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

项目摘要 感染是造血干细胞移植中发病率和死亡率的头号原因 (HSCT)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HSPC功能受损的机制，我们的实验室利用 一种禽分枝杆菌感染模型，并发现慢性感染通过损害自身 更新和促进髓系分化--通过增加髓系的激活和转录 分化基因如BATF2、FosB和Jun-通过干扰素-γ依赖的机制。 此外，实验室还表明，炎症诱导的髓系分化是由表观遗传学驱动的，因为 DNA甲基转移酶DNMT3A的敲除导致髓系分化反应的抑制。基座 根据我们小组以前研究的这些数据，我假设HSPC经历了可延展的表观遗传学 干扰素γ促进髓系分化并影响下游免疫的重编程 回应。通过定义表观遗传重新编程的程度，我试图确定保存HSC的策略 在感染压力下仍能发挥作用，从而改善HSCT结果。 鉴于我们的实验室显示干扰素γ甲基化依赖的变化有助于造血干细胞 慢性感染时的分化和耗竭，干扰素γ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中研究。因此，第一个目标是 目的是确定干扰素γ是否诱导组蛋白修饰以促进髓系分化。 具体地说，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，我会 确定哪些预先刺激的HSPC亚群通过移植M.avium而功能重新编程- 刺激HSPC亚群成为幼稚的接受者，并在3个月后用M。 阿维姆。最后，我将通过暴露鸟型支原体来研究这些表观遗传修饰的延展性。 刺激HSPC产生随后的大剂量内毒素。我将执行RNA-seq、Cut&Run-seq和atac-seq，以及 移植后免疫激发实验以确定HSPC在禽分枝杆菌感染后是否暴露于脂多糖 与单独暴露于禽类分枝杆菌的刺激相比，刺激表现出不同的分化反应。总体而言， 这项提案的工作将揭示干扰素γ促进髓系分化和造血干细胞的机制 在慢性炎症期间耗尽体力，并使预防移植物的治疗方法得以发展 移植后早期丢失。

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.