Fig. 1: Abnormal transcription is associated with open chromatin from freshly isolated mammary epithelial cells in Brca1MKO mice.
Experiments designed for isolating fresh epithelial cells from mammary tissues of WTV, WTP12 (a), MTV, and MTP12 (b) mice. The triplicated samples (n = 6 mice/group) were collected from each genotype. The RNA-seq and ChIP-seq were performed and the candidate genes from combination analysis will be followed by functional analysis. Representative IHC images with antibodies of H3K4me3 (c) and IF staining with H3K9me3 (d) on WTP12 and MT P12 mouse mammary gland tissues and percent of positive cells from 5 images for H3K4me3 and 6 images for H3k9me3 were quantified by using ImageJ. (n = 3 mice/group). The binding intensity on promoter region across -2Kb and +2Kb of the TSS sites (e) and genome-wide (f) in both WTP12 and MTP12. MACS2 tools for peak calling with default parameters were used. By default, the q-value (minimum false discovery rate) cutoff for peak detection is set to 0.05. The plot was generated by the software “deeptools” (https://deeptools.readthedocs.io/en/develop/index.html. g ChIP-seq raw data were converted into the bam file and used the “ngsplot” tool to quantify and test the intensity for H3K4me3. h The upregulated genes in WTP12 and MTP12 with scaled mean expression from normalized counts of each gene are combined first with removed duplication gene names and then extract out the same gene list from WTV group to generate the heatmap by using the R package “pheatmap” with information in Supplementary data 3. Summary of analysis of genes based on overlapping binding patterns of Flag/ERα/H3K4me3/H3K27Ac (i), binding numbers with different combinations of antibodies (j), and distributions by Venn diagram 2.1 (k) in MTP12, WTP12, and WTP12/MTP12. The ChIP-seq data were calculated using the “clusterProfiler” R package with cut off values of p < 0.05 [77]. l Visualization of ChIP binding peaks of Esr1 gene against antibodies of Flag, (l, orange color), ERα antibody (burgundy color) in MTP12 cells and ERα antibody (blue color) in WTP12 cells. The images were generated using IGB (The Integrated Genome Browser free software for distribution and exploration of genome-scale datasets) [78]. m Visualization of ChIP binding peaks of Esr1 gene against antibody of H3K4me3 in MTP12 (burgundy color) and (blue color) in WTP12 cells. n Visualization of ChIP binding peaks of Esr1 gene against antibody of H3K27Ac in MTP12 (burgundy color) and (blue color) in WTP12 cells. Differential binding peak intensities binding analysis of Esr1, Bard1, Shcbp1, Myc, Rel, Bcas3, Gadd45g, and Crebbp by ERα (o) and Η3Κ4me3 (p) between MTP12 and WTP12 cells with diffReps version 1.55.4 [79]. Using likelihood-ratio test and annotated to the closest genes. Genes associated with at least one significant genomic region (P value < 0.05, that is, -log10(P value) > 1.3 and log2 (fold change) >0.25) were denoted as statistically differentially marked. When a gene is annotated with multiple significant genomic regions, the most significant one is assigned to that gene.
