Breast cancer will constitute greater than 30% of all new cancers diagnosed in women this year. Steroid hormones, such as estrogen and progesterone, play an important role in the development and treatment of breast cancer. These receptors represent critical sensors of environmental exposures on reproduction and development while being subject to epigenetic regulation of their activity. Despite intensive efforts, the mortality resulting from this disease has not decreased significantly over the last decade. In my laboratory, the scientists have undertaken detailed analysis of the mechanism of action of the steroid receptors and clinically important steroid receptor antagonists which are used to block their action. Our experiments are focused on the role of chromatin and epigenetics that are critical to understanding their function. It is hoped that these basic research studies will provide new insight into the role that steroid hormone receptors play in breast cancer and the possible development of novel and effective treatments. Our efforts are informed by the overwhelming evidence that a full understanding of transcriptional control requires an appreciation for roles played by the chromatin structure of target genes and the molecular machines that are required to unleash the regulatory potential of steroid receptors. To achieve this we have focused our attention on the mammalian BRG1 chromatin remodeling complex and the 26S proteasome, their interactions with and regulation by the glucocorticoid and progesterone receptors. The activity of these complexes has been evaluated in the context of the chromatin within human and mouse cells with particular attention to the activity of RNA polymerase and the acetylation/methylation of the core histones. Environmental exposure to low concentration hormones can have permanent epigenetic effects in animals and humans. The consequence of long-term low-concentration glucocorticoid exposure was investigated in cell culture using glucocorticoid responsive genes organized in alternative chromatin structures. The MMTV promoter is induced by short-term glucocorticoid exposure on either an integrated (normal chromatin) or transient (unstructured chromatin) promoter. Longer hormone treatment causes a transient refractoryrepression of only the integrated promoter. Exposure to low-concentrations of hormone for several passages persistently represses the integrated MMTV and endogenous glucocorticoid responsive promoters. The glucocorticoid receptor cannot bind to persistently repressed promoters. Induction by androgens is also inhibited on the repressed MMTV promoter. Persistent repression by glucocorticoids targets glucocorticoid-responsive genes using a chromatin-dependent mechanism that disrupts binding of both GR-dependent and GR-independent transcription complexes. Steroid hormone receptors initiate a genetic program tightly regulated by the chromatin environment of the responsive regions. Using the glucocorticoid receptor (GR) as a model factor for transcriptional initiation, we classified chromatin structure through formaldehyde-assisted isolation of regulatory elements (FAIRE). We looked at dynamic changes in FAIRE signals during GR activation specifically at regions of receptor interaction. We found a distribution of GR-responsive regions with diverse responses to activation and chromatin modulation. The majority of GR binding regions demonstrate increases in FAIRE signal in response to ligand. However, the majority GR-responsive regions shared a similar FAIRE signal in the basal chromatin state, suggesting a common chromatin structure for GR recruitment. Supporting this notion, global FAIRE sequencing (seq) data indicated an enrichment of signal surrounding the GR binding site prior to activation. Brg-1 knockdown showed response element-specific effects of ATPase-dependent chromatin remodeling. FAIRE induction was universally decreased by Brg-1 depletion, but to varying degrees in a target specific manner. Taken together, these data suggest classes of nuclear receptor response regions that react to activation through different chromatin regulatory events and identify a chromatin structure that classifies the majority of response elements tested. More recently my group has embarked on an exciting new area of research that examines the fundamental nature of human embryonic stem cells by characterizing the chromatin remodeling and chromatin modifying complexes that are present in these cells. The master regulatory proteins Oct4 and Sox2 are transcription factors required for pluripotency during early embryogenesis and maintenance of embryonic stem cell (ESC) identity. Functional mechanisms contributing to pluripotency are expected to be associated with genes transcriptionally activated by these factors. Here, we show that Oct4 and Sox2 bind to a conserved promoter region of miR-302, a cluster of eight miRNAs expressed specifically in ESCs and pluripotent cells. Expression of miR-302a is dependent on Oct4/Sox2 in human ESCs, and miR-302a is expressed at the same developmental stages and in the same tissues as Oct4 during embryogenesis. Transcriptional activation of miR-302 and translational repression of its targets, such as Cyclin D1, may provide a link between Oct4/Sox2 and cell cycle regulation in pluripotent cells. The heterogeneous nature of stem cells is an important issue in both research and therapeutic use in terms of directing cell lineage differentiation pathways, as well as self-renewal properties. Using flow cytometry we have identified two distinct subpopulations by size, large and small, within cultures of human embryonic stem (hES) cell lines. These two cell populations respond differentially to retinoic acid (RA) differentiation and several endocrine disruptor compounds (EDC). The large cell population responds to retinoic acid differentiation with greater than a 50% reduction in cell number and loss of Oct-4 expression, whereas the number of the small cell population does not change and Oct-4 protein expression is maintained. In addition, four estrogenic compounds altered SSEA-3 expression differentially between the two cell subpopulations changing their ratios relative to each other. Both populations express stem cell markers Oct-4, Nanog, Tra-1-60, Tra-1-80 and SSEA-4, but express low levels of differentiation markers common to the three germ layers. Cloning studies indicate that both populations can revive the parental population. Furthermore, whole genome microarray identified approximately 400 genes with significantly different expression between the two populations (p<0.01). We propose the differential response to RA in these populations is due to differential gene expression of Notch signaling members, CoupTF1 and CoupTF2, chromatin remodeling and histone modifying genes that render the small population resistant to RA differentiation. The findings that hES cells exist as heterogeneous populations with distinct responses to differentiation signals and environmental stimuli will be relevant for their use for drug discovery and disease therapy. The nature of many of our models, human and mouse, breast cancer cells, as well as embryonic stem cells, is indicative of our active interest in women's health and breast cancer specifically. Our research plan discovers and evaluates the contributions that chromatin remodeling proteins, 26S proteasome, nuclear receptors, pluripotency factors, mirRNAs and promoter chromatin architecture make to regulate the transcriptional response to endogenous and environmental signals in normal, human embryonic stem, induced pluripotent stem and cancerous cells.
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