Altered inflammatory response associated with acquired DNMT3A mutations

项目摘要 人类基因DNMT 3A编码三种酶之一，这些酶在细胞中进行DNA甲基化。 人类具有DNMT 3A获得性突变的血细胞的克隆扩增在老年人中很常见， 发生在5%-10%的60岁或以上的健康个体中。DNMT 3A突变的携带者具有 患血液系统癌症的风险增加约10倍， 冠心病考虑到美国和世界各地人口的迅速老龄化， 血细胞中获得性DNMT 3A突变与增加的 对癌症和心血管疾病的易感性对公众健康极为重要。最近的研究 发现当DNMT 3A的直系同源物被激活时，由骨髓细胞介导的炎症增加的证据。 在动物模型中受到干扰。然而，这种现象背后的分子机制，以及是否 DNMT 3A突变影响人类骨髓细胞的炎症反应仍然知之甚少。到 为了解决这一知识空白，我们建立了一个基于髓系细胞分化的实验系统， 从人类多能干细胞。使用该系统，我们发现具有DNMT 3A的人巨噬细胞 与野生型巨噬细胞相比，突变显示出改变的炎症反应，其特征在于： 增强IL-6的表达，IL-6是一种有效的促炎细胞因子。IL 6启动子是最重要的启动子之一。 DNMT 3A突变的巨噬细胞中的显著低甲基化位点，表明直接的机制联系 DNA甲基化和炎症反应之间的联系在本申请中，我们建议 使用以下方法表征与DNMT 3A突变相关的炎症反应的分子特征： 基因定义的人类巨噬细胞和中性粒细胞，并剖析表观遗传机制的基础上， DNMT 3A介导的基因表达调控。此外，我们还将研究克隆性地窝藏 使用DNMT 3A突变扩增的血细胞对原代髓系细胞炎症反应的影响， 新的单细胞转录组学技术。我们正处于一个理想的位置，以追求这个项目， 我们开发的基于人多能干细胞的人骨髓细胞模型的可用性， 验证，我们可以访问一个代表极其多样化人群的大型生物库， 由具有互补专业知识的研究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DNMT 3A突变对炎症的影响提供重要的新见解， 并帮助我们开发新的策略来预防和治疗与DNMT 3A突变相关的病理状况。

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.