The expression profiles of the three distinct cell

The expression profiles of the three distinct cell populations show a high correlation in 3-week-old virgin mice, which represents the prepubertal immature mammary gland, but become very distinct with puberty and even more so during pregnancy, whereas consistency from retired breeders resemble those from mice in prepregnant virgin states. These results suggest that the transcriptome and the phenotype of mammary epithelial cells are mostly influenced by reproductive hormones (Asselin-Labat et al., 2010). We identified several TFs with expression restricted to CD24+CD61+CD29hi cells, many of which belong to the Id, Snai, and Foxo gene families. All of these gene families are targets of TGFβ signaling and have known roles in stem cells (Eijkelenboom and Burgering, 2013; Ruzinova and Benezra, 2003). Some of these TFs were expressed in CD24+CD61+CD29hi cells regardless of age or reproductive stage (e.g., Id4 and Snai2), implying that they play key roles in stem cell function, whereas others were only present in prepubertal virgin (e.g., Snai1) or retired breeder (e.g., Foxd3) mice, potentially reflecting stage-specific function. In contrast to the significant cell-type- and reproductive-stage-specific differences in gene expression profiles, the DNA methylation and histone modification (H3K27me3) patterns showed much less pronounced changes. For the majority of differentially expressed genes, alterations in mRNA levels were not associated with changes in DNA methylation or enrichment for the H3K27me3 mark. These findings are consistent with results of studies that investigated the DNA methylation profiles of a wide array of cell types in both mice and humans (Bock et al., 2012; Ziller et al., 2013), and with the relatively minor effects of Ezh2 deletion on mammary epithelial cell differentiation (Michalak et al., 2013; Pal et al., 2013). A few TFs (e.g., Trp73 and Foxi1) displayed both differential DNA methylation and expression among cell types or across stages, and Foxi1 expression was also induced by AzaC and VPA treatment. As none of these TFs have been analyzed in the mammary gland, the physiologic relevance of their expression and epigenetic patterns is still unclear. However, their function in other organs suggests potential roles in mammary epithelial stem or progenitor cells. CD24+CD61+CD29hi cells formed the most distinct category in both the gene expression and DNA methylation profiles. Whereas expression profiles separated CD24+CD61+CD29hi cells from all ages and reproductive stages into a unique group, clustering based on DNA methylation levels suggested that age and pregnancy induce unique changes in the DNA methylation profiles of CD24+CD61+CD29hi cells, which then may alter their self-renewal and differentiation capacity. As proliferative (i.e., stem and progenitor) cells are thought to be the cellular targets of neoplastic transformation, investigating these DNA methylation changes in further detail may reveal markers relevant to breast cancer risk. Related to this, our finding that p27 and TGFβ signaling show the most significant differential expression and activity between CD24+CD61+CD29lo cells from 9-week-old virgin and retired breeder mice correlates with our recent results describing the association of these genes with breast cancer risk in women (Choudhury et al., 2013). In summary, our data support the hypothesis that pregnancy induces permanent changes in CD24+CD61+CD29hi and CD24+CD61+CD29lo cells that are relevant to breast cancer risk, and imply that depletion of these cells may be a feasible cancer prevention strategy. Since studies in mice appear to reproduce our findings in women, they may provide a physiologically relevant preclinical model in which to test the feasibility of such a strategy.
Experimental Procedures
Author Contributions S.J.H. designed and performed the experiments and data analysis. K.C. performed bioinformatics analysis of the data. D.J., A. Merlini, S.C., and R.M. assisted with the experiments and data analysis. R.Y., F.A.R., and L.J.-G. provided the Dnmt1+/+ and Dnmt1 mice. A.C. and H.L.M. provided the mammary gland sections for the Tgfbr2+/+ and Tgfbr2−/− mice. P.B. prepared the RRBS libraries. M.H.B.-H. provided the mammary gland sections for the Tgfb1+/+ and Tgfb1+/− mice. A. Meissner and K.P. supervised the study. All authors contributed with discussions and wrote the manuscript.