Elucidating the mechanisms of intrinsic stem cell resistance to virus infection

主要的研究目标是了解和表征干细胞中干扰素（IFN）刺激基因（ISGs）内在表达的机制。维持健康的干细胞对生物体内的组织修复至关重要。然而，与分化的细胞不同，干细胞不能产生同样强大的IFN反应来对抗感染。那么干细胞是如何抵抗病毒感染的呢？我们最近的发现表明，干细胞具有高基础水平的细胞类型特异性isg亚群，这赋予了对许多病毒的有效保护。这种内在ISG表达的机制尚不清楚。为此，我建议采用综合方法，使用(1)ATAC-seq分析来检测染色质可及性(2)定制针对人转录因子（TFs）的CRISPR基因敲除筛选。结合这两种方法，我旨在鉴定干细胞中调节ISG表达的tf。

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 important implications for our understanding of stem cell biology, primordial aspects in development and cancer, and the evolution of vertebrate pathogen defense.