Environmental regulation of estrogen responsive genes in single living cells

National Institute Of Environmental Health Sciences

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Abstract

Our group is interested in somatic gene regulation in disease. Over the past decade several groups have characterized significant expression heterogeneity in stem cells, progenitors, and terminally differentiated cells across several tissues. Although the function of this heterogeneity in normal tissue has not been extensively explored, heterogeneity enables cancer to evade chemotherapeutic treatments. Moreover, genes involved in resistance and metastasis are transiently expressed. This dynamic gene expression highlights the importance of defining dysregulated mechanisms of non-genetic heterogeneity to develop more effective treatments. A great example is heterogeneity in estrogen receptor-alpha positive (ERa+) breast cancer which makes up over 70% of diagnosed breast cancers. Like other cancers, tumors are very heterogeneous in ER+ expression. Some tumors contain as low as 1% ERa+ expressing cells. Since the estrogen receptor alpha (ERa) is a transcription factor which drives several functions including cellular proliferation, targeting its response has been the primary mode of treatment. These cancers are effectively treated with ERa antagonists such as tamoxifen. However, resistance to prolonged tamoxifen treatment is common and often involves mutations in ERa, altered signaling pathways and epigenetic reprogramming. Intriguingly, knockdown of a chromatin remodeler reduced expression heterogeneity of ERa+ breast cancer and led to more sensitivity to ERa antagonists. Most of our molecular understanding of the estrogen response comes from cancer cell line studies. Within 40 minutes after the addition of estradiol, hundreds of genes are activated and repressed. This partly achieved through estrogen receptor and cofactor recruitment to distal regulatory sequences called enhancers. These sequences function through local proximity, although how exactly they aid in transcriptional initiation is unclear. Although we have a genome wide view of the estrogen transcriptional response, single cell studies have shown that only a fraction of hormone responsive genes within single cells respond to hormone. These data suggest that there is either a rate limiting factor needed for transcription initiation of hormone responsive genes or that individual gene loci exist in stable transcriptionally permissive and non-permissive states. Our goal in this project is to characterize mechanisms of enhancer and promoter chromatin states across time. This project will help us determine regulatory features of highly active or deep repressed genes by grouping genes with similar transcriptional characteristics. We have characterized enhancer and promoter activity and observe that enhancers of hypervariably expressed genes exhibit dynamic transcriptional states. We have also made significant progress developing reagents to visualize transcription of enhancer and promoters in live cells and in whole mouse mammary glands. These reagents are helping us visualize enhancer and promoter states. Lastly, we have characterized how acute exposure to high levels doses of endocrine disruptor chemicals alters cofactor recruitment and transcriptional state switching. Our work has implications into how transcriptional dysregulation at single loci alters the population response in normal and disease states.