Cell type-specific DNA methylation patterns in the human breast

Abstract

Cellular identity and differentiation are determined by epigenetic programs. The characteristics of these programs in normal human mammary epithelium and their similarity to those in stem cells are unknown. To begin investigating these issues, we analyzed the DNA methylation and gene expression profiles of distinct subpopulations of mammary epithelial cells by using MSDK (methylation-specific digital karyotyping) and SAGE (serial analysis of gene expression). We identified discrete cell-type and differentiation state-specific DNA methylation and gene expression patterns that were maintained in a subset of breast carcinomas and correlated with clinically relevant tumor subtypes. CD44+ cells were the most hypomethylated and highly expressed several transcription factors with known stem cell function including HOXA10 and TCF3. Many of these genes were also hypomethylated in BMP4-treated compared with undifferentiated human embryonic stem (ES) cells that we analyzed by MSDK for comparison. Further highlighting the similarity of epigenetic programs of embryonic and mammary epithelial cells, genes highly expressed in CD44+ relative to more differentiated CD24+ cells were significantly enriched for Suz12 targets in ES cells. The expression of FOXC1, one of the transcription factors hypomethylated and highly expressed in CD44+ cells, induced a progenitor-like phenotype in differentiated mammary epithelial cells. These data suggest that epigenetically controlled transcription factors play a key role in regulating mammary epithelial cell phenotypes and imply similarities among epigenetic programs that define progenitor cell characteristics.

Fig. 1.

Enrichment and phenotype of assorted mammary epithelial cells. (A) Dendrogram depicting relatedness of SAGE libraries prepared from CD44+, CD10+, MUC1+, and CD24+ cells from multiple independent cases. Selected portions of the clustering heat map are shown representing genes enriched in luminal (CD24+ and MUC1+) and basal/progenitor-like (CD44+ and CD10+) cells. Each row represents a tag and is labeled with the symbol of the gene that best matches that tag. Red and green indicate high and low expression levels, respectively. (B) Gene ontology enrichment analysis of genes differentially expressed between CD44+ cells and each of the other three cell types. Ontology terms for genes highly expressed in CD44+ relative to CD24+ (blue), CD10+ (green), and MUC1+ (yellow) cells were compared with those for the rest of genes expressed in CD44+ cells by using the Fisher Exact test. Significantly enriched ontology terms are shown with relative enrichment fold as negative log of P value. Immunofluorescence analyses of cell type-specific markers in representative colonies of other (C) and CD44+ (D) cells in 2D culture conditions.

Fig. 2.

Cell type-specific DNA methylation patterns and their functional significance. (A) Arbitrary hypomethylation score of each cell type analyzed by MSDK. (B) Gene ontology enrichment analysis of genes differentially methylated between CD44+ cells and each of the other three cell types. Ontology terms for genes hypomethylated in CD44+ relative to CD24+ (blue bars), CD10+ (green bars), and MUC1+ (yellow bars) cells were compared with those for the rest of genes expressed in CD44+ cells by using the Fisher Exact test. Significantly enriched ontology terms are shown with relative enrichment fold as negative log of P value. The dotted gray line marks statistical significance (1.3). (C) Validation of MSDK results by qMSP for selected genes by using bisulfite-treated DNA from CD44+ (red bars), CD10+ (green bars), MUC1+ (yellow bars), and CD24+ (blue bars) cells. Each column represents a different individual. y-axis indicates relative methylation levels normalized to ACTB. P values indicate the statistical significance of the observed differences in DNA methylation among the cell types. (D) qMSP analysis of the methylation of the indicated genes in CD44+ and CD24+ cells isolated from breast tumors and normal breast. The y-axis indicates the ln ratio of qMSP values in CD24+ and CD44+ cells. PE, pleural effusion, IDC, invasive ductal carcinoma. Colors indicate different samples.

Fig. 3.

Expression of differentially methylated genes. (A) qRT-PCR analysis of the indicated genes in the same set of cells as used for methylation studies. Colors denote cell types as described in Fig. 2. Relative expression levels normalized to RPL19 are indicated on the y-axis. “U” (unmethylated) and “M” (methylated) indicate the methylation status of the gene in each of the cell types, and p-values indicate the statistical significance of the observed difference in expression levels among the cell types analyzed. Numbers next to gene names indicate AscI site location relative to transcription start site. “CpG island” indicates that the AscI site analyzed is within a predicted CpG island. (B) Relationships between genes highly expressed in CD44+ compared with CD24+ cells and Suz12 targets. SAGE expression ratios are represented by shades of red and green depicting up and down-regulated genes, respectively. Yellow bars mark threshold ratios (2-fold difference). Genes are ordered according to their fold difference. Suz12 target genes are indicated by black bars with right-hand panels showing their density and corresponding p-values along the gene list. (C) Immunohistochemical analysis of the expression of the indicated genes in normal human breast tissue. (D) Dual immunohistochemical staining for HOXA11 and CD44v6 expression to demonstrate coexpression of the two markers in the same cells (red arrows). Inset shows higher magnification image.

Fig. 4.

The role of FOXC1. (A) Morphology of MCF12A mammary epithelial cells and expression of E-cadherin after infection with control or FOXC1-expressing retrovirus. (B) FOXC1 expression increases cell migration and invasion in MCF12A cells. (C) qRT-PCR analysis of the indicated genes in FOXC1-expressing MCF12A cells and in normal CD44+ progenitor-like cells. The y-axis indicates fold induction of the indicated genes in FOXC1+ and CD44+ cells compared with control and CD24+ cells, respectively.