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Elucidating the mechanisms of intrinsic stem cell resistance to virus infection

阐明内在干细胞抵抗病毒感染的机制

基本信息

批准号：
10373121
负责人：
Xianfang Wu
金额：
$ 2.7万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-02 至 2022-08-05
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10373121
关键词：
ATAC-seq Automobile Driving Binding Bioinformatics Biological Assay CRISPR/Cas technology Cell Differentiation process Cell Maintenance Cells Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Complement Custom Data Data Set Development ETS1 gene Epigenetic Process Evolution Gene Expression Gene Expression Regulation Genes Genetic Transcription Goals Growth Hematopoietic Hematopoietic stem cells Hepatic Hepatocyte Human In Vitro Infection Interferons Knock-out Maintenance Malignant Neoplasms Mentors Methods Modification Natural regeneration Neurons Organism Phase Pluripotent Stem Cells Regulation Research Resistance SPI1 gene Signal Transduction Techniques Testing Time Tissues Training Transcriptional Regulation Transposase Virus Virus Diseases Work base cell type combat differential expression experimental study hematopoietic stem cell differentiation hematopoietic stem cell self-renewal human pluripotent stem cell in vivo knockout gene loss of function mouse model pathogen promoter repaired response stem cell biology stem cell differentiation stem cell fate stem cells stemness tissue repair tissue stem cells transcription factor transcriptome

项目摘要

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.

主要研究目标是了解和表征干细胞中干扰素 (IFN) 刺激基因 (ISG) 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，与分化细胞不同，干细胞不会产生同样强大的干扰素反应来对抗感染。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 子集，可提供针对多种病毒的有效保护。这种内在 ISG 表达的机制仍然难以捉摸。为此，我建议采用综合方法，通过 (1) ATAC-seq 分析来检查染色质可及性，以及 (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，我的目的是鉴定调节干细胞中 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分析方面的培训。我们对原代人类造血细胞（即造血干细胞 (HSC) 和分化的后代）进行了 ATAC-Seq 分析。我们的分析表明染色质可及性与基因表达之间存在很强的相关性。通过关注这些可及区域内的 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，我们还鉴定了一组参与 HSC 自我更新和分化调节的 TF。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 ISG 和干细胞身份之间存在共同的转录调控。在R00独立阶段，我们建议继续致力于ATAC-Seq和CRISPR敲除分析。为了进一步检验我们的假设，我们将重点关注 HSC 中 ATAC-Seq 分析中新鉴定的一组 TF，旨在阐明它们在 HSC 中调节 ISG 和细胞身份基因的机制（目标 1）。我们的初步实验还表明，不同类型的干细胞中存在细胞类型特异性的 ISG 调节机制（即使是相同的 ISG）。我们将重点关注肝和神经谱系的干细胞，并使用 K99 研究建立的类似 ATAC-Seq 流程来识别调节这些干细胞中 ISG 表达的 TF（目标 2）。最后，根据我们的试点实验收集的数据，我们稍微修改了我们的淘汰策略。利用“每孔一个基因”的CRISPR敲除方法，我们在多能干细胞（hPSC）中鉴定出了调节ISG表达的TF和控制干细胞识别的TF。与我们在 HSC 中的观察类似，我们发现一些 TF 可能在调节 ISG 表达和 hPSC 维持方面发挥双重功能。在目标 3 中，我们将继续针对本研究中包含的干细胞中已知的人类 TF 进行修改后的敲除分析。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产生重要影响。