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Recent characterization of mammary stem and progenitor cells has improved our understanding of the transcriptional network that coordinates mammary development; however, little is known about the mechanisms that enforce lineage commitment and prevent transdifferentiation in the mammary gland. The E‐twenty six transcription factor Elf5 forces the differentiation of mammary luminal progenitor cells to establish the milk producing alveolar lineage. Methylation of the  Elf5  promoter has been proposed to act as a lineage gatekeeper during embryonic development. We used bisulphite sequencing to investigate in detail whether  Elf5  promoter methylation plays a role in lineage commitment during mammary development. An increase in  Elf5  expression was associated with decreasing  Elf5  promoter methylation in differentiating HC11 mammary cells. Similarly, purified mammary epithelial cells from mice had increased  Elf5  expression and decreased promoter methylation during pregnancy. Finally, analysis of epithelial subpopulations revealed that the  Elf5  promoter is methylated and silenced in the basal, stem cell‐containing population relative to luminal cells. These results demonstrate that  Elf5  promoter methylation is lineage‐specific and developmentally regulated in the mammary gland in vivo, and suggest that loss of  Elf5  methylation specifies the mammary luminal lineage, while continued  Elf5  methylation maintains the stem cell and myoepithelial lineages. S TEM  C ELLS  2011;29:1611–1619

Lineage Specific Methylation of the Elf5 Promoter in Mammary Epithelial Cells