During this reporting period the Laboratory of Genetics and Physiology has made progress and elucidated mechanisms by which cytokines control mammary development during pregnancy through the transcription factor STAT5. In particular, we identified that genomic binding of STAT5 to mammary-specific genes primes them for activation and that it controls gene expression in synergy with the transcription factor NFIB. In addition, we investigated the role of cytokine-induced miRNA genes in mammary gland development. Mammary development Integrating mouse genetics with large-scale genomic analyses led to the discovery that the sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on the concentration of STAT5A/B (Yamaji et al., 2013). Current studies have focused on mechanisms used by STAT5 to activate mammary-specific genetic programs. Genetic and genomic studies, including ChIP-seq and RNA-seq, have demonstrated that mammary-specific gene activation is defined by progressive recruitment of STAT5 during pregnancy and the establishment of H3K4me3 marks (kang et al., 2014). Based on these studies we suggest that binding of STAT5 to the approximately 700 mammary-specific genes that are activated during pregnancy is an early molecular event in the differentiating mammary epithelial lineage. Future studies will address the question whether STAT5 is a pioneer factor in the mammary lineage or whether its activity is preceded by the binding of additional transcription factors and specific histone modifications. STAT5 is being used by a number of cytokines, including interleukins, prolactin and growth hormone, to execute their biology in a diverse set of cell types. At this point it is not clear how distinct cytokines can convey cell specificity through a common transcription factor. One possibility would the restricted expression of the corresponding cytokine receptor, but this does not appear to be the guiding principle. Alternatively, it could be hypothesized that cooperativety between STAT5 and additional transcription factors could contribute to cell specific genetic programs. A bioinformatics approach had revealed the presence of consensus sequences for the transcription factor NFIB in the vicinity of STAT5 binding sites in one third of mammary-specific genes. Using mouse genetics we could demonstrate that STAT5 and NFIB synergistically active mammary-specific genes (Robinson et al., 2014). Specifically, we analyzed mice from which either Nfib or Stat5 were inactivated specifically in the mammary epithelial lineage or both genes together. This study revealed that NFIB and STAT5 coregulate genes. ChIP-seq analyses validated the juxtaposed binding of STAT5 and NFIB to regulatory units of these genes. While STAT5 is generally considered to be an activating transcription factor, a collaborative study has revealed that it can suppress expression of specific target genes. Through the integration of large-scale data gauging genome-wide STAT5 binding and various histone modifications Trp63 was identified as a gene suppressed by STAT5 (Assefnia et al., 2014). In addition, Trp63 was identified as a predictive marker of basal breast cancer cells. Micro RNAs and mammary physiology Micro RNAs (miRNAs) are believed to be important post transcriptional regulators of messenger RNAs and that by controlling their stability and translation they modulate the physiology of cells. LGP scientists have identified several miRNAs that are under cytokine-STAT5 control, suggesting a role for them in mammary epithelium during pregnancy (Feuermann et al., 2012). The biology of specific STAT5-inducible miRNAs was studied in gene knockout mice. In the current reporting period we have investigated the biology of miR-21 in respective germ-line knock-out mice (every cell of the mouse lacks miR-21). In addition, we have inactivated the locus containing miR-193b and miR-365-1, both in the germ-line and in specific cell types using Cre-loxP technology. Although miR-21 is an abundant miRNA and its expression is induced by cytokines, including prolactin during pregnancy, through STAT5, mammary development and function were overtly unimpaired in its absence. This suggests that miR-21 does not contribute in a measurable way to the physiology of mammary tissue. In an earlier reporting period we had demonstrated that the miR17-92 cluster is also dispensable for mammary development and function (Feuermann et. al., 2012). These two studies suggest that miRNAs might have less of a prominent role in the biology of mammary epithelium than previously thought. In a landmark study published in Nature Cell Biology scientists had reported data from in vitro cell culture studies that demonstrated a critical role for miR-193b and miR-365-1 for the development and function of brown adipose tissue (BAT). However, using genuine gene knock-out mice that lack miR-193b and miR-365-1 we could not detect any aberrations in the development and function of BAT (Feuermann et al., 2013). This study highlighted differences between in vitro and in vivo biology and suggests that the conclusions of in vitro studies might not match those from genuine in vivo experiments.
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