Figure 2: ESR1 regulation of enhancer sites commonly hypermethylated in endocrine-resistant cell models.

(a) A bar plot showing the association of HM450 probes that were more heavily methylated in endocrine-resistant cell models (compared with MCF7 cells) and also specifically located in enhancer regions, across ESR1-, FOXA1- and GATA3-binding sites in MCF7 cells. The height of the bars represents the enrichment measured as a ratio between the frequency of hypermethylated probes in enhancers overlapping a transcription factor binding site over the expected frequency if such overlaps were to occur at random across the genome (*P value<<0.0001; hypergeometric test). The numbers of commonly hyper/hypomethylated probes located within each specific region are presented in the columns. (b) A Venn diagram showing the overlap of enhancer-specific HM450 methylation probes that are more heavily methylated in multiple endocrine-resistant cell models (compared with MCF7 cells) across ESR1-, FOXA1- and GATA3-binding sites. (c) A box plot showing the log-fold change (logFC) in ESR1 binding signal at ESR1-enhancer sites that contain at least one commonly hypermethylated probe (yellow box) and all other ESR1-enhancer sites that overlap a HM450 probe (grey box) in TAMR cells compared with the parent MCF7 cells. The mean logFC in ESR1 binding at hypermethylated ER-enhancer sites is −2.29 and the mean logFC of all other ESR1-enhancer sites is −0.52 (*P<<0.0001; t-test). (The whiskers of the box plot extend to the most extreme data point, which is no more than 1.5 × interquartile range from the box). (d) IGV screen shots to illustrate the loss of ESR1 binding in TAMR cells compared with the parent MCF7 cells in enhancer regions that overlap methylation probes that are more heavily methylated in the endocrine-resistant cell models. The MCF7 ChromHMM regions are colour coded as follows—blue, enhancer; yellow, transcribed; green, promoter; light blue, CTCF; and burgundy, transcribed. The HM450 β values are shown for the MCF7 (green), MCF7X (burgundy), TAMR (orange) and FASR cells (red) and are representative of biological duplicates. ESR1 ChIP data (blue) is presented in duplicate for both MCF7 and TAMR cells. The ESR1 enhancers that overlap the regions of endocrine-resistant-specific hypermethylation are highlighted by the blue boxes.