER binding sites, and are associated with altered expression of ER- regulated genes, consistent with dynamic remodelling of ER pathways accompanying the development of endocrine resistance. We observe that loss of 3D chromatin interactions often occurs coincidently with hypermethylation and loss of ER binding. Alterations in active A and inactive B chromosomal compartments are also associated with decreased ER binding and atypical interactions and gene expression. Together, our results suggest that 3D epi- genome remodelling is a key mechanism underlying endocrine resistance in ER+ breast cancer. 1 Epigenetics Research Laboratory, Genomics and Epigenetics Theme, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia. 2 St. Vincent’s Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW 2010, Australia. 3 Cancer Theme, The Kinghorn Cancer Centre, Sydney, NSW 2010, Australia. 4 Department of Translational Molecular Medicine, John Wayne Cancer Institute, Santa Monica, CA, USA. 5 Breast Cancer Molecular Pharmacology Group, School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Wales CF10 3NB, UK. *email: [email protected] NATURE COMMUNICATIONS | (2020) 11:320 | https://doi.org/10.1038/s41467-019-14098-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-14098-x nappropriate reprogramming of the estrogen receptor (ER) MCF7 data17 cluster away from the resistant derivatives TAMR signalling network in mammary epithelial cells initiates neo- and FASR (Supplementary Fig. 1a), irrespective of the time in I + plastic transformation and drives ER-positive (ER ) breast culture. cancer1.ER+ breast cancer patients will receive long-term Next to identify the differential chromatin interactions between endocrine therapies, including tamoxifen and fulvestrant, to the parental MCF7 cells and endocrine-resistant cells we used the inhibit the ER-signalling pathways on which their tumours are diffHiC method18, and found 981 significantly different interac- dependent2. Tamoxifen is one of the selective estrogen receptor tions between MCF7 and tamoxifen-resistant TAMR cells modulators (SERM) and acts as an antiestrogen in the mammary (diffHiC, FDR < 0.05, Supplementary Data 1) and 2596 signifi- tissue whereas fulvestrant is a selective estrogen receptor cantly differential interactions between MCF7 and fulvestrant- degrader (SERD) and acts by binding to the estrogen receptor resistant FASR cells (diffHiC, FDR < 0.05, Supplementary Data 2) and destabilising it3. Endocrine treatment significantly reduces at 20 kb resolution. Differential interactions were more often lost the relapse rate by almost 50%; however, around 33% of patients with the development of fulvestrant resistance (62% are MCF7- treated with endocrine therapy develop endocrine resistance and specific), while there were similar numbers of differential relapse within 15 years of first receiving treatment4. The mole- interactions lost and gained in the tamoxifen-resistant cells cular factors that define endocrine response in ER+ breast (46% are TAMR-specific) (Fig. 1b). The majority of differential cancer patients remain poorly understood. Recent research interactions detected in TAMR cells were not present in FASR suggests that global reprogramming of estrogen-responsive cells (Fig. 1b), potentially consistent with the different mode of regions of the genome can modulate endocrine sensitivity and action between tamoxifen and fulvestrant and the different contribute to the onset of ER+ breast cancer and the acquisition pathways to development of endocrine resistance in these two of endocrine resistance5–7. We also reported that differential models15,16. DNA methylation at estrogen-responsive enhancers is associated Since 3D chromatin interactions bring distal regulatory with endocrine response in breast cancer6, raising the possibility elements, such as enhancers into close proximity of their target that dynamic three-dimensional (3D) chromatin remodelling of genes, we explored whether differential interactions gained and ER-mediated enhancer−promoter interactions could potentially lost in endocrine resistance include direct enhancer−promoter underlie the development of endocrine resistance in breast interactions. We integrated the differential chromatin interac- cancer. However, these and related studies6,8 did not directly tion data with chromatin state information based on five ChIP- interrogate the role of 3D genome architecture in association seq marks (H3K27ac, H3K4me1, H3K4me3, H2AZac and with such alterations. H3K27me3) using chromHMM19. Interestingly, all differential Chromosome conformation capture (Hi-C) interaction maps interactions were significantly enriched for enhancer and provide information on multiple levels of 3D chromatin structure promoters, as well as CTCF sites, regardless of the TAMR or and three main layers of genome organisation have been descri- FASR treatment regime (Fig. 1c). However, gained chromatin bed to date9,10. First, at the level of single genes, the genome is interactions in TAMR and FASR cells showed higher enrich- organised into local enhancer–promoter interactions. Second, the ment of active enhancer marks (H3K4me1 and H3K27ac), genome is segmented into topologically associated domains compared to lost interactions (Supplementary Fig. 1b). Similarly (TADs) that are ~1 Mb in size and encompass multiple genes and there was increased enrichment of the active promoter mark regulatory elements10,11. TADs are conserved and largely invar- H3K4me3 at gained interactions in TAMR and FASR cells iant between different cell types, while the chromatin interactions relative to MCF7 cells (Supplementary Fig. 1b). within TADs are more tissue specific12–14. Finally, at the higher Differential interactions are frequently associated with altered level, chromatin interactions and TADs are organised into expression of the genes they connect20,21. Therefore, to address functionally distinct compartments, comprising large regions that whether differential interactions present in endocrine-resistant are either A-type (active) or B-type (inactive)9. cells are associated with deregulation of gene expression, we Here, we characterise the 3D chromatin organisation in identified genes located at anchors of differential interactions and endocrine-sensitive and endocrine-resistant ER+ breast cancer compared their expression between MCF7 cells and TAMR and cell lines. We show that 3D epigenome remodelling is a key FASR cells (Supplementary Data 3). We found that differentially mechanism associated with endocrine resistance that consists of expressed genes were enriched for pathways known to be aberrant DNA methylation and differential ER-bound enhancer associated with endocrine resistance (e.g. estrogen response) −promoter interactions. and cancer (e.g. EMT, angiogenesis) (Supplementary Fig. 1c). Differential interactions in FASR cells overlapped promoters of 2069 genes and loss of interactions in these cells was associated Results with decreased expression of 213 genes (Fig. 1d). Gain of Differential interactions associate with altered expression.To interactions was associated with increased expression of 170 initially address if 3D epigenetic remodelling is associated with genes (Fig. 1e). In TAMR cells, 500 genes were located at endocrine resistance, independent of the class of endocrine differential chromatin interactions. Loss of interactions resulted therapy, we performed in situ Hi-C experiments in parental in significant decrease in gene expression, with 50 genes endocrine-sensitive ER+ MCF7 cells, tamoxifen-resistant downregulated (Supplementary Fig. 1d), while gained interactions (TAMR)15 cells and fulvestrant-resistant (FASR)16 cells (Sup- were associated with increased expression of 21 genes (Supple- plementary Table 1). Multidimensional scaling analysis of Hi-C mentary Fig. 1e). Overall, in both TAMR and FASR cells, lost and contact maps revealed high dissimilarity between MCF7, TAMR gained interactions were enriched for differentially expressed and FASR genomes (Fig. 1a). As a control to ensure that the genes (FDR < 0.05) (chi-square test P < 0.001) with most of the differences in the chromatin contacts were associated with genes located at anchors of lost interactions being downregulated, endocrine resistance, and not due to long-term culture, we per- and genes located at ectopic/gained