Mechanistic modeling of epigenetic modifier mutations in human pluripotent stem cell-derived immune cells

项目摘要 不确定潜能的克隆性造血（CHIP）是指存在扩增的血细胞克隆 有一个或多个体细胞突变，没有其他血液学异常。CHIP在老年人中很常见， 影响超过10%的65岁以上的人。CHIP与10倍的风险增加有关， 血液恶性肿瘤和动脉粥样硬化性心血管疾病的风险增加一倍，导致 全因死亡率增加。CHIP中两个最常见的突变基因是DNMT3A和TET2，两者都是 其中编码表观遗传修饰物。最近的研究发现炎症增加的证据， 当TET2的小鼠直系同源物在体内髓样细胞中受到干扰时，动脉粥样硬化恶化。然而，在这方面， 这种表型背后的特定表观遗传机制以及DNMT3A和TET2突变是否 针对相同的生物途径尚不清楚。此外，仍然无法解释为什么功能丧失 DNMT3A和TET2中的突变共享临床结果，尽管这两种基因编码的酶具有 相反的生物化学功能（DNA甲基化与去甲基化）。为了弥补这一知识差距，我们 开发了DNMT 3A-和TET 2-的人多能干细胞（hPSC）衍生的巨噬细胞模型， 单倍不足hPSC衍生的巨噬细胞是取之不尽、可扩展的，并且适合遗传修饰。 操作，提供了一个强大的体外模型非常适合机制的研究。在本提案中，我们 提出使用该模型来（1）定义DNMT3A和TET2的独特和共享的表观遗传特征 单倍不足，和（2）鉴定驱动DNMT3A-1中改变的免疫基因表达的转录因子。 和TET2-单倍不足的免疫细胞。这个项目是建立在我们以前的工作，我们预计， 这项研究的结果将指导未来的研究，针对一个共同的表观遗传脆弱性， DNMT3A和TET2突变的细胞以及对CHIP的其他驱动因素的机制研究。

PROJECT SUMMARY Clonal hematopoiesis of indeterminate potential (CHIP) refers to the presence of expanded blood cell clones with one or more somatic mutations without other hematologic abnormalities. CHIP is common in the elderly, affecting more than 10% of individuals over 65 years. CHIP is associated with a 10-fold increase risk of hematologic malignancies and a doubled risk of atherosclerotic cardiovascular disease, contributing to an increase in all-cause mortality. The two most commonly mutated genes in CHIP are DNMT3A and TET2, both of which encode epigenetic modifiers. Recent studies have found evidence of increased inflammation and worsened atherosclerosis when the murine ortholog of TET2 was perturbed in myeloid cells in vivo. However, the specific epigenetic mechanism underlying this phenotype and whether DNMT3A and TET2 mutations target the same biological pathway are unclear. Furthermore, it remains unexplained why loss-of-function mutations in DNMT3A and TET2 have shared a clinical outcome despite the two genes encode enzymes with opposite biochemical functions (DNA methylation vs. de-methylation). To address this knowledge gap, we developed human pluripotent stem cell (hPSC)-derived macrophage models of DNMT3A- and TET2- haploinsufficiency. hPSC-derived macrophages are inexhaustible, scalable, and amenable to genetic manipulation, offering a powerful in vitro model well-suited for mechanistic studies. In this proposal, we propose the use of this model to (1) define unique and shared epigenetic features of DNMT3A and TET2 haploinsufficiency, and (2) identify transcription factors driving altered immune gene expression in DNMT3A- and TET2-haploinsufficient immune cells. This project is built on our previous work, and we anticipate that findings from this study will guide future studies on targeting an epigenetic vulnerability shared between DNMT3A- and TET2-mutated cells as well as mechanistic studies on other drivers of CHIP.