PMC

Proximal Estradiol-responsive genes and carrier ERα ChIP-seq enrichment. A. Estradiol up- (white) and down-regulated (black) genes were interrogated for proximal ERα binding events under saturated conditions with 20*10⁶ cells (right), as well as ChIP-seq on 10,000 cells without (left) and with mRNA/histones carrier (middle). The percentage of E2-reponsive genes with a proximal ERα binding site is shown. B. ERα binding events with mRNA/histones carriers were ranked and proximal E2-upregulated genes were determined for the top 10 highest intensity binding sites. Heatmap indicates tag count.

Carrier-optimized ChIP-seq in MCF7 cells. A. ChIP-qPCR on pS2/TFF1 promoter for ERα with differential carrier conditions (none, glycogen, mRNA/histones, glycogen/mRNA/histones). Data were normalized over negative control region and input. Bars show standard deviation from triplicate measurements. B. Genome browser snapshot of ERα ChIP-seq, depicting the pS2/TFF1 promoter and enhancer region for 10,000 cells without (top) or with mRNA/histones carrier (middle). Data was compared to a saturated ERα ChIP-seq from 20*10⁶ cells (bottom). Genomic coordinates and tag count are indicated. C. Heatmap visualization of ERα ChIP-seq data, depicting all binding events centred on the peak region within a 5 kb window around the peak. All peaks for the 20*10⁶ cells ChIP-seq condition were ranked on intensity, and the data from both 10,000 cells-conditions were plotted in identical order.

Carrier ChIP-seq analyses on breast tumour core needle biopsies. A. Genome browser snapshots of ERα ChIP-seq on MCF7 cells (black) and breast cancer core needle biopsy samples with glycogen (blue) or mRNA/histones (red) as carriers. Tag count is shown on the Y-axis. Genomic locations are indicated. B. Genomic distributions of ERα binding events in MCF7 cells (top) and breast tumour biopsy samples with mRNA/histones as carrier (bottom). C. Motif analysis of ERα binding events on a core needle biopsy sample, with mRNA/histones as carrier. Indicated are the top 3 enriched motifs and p-values. D. Luminal enrichment for ERα carrier ChIP-seq genes. Genes, identified as ‘luminal’ or ‘basal’ signatures by Perou et al. [2] were analysed for proximal ERα binding events. Enrichment of ‘luminal’ over ‘basal’ genes was found. E. Genes, identified in D., were tested for correlation with recurrence-free survival, using Estrogen Receptor positive samples from a meta-analysis of breast cancer patients []. Expression of identified genes correlated with a favourable outcome after endocrine treatment.