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Lineage Specific Methylation of the Elf5 Promoter in Mammary Epithelial Cells

From the October Edition of Stem Cells
By Stuart P. Atkinson

The E-twenty six transcription factor, Elf5, has been previously identified as an important regulator of mammary alveolar development (Oakes et al and Choi et al). Elf5 is not expressed in the stem cell-enriched compartment of the mammary gland, but is expressed in both luminal progenitors and mature luminal cells and is required for the differentiation of luminal progenitor cells toward the alveolar lineage. Few studies have sought to understand the role of epigenetic regulation of gene expression in the mammary gland (Rinkels et al, Bloushtain-Qimron et al, Gu et al and Pietersen et al) and, as the epigenetic regulation of Elf itself is known to be very important during development (Ng et al), DNA methylation studies may allow a better understanding the epigenetic control of mammary development. This was the focus of a recent study published in the October edition of Stem Cells from the laboratories of Susan J. Clark and Christopher J. Ormandy at the Garvan Institute of Medical Research, New South Wales, Australia. Using bisulphite sequencing, the authors show that Elf5 expression and DNA methylation are correlated and so demonstrate that Elf5 promoter methylation is lineage-specific and developmentally regulated in the mammary gland in vivo (Lee and Hinshelwood et al).

Initial studies into Elf5 expression utilised the HC11 mouse mammary cell line as an in vitro model of mammary cell differentiation (Ball et al). Differentiation if these cells can be induced by the removal of Epidermal Growth Factor on day 3 of culture and the addition of DMX and prolactin on days 4 to 8. Differentiation can be confirmed through the increase expression of milk proteins (a-casein, and b-casein) as early as day 6. Analysis of Elf5 expression demonstrated an upregulation preceding b-casein and whey acidic protein (Wap) expression, while siRNA-mediated downregulation of Elf5 on day one of the differentiation protocol led to the decreased expression of b-casein and Wap, significantly so by day 6. Further, Elf5 overexpression led to the increase of Wap expression suggesting a link between Elf5 expression and mammary cell differentiation. DNA methylation of the Elf5 promoter was high in undifferentiated HC11 cells and decreased throughout the differentiation period, suggesting this to be a major regulatory mechanism behind Elf5 expression.

To move this research towards in vivo analysis, mammary epithelial cells (MECs) were purified from virgin and pregnant mice. Relative to MECs in virgin mice, Elf5 expression was increased 18 days postcoitus (dpc) and was again linked to changes in DNA methylation status of promoter regions, although not as definitively as was observed in HC11 cells. Next the expression patterns of Elf5 during development were studied through detailed immunohistochemical analysis, which revealed that while around half of the luminal epithelial cells stained positive for Elf5 in virgin mice at both oestrus and dioestrus, by 4 dpc the majority of luminal cells stained positive for Elf5, while the myoepithelial cells remained negative and this pattern was maintained throughout pregnancy to 1 day post partum (dpp). By late pregnancy, the Elf5 positive luminal cells greatly outnumbered the Elf5 negative myoepithelial cells.

To further investigate cell-specific Elf5 expression patterns, flow assisted cell sorting (FACs) was used to separate out basal cells including myoepithelial and stem cells (CD24+CD29hi), luminal progenitor cells (CD24+CD29loCD61+) and mature luminal cells (CD24+CD29loCD61-) from virgin mouse mammary tissue. While Elf5 expression was low in basal cells and greater in mature luminal cells, it was significantly increased in the luminal progenitor cells, while DNA methylation of the promoter was highest in the basal cells with a reduction observed in the luminal cells. However, it was noted that the proportion of Elf5+ cells and DNA methylation levels were not dramatically different between the two luminal populations suggesting a further mode of control over Elf5 expression. This analysis was repeated during pregnancy, comparing the basal (CD24+CD29hi) with the total luminal (CD24+CD29lo) population, due to the scarcity of luminal progenitor cells during pregnancy, and showed similar results to that observed in virgin mice. However, Elf5 was higher in the luminal cells from pregnant mice, reflecting an increased proportion of luminal cells expressing Elf5 during pregnancy, while the Elf5 promoter showed a decrease in its DNA methylation level. While there was no alteration in Elf5 expression observed in the basal populations, the pregnant mice also showed also a decrease in Elf5 promoter DNA methylation.

Overall, the data suggests that suggest that loss of Elf5 methylation specifies the mammary luminal lineage, while continued Elf5 methylation maintains the stem cell and myoepithelial lineages, the first example of a lineage specific epigenetic mark to be associated with a transcription factor that governs mammary cell fate.

 

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