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. The chromatin and gene expression sections primary scientific focus is the mechanisms by which gene expression is initiated in response to physiological and environmental signals and how those signals are mediated by steroid 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 interest in breast cancer. ? ? To this end we have pursued two highly interactive objectives as part of two specific aims. The first is to provide a molecular definition of the relationship between nuclear receptors, chromatin remodeling machines and promoter chromatin structure in the regulation of steroid receptor activity. Within this objective we will continue to make use of the Mouse Mammary Tumor Virus (MMTV) system, see below, as the preeminent model for a steroid hormone activated promoter in the context of chromatin. The wealth of prior information, extensive reagents and resources allow us pursue a series of goals that are not possible in other systems. The second objective is the development of additional model systems to understand glucocorticoid, progesterone and estrogen receptors (GR, PR, ER). This objective has resulted in initial characterization of the human P21, PLZF and Inhibitor of Nuclear Kappa B alpha genes. More recently we have begun to develop mouse models that interrogate the specific role played by chromatin remodeling proteins focusing on a member of the human SWI/SNF complex, BRG1 associated factors.? ? 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. The approach has been bidirectional with efforts geared to understanding transacting proteins and the protein architecture of chromatin that is subject to post-translational modifications. To achieve this we have focused our attention on the mammalian BRG1 chromatin remodeling complex that is the homologue of the yeast SWI/SNF complex and its interactions and regulation by the glucocorticoid and progesterone receptors. The activity of this complex has been evaluated in the context of the chromatin within human and mouse cells. Using the MMTV promoter as our primary model system, we have paid particular attention to the phosphorylation of histone H1 and the acetylation/methylation of the core histones.? ? Previous studies have shown that proteasome inhibition increases GR, but decreases ER-mediated gene expression. At the gene expression level this divergent role of the proteasome in receptor-dependent transcriptional regulation is not well understood. 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 treated with dexamethasone or 17beta-estradiol, the proteasome inhibitor MG132 or MG132 and either hormone for 24 h. 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 upregulated and downregulated after proteasome inhibition. In addition, proteasome inhibition modulates receptor-dependent genes involved in the etiology of a number of human pathological states, including multiple myeloma, leukemia, breast/prostate cancer, HIV/AIDS, and neurodegenerative disorders. 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 chromatin modifying enzymes is intriguing in this era of epigenetic therapy.? ? In pursuit of our third specific aim, my group has recently embarked on an exciting new area of research that examines the fundamental nature of human embryonic stem cells. We have begun to characterize the chromatin remodeling and chromatin modifying complexes that are present in these cells. This is complimented by analyses of the master regulatoryproteins OCT4, SOX2 and Nanog to determine the genetic and epigenetic targets that underlie the biological program for stem cells. 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. 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 nature of many of our models, human and mouse beast cancer cells as well as embryonic stem cells, is also indicative of our active interest in women's health and breast cancer specifically. Our research plan is to assess the contributions that chromatin remodeling proteins, nuclear receptors, pluripotency factors, miRNAs and promoter chromatin architecture make to regulate the transcriptional response to endogenous and environmental signals in normal, embryonic and cancerous cells.
Showing the most recent 10 out of 61 publications
