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

项目总结 不确定潜能克隆性造血(CHIP)是指扩增的血细胞克隆的存在 有一个或多个没有其他血液异常的体细胞突变。芯片在老年人中很常见， 影响超过10%的65岁以上的人。芯片与增加10倍的风险有关 恶性血液病和动脉粥样硬化性心血管疾病的风险增加一倍，导致 全因死亡率上升。CHIP中最常见的两个突变基因是DNMT3A和TET2，两者都是 其中编码表观遗传修饰因子。最近的研究发现，有证据表明炎症和 当小鼠的TET2同源基因在体内的髓系细胞中受到干扰时，会加剧动脉粥样硬化。然而， 这种表型背后的特殊表观遗传机制以及DNMT3A和TET2突变 靶向相同的生物途径尚不清楚。此外，为什么功能丧失仍未得到解释 DNMT3A和TET2的突变具有相同的临床结果，尽管这两个基因编码的酶与 相反的生化功能(DNA甲基化与去甲基化)。为了解决这一知识差距，我们 人多能干细胞来源的巨噬细胞DNMT3A-和TET2-模型的建立 单倍体功能不全。HPSC来源的巨噬细胞取之不尽用之不竭，可伸缩，并服从于基因 操控，提供了一个强大的体外模型，非常适合于机械学研究。在这项提案中，我们 建议使用该模型来(1)定义DNMT3A和TET2独特且共享的表观遗传特征 单倍体不足，以及(2)确定驱动DNMT3A免疫基因表达改变的转录因子- 和TET2单倍体不足的免疫细胞。这个项目是建立在我们以前的工作基础上的，我们预计 这项研究的发现将指导未来针对表观遗传易感性的研究 DNMT3A和TET2突变细胞以及其他芯片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.