Epigenetic modification of hematopoietic stem and progenitor cells in inflammation-induced differentiation

项目概要 感染是造血干细胞移植中发病率和死亡率的首要原因 （造血干细胞移植）患者。感染导致造血干细胞和祖细胞植入延迟或失败 细胞（HSPC）。为了研究感染相关的 HSPC 功能受损的机制，我们的实验室利用 鸟分枝杆菌感染模型，发现慢性感染通过损害自身 更新和促进骨髓分化——通过增加骨髓的激活和转录 分化基因，如 Batf2、Fosb 和 Jun——通过干扰素γ (IFNγ) 依赖性机制。 此外，实验室表明炎症诱导的骨髓分化是表观遗传驱动的，因为 DNA甲基转移酶DNMT3A的敲除导致骨髓分化反应的抑制。基于 根据我们小组之前研究的这些数据，我假设 HSPC 经历了可塑的表观遗传 响应 IFNγ 的重编程促进骨髓分化并影响下游免疫 回应。通过定义表观遗传重编程的程度，我寻求确定保护 HSC 的策略 尽管存在感染应激，但仍能发挥功能，从而改善 HSCT 的结果。 而我们的实验室表明，DNA 甲基化的 IFNγ 依赖性变化有助于 HSC 慢性感染期间的分化和衰竭、IFNγ刺激对组蛋白修饰的影响、 表观遗传调控的另一个关键机制尚未在 HSPC 中进行研究。因此，第一个目标 目的是确定 IFNγ 是否诱导 HSPC 中的组蛋白修饰以促进骨髓分化。 具体来说，我将使用表观基因组测序技术 CUT&RUN-seq 和 ATAC-seq 来确定 炎症应激下 HSPC 中组蛋白修饰和染色质可及性的变化。接下来，我将 确定哪些预刺激的 HSPC 亚群通过移植 M. avium 进行功能重编程 将 HSPC 亚群刺激到初始接受者中，并在 3 个月后用 M. 挑战接受者。 鸟。最后，我将通过暴露 M. avium 来研究这些表观遗传修饰的可塑性- 刺激 HSPC 产生随后的高剂量 LPS。我将执行 RNA-seq、CUT&RUN-seq 和 ATAC-seq，并且 移植后免疫激发实验以确定 HSPC 在鸟分枝杆菌后是否暴露于 LPS 与单独暴露于鸟分枝杆菌相比，刺激显示出不同的分化反应。总体而言， 该提案中的工作将揭示 IFNγ 促进骨髓分化和 HSC 的机制 慢性炎症期间的疲惫，并能够开发预防移植的治疗方法 移植后早期的损失。

Project Summary Infections are the number one cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) patients. Infections contribute to delayed or failed engraftment of hematopoietic stem and progenitor cells (HSPCs). To study the mechanism underlying infection-related impaired HSPC function, our lab has utilized a Mycobacterium avium infection model and discovered that chronic infection depletes HSCs by impairing self- renewal and promoting myeloid differentiation – through increased activation and transcription of myeloid differentiation genes such as Batf2, Fosb, and Jun– via an interferon-gamma (IFNγ)-dependent mechanism. Further, the lab showed that inflammation-induced myeloid differentiation is epigenetically driven, as the knockout of DNA methyltransferase DNMT3A led to suppression of the myeloid differentiation response. Based on these data from our group’s previous studies, I hypothesize that HSPCs undergo a malleable epigenetic reprogramming in response to IFNγ that promotes myeloid differentiation and affects downstream immune responses. By defining the extent of epigenetic reprogramming, I seek to identify strategies to preserve HSC function despite infectious stress and thereby improve HSCT outcomes. Whereas our lab showed that IFNγ-dependent changes in DNA methylation contribute to HSC differentiation and exhaustion during chronic infection, the impact of IFNγ stimulation on histone modifications, another key mechanism of epigenetic regulation, has not been studied in HSPCs. Therefore, the first objective is to determine whether IFNγ induces histone modifications in HSPCs to promote myeloid differentiation. Specifically, I will use epigenomic sequencing techniques CUT&RUN-seq and ATAC-seq to determine the changes in histone modifications and chromatin accessibility in HSPCs under inflammatory stress. Next, I will identify which pre-stimulated HSPC subpopulations are functionally reprogrammed by transplanting M. avium- stimulated HSPC subpopulations into naïve recipients and challenging the recipients 3 months later with M. avium. Finally, I will investigate the malleability of these epigenetic modifications by exposing M. avium- stimulated HSPCs to subsequent high-dose LPS. I will perform RNA-seq, CUT&RUN-seq, and ATAC-seq, and immune challenge experiments post-transplant to determine whether HSPCs exposed to LPS after M. avium stimulation show altered differentiation responses compared to those exposed to M. avium alone. Overall, the work in this proposal will uncover the mechanisms by which IFNγ promotes myeloid differentiation and HSC exhaustion during chronic inflammation and enable the development of therapeutic approaches to prevent graft loss in the early post-transplant period.