Altered inflammatory response associated with acquired DNMT3A mutations

项目总结 人类基因DNMT3A编码执行DNA甲基化的三种酶之一 人类。带有获得性DNMT3A突变的血细胞克隆性扩增在老年人中很常见， 在60岁或以上的健康人中，有5%-10%的人会发生这种情况。DNMT3A突变携带者有 患血液病的风险大约增加10倍，罹患血液病的可能性增加两倍 冠心病。鉴于美国和世界各地迅速老龄化的人口，理解 血细胞获得性DNMT3A突变与增高性疾病相关性的机制基础 对癌症和心血管疾病的易感性对公共健康极其重要。最新研究 当DNMT3A的同源基因被发现时，发现了髓系细胞介导的炎症增加的证据 对动物模型心烦意乱。然而，这种现象背后的分子机制以及是否 DNMT3A突变影响人类髓系细胞的炎症反应仍然知之甚少。至 针对这一认识空白，我们建立了基于髓系细胞分化的实验系统 来自人类多能干细胞。使用该系统，我们发现携带DNMT3A的人巨噬细胞 与野生型巨噬细胞相比，突变显示出炎症反应的改变，其特征是 IL-6的表达增强，这是一种强有力的促炎细胞因子。IL6的启动子是最多的 DNMT3A突变的巨噬细胞中显著低甲基化的基因座，提示有直接的机制联系 在我们的模型中，DNA甲基化和炎症反应之间的关系。在本申请中，我们建议 研究与DNMT3A突变相关的炎症反应的分子特征 从基因上定义人类巨噬细胞和中性粒细胞，并剖析潜在的表观遗传机制 DNMT3A介导的基因表达调控。此外，我们将研究克隆人窝藏的影响 扩增的DNMT3A突变血细胞对原代髓系细胞炎症反应的影响 新的单细胞转录切割技术。我们处于一个理想的位置来推进这个项目，因为 我们开发的基于人类多能干细胞的人髓系细胞模型的可用性 经过验证，我们可以访问代表极其不同人群的大型生物库，并组装一个 由具有互补专业知识的调查人员组成的强大的科学团队。建议的调查结果 这项研究将为获得DNMT3A突变对炎症的后果提供关键的新见解。 并帮助我们开发新的策略来预防和治疗与DNMT3A突变相关的病理情况。

PROJECT SUMMARY The human gene DNMT3A encodes one of the three enzymes that carry out DNA methylation in humans. Clonal expansion of blood cells with acquired mutations in DNMT3A is common in older adults, occurring in 5-10 % of healthy individuals aged 60 or above. Carriers of DNMT3A mutations have an approximately tenfold increased risk of developing hematologic cancers and are twice as likely to develop coronary heart disease. Given the rapidly aging population in the United States and worldwide, understanding the mechanistic basis of the association between acquired DNMT3A mutations in blood cells and increased susceptibility to cancer and cardiovascular disease is tremendously important for public health. Recent studies found evidence of increased inflammation mediated by myeloid cells when the ortholog of DNMT3A was perturbed in animal models. However, the molecular mechanisms underlying this phenomenon and whether DNMT3A mutations affect the inflammatory response of human myeloid cells remain poorly understood. To address this gap in knowledge, we established an experimental system based on myeloid cells differentiated from human pluripotent stem cells. Using this system, we found that human macrophages with DNMT3A mutations displayed altered inflammatory response compared to wild-type macrophages, characterized by augmented expression of IL-6, a potent proinflammatory cytokine. The IL6 promoter was one of the most significantly hypomethylated loci in DNMT3A-mutated macrophages, suggesting a direct mechanistic link between DNA methylation and inflammatory response in our model. In this application, we propose to characterize the molecular signature of the inflammatory response associated with DNMT3A mutations using genetically defined human macrophages and neutrophils and to dissect the epigenetic mechanisms underlying DNMT3A-mediated gene expression regulation. In addition, we will examine the impact of harboring clonally expanded blood cells with DNMT3A mutations on the inflammatory response of primary myeloid cells using a novel single-cell transcriptomic technique. We are in an ideal position to pursue this project given the availability of human pluripotent stem cell-based human myeloid cell models that we have developed and validated, our access to a large biobank representing extremely diverse populations, and the assembly of a strong scientific team consisting of investigators with complementary expertise. Findings from the proposed study will provide critical new insights into the consequence of acquiring DNMT3A mutations on inflammation, and help us develop novel strategies to prevent and treat pathologic conditions related to DNMT3A mutations.