Fig. 4: Reactivation of macro-Bound Enhancers associates with oncogenic programs.

a Immunoblot of chromatin extracts were probed for mH2A1 and mH2A2 across a panel of breast cancer cell lines including the three different major sub-types (Luminal, HER2 positive and triple negative), and non-tumorigenic cells (human mammary epithelial cells, HMEC; and immortalized mammary cells MCF10A). Mutational status defined above. Amido Black of core histones used as loading control. b Immunohistochemistry (IHC) from normal breast tissue, ductal carcinoma in situ (DCIS) and grade III invasive tumors for mH2A2. Histone H3 IHC was used as a control (top right). Quantification of mH2A2 scoring for TMA and ISMMS patient samples according to the tumor grade or sub-class (below). B – Benign tissue, D – DCIS, I – Invasive. Column bar represents mean and SE. Unpaired (two tailed) student’s t-test *p < 0.05, **p < 0.005. c Principal component analysis of H3K27ac ChIP-seq signal in HMEC macro-Bound enhancers (left) and all CREs (right) in 12 breast cancer cell lines from Franco et al.⁴¹. d Volcano plot showing enrichment of breast cancer risk variants from GWAS studies (GWAS p-value < 5 × 10⁻⁸), in each CRE class in HMEC, MCF7 and 231 L cells, enrichment p-value and odds ratio calculated using GARFIELD. e Immunoblots for mH2A2 from chromatin extracts in MCF7 clones. H3 and histones (amido black) used as loading controls. f Proliferation of MCF7 clones (wild type and mH2A2 KO) transduced with H2A-GFP and analyzed by number of GFP cells using Incucyte. Data represented are mean with SE (n = 3). g Proliferation of MDA-MB-231L cells with over-expression of mH2A-GFP constructs (and H2A-GFP as control) determined by the number of GFP positive cells on Incucyte. Data are mean with SD (n = 9). Proliferation of cells over-expressing of mH2A1.1-GFP is significantly lower than control starting at 120 hours, mH2A1.2-GFP at 108 hours and mH2A2-GFP at 84 h (Two-way ANOVA with Dunnett’s multiple comparison test, *p < 0.05). h Tumorsphere formation assessed by number of cells upon mH2A2-GFP induction (Unpaired two tailed student’s t-test, p = 0.0428). Data represented are mean with SE (n = 3).