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. While the SWI/SNF (switching/sucrose nonfermenting)-dependent chromatin remodeling complex has been show to establish coordinated gene expression programs during development, important functional details remain to be elucidated. In newly published studies we showed that the Brg1 (Brahma-related gene 1; Smarca4) ATPase was globally expressed at high levels during postimplantation development and its conditional ablation, beginning at gastrulation, resulted in increased apoptosis, growth retardation, and, ultimately, embryonic death. Global gene expression analysis revealed that genes upregulated in Rosa26CreERT2; Brg1(flox/flox) embryos (here referred to as Brg1(d/d) embryos to describe embryos with deletion of the Brg1(flox/flox) alleles) negatively regulate cell cycle progression and cell growth. In addition, the p53 (Trp53) protein, which is virtually undetectable in early wild-type embryos, accumulated in the Brg1(d/d) embryos and activated the p53-dependent pathways. These results provide important new insights into the mechanisms by which Brg1 functions, which is in part via the p53 program, to constrain gene expression and facilitate rapid embryonic growth. It is now widely accepted that microRNAs and chromatin remodeling complexes represent powerful epigenetic mechanisms that regulate the pluripotent state. Our previous work established that miR-302 is a strong inducer of pluripotency, which is characterized by a distinct chromatin architecture. This suggests that miR-302 regulates global chromatin structure; however, a direct relationship between miR-302 and chromatin remodelers has not been established. Our latest investigations provide data to show that miR-302 regulates Brg1 chromatin remodeling complex composition in human embryonic stem cells (hESCs) through direct repression of the BAF53a and BAF170 subunits. Overexpression of BAF170 in hESCs led to biased differentiation toward the ectoderm lineage during EB formation and severely hindered directed definitive endoderm differentiation. Taken together, these data uncover a direct regulatory relationship between miR-302 and the Brg1 chromatin remodeling complexthat controls gene expression and cell fate decisions in hESCs and suggests that similar mechanisms are at play during early human development. Prior work in my laboratory demonstrated that BRG1, the central ATPase of the human SWI/SNF complex, was critical nuclear receptor (NR)-regulated transcription. Analysis of BRG1 mutants demonstrated that functional motifs outside the ATPase domain are important for transcriptional activity. In the course of experiments examining protein interactions mediated through these domains, Ku70 (XRCC6) was found to associate with a BRG1 fragment encompassing the conserved helicase-SANT-associated (HSA) and BRK domains of BRG1. Subsequent transcriptional activation assays and chromatin immunoprecipitation studies showed that Ku70/86 and components of the topoisomerase II (TOP2)/poly(ADP ribose) polymerase 1 (PARP1) complex are necessary for NR-mediated SWI/SNF-dependent transcriptional activation from endogenous promoters. Taken together, these results suggest that a direct interaction between Ku70/86 and BRG1 brings together SWI/SNF remodeling capabilities and TOP2 activity to enhance the transcriptional response to hormone stimulation. While evaluating gene expression changes in response to 26S proteaosome inhibition in breast cancer cells, we identified the evolutionarily conserved RNA-binding protein Lin28. This was interesting because while LIN28 was known to have critical functions in development and cancer, the molecular mechanisms underlying LIN28's oncogenic properties are yet to be described. We used RNA-protein immunoprecipitation coupled with genome-wide sequencing (RIP-Seq) analysis to reveal significant LIN28 binding within 843 mRNAs in breast cancer cells. Many of the LIN28-bound mRNAs are implicated in the regulation of RNA and cell metabolism. We identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein with multiple roles in mRNA metabolism, as a LIN28-interacting partner. Subsequently, we used a custom computational method to identify differentially spliced gene isoforms in LIN28 and hnRNP A1 small interfering RNA (siRNA)-treated cells. The results demonstrated that these proteins regulate alternative splicing and steady-state mRNA expression of genes implicated in aspects of breast cancer biology. Intriguingly, analysis of publicly available array data from the Cancer Genome Atlas (TCGA) discloses that LIN28 expression in the HER2 subtype is significantly different from that in other breast cancer subtypes. Collectively, our data suggest that LIN28 may regulate splicing and gene expression programs that drive breast cancer subtype phenotypes. Previous studies focusing primarily on antisense transcription initiating upstream of genes established Antisense transcription is a prevalent feature at mammalian promoters. In recently published studies we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We found extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription initiated between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.
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