The primary research goal is to understand and characterize the mechanisms underlying intrinsic expression of interferon (IFN) stimulated genes (ISGs) in stem cells. Maintenance of healthy stem cells is essential for tissue repair within an organism. Unlike differentiated cells, however, stem cells do not produce the same robust IFN response to combat infection. Then how do stem cells resist viral infection? Our recent discoveries demonstrate that stem cells have high basal levels of cell type-specific subsets of ISGs that confer potent protection against a number of viruses. The mechanism underlying this intrinsic ISG expression remain elusive. To this end, I proposed to use comprehensive approaches by using (1) ATAC-seq analysis to examine chromatin accessibility and (2) customized CRISPR gene knockout screens targeting human transcription factors (TFs). Combining these two methods, I aimed to identify TFs that regulate ISG expression in stem cells. In the K99 mentored phase, I have received training in epigenetic techniques, CRISPR knockout screen, and bioinformatic analysis. We performed ATAC-Seq analysis on primary human hematopoietic cells (i.e. hematopoietic stem cells (HSCs) and differentiated progeny). Our analysis suggests a strong correlation between chromatin accessibility and gene expression. By focusing on TF binding motif within these accessible regions, we identified a group of TFs likely regulating ISG expression in HSCs. Using similar strategies, we also identified a group of TFs involved in regulation of HSC self-renewal and differentiation. We found there is a substantial overlap between these two TF groups, in line with our hypothesis that there exists common transcriptional regulation between ISGs and stem cell identity. In the R00 independent phase, we propose to continue our effort in ATAC-Seq and CRISPR knockout analyses. To further test our hypothesis, we will focus on a group of newly identified TFs from ATAC-Seq analysis in HSCs and aim to elucidate their mechanisms in regulation both ISG and cell identity genes in HSCs (Aim 1). Our pilot experiments also suggest that there are cell type-specific mechanisms for ISG regulation (even the same ISGs) in different types of stem cells. We will focus on stem cells form hepatic and neuronal lineages and use similar ATAC-Seq pipelines established from K99 study to identify TFs regulating ISG expression in these stem cells (Aim 2). Finally, based on data collected from our pilot experiments, we slightly modified our knockout strategy. Using “one gene per well” CRISPR knockout method, we have identified TFs regulating ISG expression and TFs controlling stem cell identify in pluripotent stem cells (hPSCs). Similar to our observation in HSCs, we found several TFs might exert dual function in regulating ISG expression and hPSC maintenance. In Aim 3, we will continue this modified knockout analysis targeting known human TFs in stem cells included in this study. Our study will have ...