Epigenetic and environmental factors of ACE2 and TMPRSS2 co-expression

National Institute Of Environmental Health Sciences

Investigators

Abstract

Viruses use host components for their entire life cycle, from transcription and RNA processing of viral components to export and reinfection of new cells. ACE2 is an estrogen responsive cell receptor used by COVID19 to dock and enter host cells. TMPRSS2 is also an estrogen and androgen responsive serine protease which is required for S protein priming of coronaviral spike protein and is also necessary for host cell entry. Both ACE2 and TMPRSS2 are hypervariably expressed between people and in the epithelium of several tissues. Interestingly, COVID19 patients display inflammation in several tissues including the small intestine, kidney, lung and nasal passages; indicating a general and broad mode of pathogenicity. Additionally, ACE2 and TMPRRS2 show little co-expression in the same cells suggesting that a small fraction of ACE2 and TMPRSS2 expressing cells are infected. Given these data our major goals have been to characterize ACE2 and TMPRSS2 gene regulation in the single cells, as well as develop assays that allow us to visualize their coregulation over time. We have used single molecule RNA fluorescence in situ hybridization (smFISH) assays which are highly sensitive to low mRNA copy number and give discrete RNA counts. These qualities are preferable over single cell RNA-seq assays given that ACE2 and TMPRSS2 are low expressed in cell lines and tissue. During the initial characterization of ACE2 and TMPRSS2, we used several hormones described in the literature to alter ACE2 and TMPRSS2 expression. We observe both activation and repressive roles for different hormone pathways on the expression of these two genes. Additionally, we observe hierarchal functions where some hormonal action is upstream of others. We have also tested a handful of hormone pathway inhibitor compounds recently identified in the literature as altering ACE2 or TMPRSS2 expression. However, we have not observed changes to expression, suggesting a cell type specific effect. These results taken together suggest that hormone pathways and levels which change as a function of aging and health, could alter the expression of these genes, which are important for SARS-COV-2 entry, differentially in specific tissues. In addition to these characterizations, we have worked to adapt smFISH assays to both medium-throughput microscope and high-content imaging systems. We are screening epigenetic, chromatin modifying, and hormone pathway proteins using small molecule inhibitor libraries. We have successfully adapted our protocols and automated software analysis. We have currently screened an epigenetic factor inhibitor library and will continue to screen additional libraries including endocrine disrupting compounds (EDC) such as flavonoids. These compounds are commonly found in our food, water, plastics and can epigenetically alter the expression of hormone regulated genes. Lastly, we are developing an immuno-smFISH viral entry assay which uses pseudotyped spike protein lentivirus to visualize viral entry into host cells by the presence of cellular GFP. We are primed for the identification of molecular pathways and environmental exposures which affect the expression of two factors important for SARS-CoV-2 host entry. Our future experiments will validate and characterize our hits to better understand how these two genes are regulated at the single cell level, and whether variability in their expression plays a role in differentially susceptibility observed in the population. We are also going to develop the live cell component of our work which will enable us to visualize the coregulation of ACE2 and TMPRSS2 in living cells.