Wellberg, Elizabeth (2009-05). Regulation of Mammary Lactogenic Differentiation by Singleminded-2s. Doctoral Dissertation.
Sim2s is a basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor. In Drosophila, the Sim2 homolog, sim, is necessary for cell fate determination during central nervous system (CNS) development. In mammals, both Sim2 isoforms are involved in development of various tissues, including muscle, cartilage, and mammary gland. Loss-of-function studies revealed a role for Sim2s in specifying epithelial cell fate during mammary development and inhibiting growth and invasion of aggressive breast cancer cells. This study determined the role of Sim2s in mammary epithelial cell differentiation. Our hypothesis is that Sim2s is sufficient to promote lactogenic differentiation in vivo, characterized by expression of lactation-specific genes. Two models were used to test this hypothesis: (1) a transgenic mouse, expressing Sim2s under control of the MMTV-LTR, and (2) the mouse mammary epithelial cell line HC11. Together, these models allow analysis of the effect of Sim2s on global mammary gland differentiation and the mechanism through which it accomplishes this in a relatively homogenous population of cells. We determined that precocious expression of Sim2s in vivo is associated with upregulation of a subset of milk protein genes in nulliparous females. During early pregnancy, Sim2s regulation of lactogenic differentiation extended to a larger group of genes. Following pup removal, Sim2s appears to promote survival of alveolar epithelial cells. In vitro, Sim2s expression is necessary for maximal Csn2 expression, as determined by loss-of-function studies. Overexpression of Sim2s is sufficient to enhance prolactin-mediated Csn2 expression. Chromatin immunoprecipitation assays performed in HC11 cells revealed enhanced recruitment of Stat5a and RNA Polymerase II (RNAPII) to the regulatory region of Csn2 in the presence of Sim2s. In addition, Sim2s and RNAPII were found in a complex that was localized to both the promoter and coding region of the Csn2 gene. These studies support the idea that Sim2s is upregulated in a developmental stage-specific manner in the mouse mammary gland to promote the survival and differentiation of alveolar epithelial cells expressing high levels of milk protein genes. Further, Sim2s may regulate the function of a specific subset of alveolar cells by targeting the RNAPII holoenzyme complex to genes expressed during lactogenic differentiation.