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. Of the treatments currently available, hormone therapy remains one of the most effective means of clinical intervention. 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. As major determents of homeostasis and development, the steroid receptors are particularly important targets for environmental endocrine disruptors that have many human health consequences. While these agents are extremely diverse in structure, activity and bioavailability, many if not all activities are mediated via these receptors. Consequently a molecular description of the mechanism of action of these receptors will provide a precise underpinning to evaluate their physiological and clinical impact with a specific relevance to breast cancer. 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. Previous studies have shown that proteasome inhibition increases GR, but decreases ER-mediated gene expression. We have used a genomic approach to examine the impact of proteasome activity on GR and ER-mediated gene expression in MCF-7 breast cancer cells. Transcript profiling reveals that inhibiting proteasome activity modulates gene expression by GR and ER in a similar manner in that several GR and ER target genes are up-regulated and down-regulated after proteasome inhibition. Importantly, our analysis reveals that a number of transcripts encoding histone and DNA modifying enzymes, prominently histone/DNA methyltransferases and demethylases, are altered after proteasome inhibition. As proteasome inhibitors are currently in clinical trials as therapy for multiple myeloma, HIV/AIDs and leukemia, the possibility that some of the target molecules are hormone regulated and by chromatin modifying enzymes is intriguing in this era of epigenetic therapy. 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 refractory repression 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. Dermatomyositis (DM) is an auto-immune disease which is often accompanied by the development of disease-specific auto-antibodies directed against the SNF2-superfamily helicase, Mi-2. Recent evidence suggests that ultraviolet radiation exposure may be an important risk factor for the development of not only the disease but also specific autoimmunity against Mi-2. Consequently, we investigated the effects of ultraviolet radiation on Mi-2 protein expression. We observed an increase in protein levels upon ultraviolet radiation exposure in cell culture systems. These changes in expression occur quite rapidly, are maximized just 1 hour following exposure, and are unique to Mi-2 when compared to other members of the NuRD complex. Taken together, we describe a system by which Mi-2 protein expression can be quickly increased following UV exposure and then maintained up to 16 hours later. These data provide a novel regulation of an important transcriptional regulator and provides insight into the possible mechanisms of the development of DM and associated autoantibodies 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. MiR-302a is predicted to target many cell cycle regulators, and expression of miR-302a in primary and transformed cell lines promotes an increase in S phase and a decrease in G1 phase cells, reminiscent of an ESC-like cell cycle profile. Moreover, we show that miR-302a represses the productive translation of an important G1 regulator, Cyclin D1, in human ESCs. 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. Our ongoing studies continue to focus on three major areas of molecular carcinogenesis: evaluating how chromatin remodeling complexes and transcription factors function in transcription from chromatin;analyzing the ubiquitin proteasome system interface with chromatin remodeling, epigenetics and hormone signaling;and exploring how chromatin and epigenetics contribute to human ES cell pluripotency. We will continue to exploit biochemical, molecular biological, cell biological, genomic and genetic approaches for these studies. The nature of many of our models, human and mouse beast cancer cells as well as embryonic stem cells, is indicative of our active interest in women's health and breast cancer specifically
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