Environmental regulation of estrogen responsive genes in single living cells

National Institute Of Environmental Health Sciences

Investigators

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Abstract

We are interested in understanding how the estrogen response regulates cell fate decisions using both cell lines and the mammary gland as models for investigation. One of the most fascinating aspects of the mammary gland is that its development completes postnatally after puberty. Moreover, the mammary gland undergoes dynamic morphological changes during the estrous cycle and pregnancy. Both processes require coordination between hormone and paracrine signaling among different cell types. Intriguingly, multiple cell markers have identified populations of cells with varying degrees of mammary gland repopulation potential. Several single cell studies of the mammary gland have revealed extensive heterogeneity in mammary gland epithelial cells suggesting that a continuum of cell states exists. What role progenitor and epithelial cell heterogeneity has in mammary gland development and homeostasis is unknown. Given that cancer is a heterogenous disease, we believe the single cell perspective is crucial to our understanding of disease emergence and progression. We aim to understand the role of expression heterogeneity in cell fate decisions, define the mechanisms that regulate it, and determine how endocrine disruptor chemicals (EDCs) perturb these processes. Over the past year we have made progress on several fronts. One of our major efforts has been to understand how EDCs which are found in our water, food, and plastics, alter mammary gland function and tumorigenesis. Our approach has been to characterize how EDCs affect single cell gene expression mechanisms during acute and low dose chronic exposure in breast cancer cell lines. We used live cell transcriptional imaging and single molecule imaging of transcription factors to determine how EDCs alter single locus gene activation. We observe differences in transcriptional activation at individual alleles in response to acute exposure. Interestingly, we observe increases in breast cancer cell heterogeneity as a result of 180-day chronic exposure to EDCs. These data suggest that exposure to EDCs may alter in vivo cancer cellular expression programs and heterogeneity. Given that heterogeneity is a hallmark of cancer, and is important for its plasticity to chemotherapeutics, our results shed light into how differential environmental exposures might aid in tumorigenesis. We have also made significant progress in our efforts to visualize mammary gland expression programs at the single cell level. We have developed several methods including imaging windows and chambers for in vivo and 3D tissue visualization of gene expression. We successfully 3D-visualized immunofluorescence and RNA FISH in a whole mount mammary gland. This protocol necessitated the development of a 3D printed chamber. Lastly, we have made a significant effort to identify and characterize mechanistic factors that coordinate or enhance expression heterogeneity. Some of these factors are known in the literature to be heterogeneously expressed. We are using fluorescence-activated cell sorting to sort for different populations of cells with high and low levels of proteins and determine how they alter the transcriptome of the sorted populations. However, we have also setup a medium throughput approach to screen for factors that may affect the estrogen response, expression and functional heterogeneity. This approach combines single molecule RNA fluorescence in situ hybridization which is a highly sensitive approach, and high-content imaging which enables us to screen hundreds of inhibitors or factors in a few hours.