Computational protocol: Epigenetic Regulation of Cell Type–Specific Expression Patterns in the Human Mammary Epithelium

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Protocol publication

[…] We generated a total of eight ChIP-Seq libraries for K4 and K27 from CD44+ and CD24+ cells, four from one individual (sample 1) and four others from two individuals (samples 2 and 3). We mainly used data from sample 1 for follow up analyses as in this case both K4 and K27 data were available for both cell types. In addition, we also generated ChIP-Seq libraries using input DNA from each of the six cell populations analyzed and these were used as background to define K27 and K4 enriched regions. We mainly focused on K27-enriched genes due to the importance of PRC2 in ESCs , , , . ChIP-Seq demonstrated clear differences of histone methylation patterns in the two cell types for known stem and luminal epithelial cell-specific genes ().To investigate cell type–specific histone methylation profiles, we employed a spatial clustering approach for the identification of ChIP-enriched regions using the SICER algorithm . We identified 7,336 and 19,358 significantly K27-enriched islands in CD44+ and CD24+ cells from sample 1, respectively, using default conditions and FDR<0.001 as cutoff (details in Protocol S1). To examine the histone modification profiles of RefSeq genes, we analyzed the promoter regions of genes for overlap with K27- or K4-enriched islands. Using this approach we identified 1,182 K27-enriched genes in CD44+ cells, 716 (60.6%) of which was also K27-enriched in CD24+ cells, whereas 466 genes lost and 1,502 genes gained K27 mark during luminal lineage commitment ( and ).Genes enriched for K27 mark in both or in each of the two cells types were functionally distinct based on DAVID bioinformatics () and MetaCore (). Several of the highest ranked pathways and processes unique for genes enriched for K27 only in CD24+ cells (CD24+/K27+) are related to stem cell function such as cyclic AMP, WNT, and TGFb signaling. These results indicate that K27 modifications regulate key signaling pathways in the two cell types relevant to progenitor and luminal epithelial cell functions.To investigate whether genomic regions enriched for K27 or K4 marks only in CD24+ or CD44+ cells may contain binding sites for cell type–specific transcriptional regulators, we performed motif-search using Cistrome Analysis Pipeline Module (http://cistrome.dfci.harvard.edu/). We did not find any motifs significantly enriched in K4-enriched regions and found only a few motifs enriched in K27-marked regions (for example, TEAD1, SOX4, TCF3, and ZEB1 binding motifs are enriched in CD24+ specific K27 enriched regions) but the significance (z-score and p-value) for these motifs was low and none of them appeared to be cell type–specific. This result is not surprising, since ChIP-Seq data for multiple important transcriptional regulators (e.g., FoxA1) have demonstrated enrichment of binding sites in enhancer regions marked by histone H3K4me2 marks . […]

Pipeline specifications

Software tools SICER, MetaCore, Cistrome
Application ChIP-seq analysis
Organisms Homo sapiens
Chemicals Phenobarbital