Elucidating the mechanisms of intrinsic stem cell resistance to virus infection

主要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于生物体内的组织修复至关重要。然而，与分化的细胞不同，干细胞不会产生同样强大的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，其赋予对许多病毒的有效保护。这种内在ISG表达的机制仍然难以捉摸。为此，我建议使用综合方法，通过使用（1）ATAC-seq分析来检查染色质可及性和（2）针对人类转录因子（TF）的定制CRISPR基因敲除筛选。结合这两种方法，我的目的是确定调节干细胞中ISG表达的TF。 在K99指导阶段，我接受了表观遗传技术、CRISPR敲除筛选和生物信息学分析的培训。我们对原代人造血细胞（即造血干细胞（HSC）和分化的后代）进行ATAC-Seq分析。我们的分析表明，染色质可及性和基因表达之间有很强的相关性。通过关注这些可接近区域内的TF结合基序，我们鉴定了一组可能调节HSC中ISG表达的TF。使用类似的策略，我们还确定了一组转录因子参与调控HSC的自我更新和分化。我们发现这两个TF组之间存在大量重叠，这与我们的假设一致，即ISGs和干细胞身份之间存在共同的转录调控。 在R 00独立阶段，我们建议继续进行ATAC-Seq和CRISPR敲除分析。为了进一步验证我们的假设，我们将集中在一组新发现的TF从ATAC-Seq分析在HSC中，并旨在阐明其在调控HSC中的ISG和细胞身份基因的机制（目的1）。我们的初步实验还表明，在不同类型的干细胞中，ISG调节（即使是相同的ISG）存在细胞类型特异性机制。我们将专注于来自肝脏和神经元谱系的干细胞，并使用从K99研究建立的类似ATAC-Seq管道来鉴定调节这些干细胞中ISG表达的TF（目的2）。最后，根据从我们的试点实验收集的数据，我们稍微修改了我们的敲除策略。使用“每孔一个基因”的CRISPR敲除方法，我们已经鉴定了调节ISG表达的TF和控制多能干细胞（hPSC）中的干细胞鉴定的TF。与我们在HSC中的观察类似，我们发现几种TF可能在调节ISG表达和hPSC维持中发挥双重功能。在目标3中，我们将继续进行这种改良的敲除分析，靶向本研究中包括的干细胞中的已知人TF。我们的研究将对我们理解干细胞生物学、发育和癌症的原始方面以及脊椎动物病原体防御的进化具有重要意义。

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.