8#and #and treated with or without PRL for 1 h. STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling. delays cancer progression driven by simian virus 40 T antigen in Pseudolaric Acid A the mouse mammary gland (22). In human breast cancer cell lines, STAT5 promotes cell survival and anchorage-independent growth (23). Given the role of PRL and STAT5 in breast cancer pathogenesis, the factors regulating PRL-induced, STAT5-mediated gene expression merit close investigation. Although STAT5 activation is critical in mediating the effects of PRL, transcription factor activation alone is not enough to translate cellular signals into the proper gene expression patterns. Upon activation, transcription factors only bind to a small fraction of their consensus elements in the genome, typically occupying 1% of their potential binding sites based on DNA sequence alone (24). Therefore, a significant question remaining in the study of transcriptional regulation is how certain consensus elements become accessible for transcription factor binding, whereas others remain unbound. Chromatin structure appears to be a major determinant of transcription factor binding patterns. In genome-wide studies, transcription factor consensus elements that are located within accessible chromatin, marked by DNase I hypersensitivity or active histone modifications, are preferentially bound by their respective transcription factors following activation (24,C26). In the mammary epithelium, chromatin accessibility plays a distinct role in determining cell type-specific and context-specific patterns of transcription factor binding and gene expression. STAT5-regulated genes that are expressed mainly in the mammary epithelium are not recognized by STAT5 in cell types where these genes are not expressed (27). These findings indicate that STAT5 loci exhibit cell type-specific patterns of accessibility, enabling STAT5 binding only in the proper context and cell type. Rabbit polyclonal to PCDHB16 Correspondingly, mammary-specific genes have been shown to exhibit an open chromatin organization that is specific not just to mammary tissue Pseudolaric Acid A but also to the appropriate developmental stage (28). One study has shown that DNA methylation in particular is associated with impaired STAT5 recruitment (29). However, additional chromatin-remodeling events that enable or prevent STAT5 recruitment have Pseudolaric Acid A not been well characterized. Our laboratory has previously shown that the chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) promotes PRL-induced, STAT5-mediated transcription (30). Following PRL stimulation, HMGN2 is Pseudolaric Acid A recruited to the promoter of the cytokine-inducible SH2-containing protein (promoter. HMGN2 facilitates the displacement of H1, and H1 loss is necessary to allow full STAT5 binding and transcriptional activation. Finally, H1 was found to antagonize STAT5 in promoting the proliferation of breast cancer cells. This is the first report to identify H1 occupancy as a critical regulator of STAT5-mediated transcription, specifically linking chromatin remodeling to STAT5 recruitment and biological function. Results STAT5 Inhibition Results in Decreased Proliferation of Breast Cancer Cells Because the transcription factor STAT5 is a critical mediator of PRL-induced signaling, we first assessed the role of STAT5 in mediating the biological effects of PRL in breast cancer cells. T47D breast cancer cells were chosen based on their high endogenous levels of PRLR expression (18). To assess the role of STAT5, a chemical inhibitor of STAT5 (CAS 285986-31-4) was utilized (33). This compound prevents STAT5 phosphorylation and DNA binding by selectively targeting the Src homology 2 domain of STAT5, which mediates protein-protein interactions of STAT5 both with activated receptors and in dimer formation (33). In the absence of STAT5 inhibition, PRL treatment resulted in phosphorylation of STAT5 in the cytoplasmic fraction and increased levels of both phosphorylated and total STAT5 in the nuclear fraction, indicative of nuclear translocation of the activated protein (Fig. 1test assuming equal sample variance, comparing without with PRL (versus + 0.05; **, 0.01. PRL Treatment Induces Chromatin Decompaction and Promotes Binding of the Transcriptional Machinery Because STAT5 is an important mediator of the biological effects of PRL, we sought to elucidate the molecular mechanisms regulating STAT5 recruitment and PRL-induced gene expression. We first assessed whether PRL signaling induces chromatin remodeling at the promoters of immediate early genes or Pseudolaric Acid A whether PRL-induced genes are poised with an open chromatin structure and bound RNA polymerase II (RNAPII). To begin the analyses, we focused on the PRL-induced immediate early gene transcription has been well characterized (10, 30, 35), making it a robust model for initial.