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（HSPCs）。为了研究感染相关的HSPC功能受损的机制，我们实验室利用 一个鸟分枝杆菌感染模型，发现慢性感染通过损害自身免疫系统， 更新和促进髓样分化-通过增加髓样细胞的激活和转录 通过干扰素-γ（IFNγ）依赖性机制，可以诱导分化基因如Batf 2、Fosb和Jun的表达。 此外，该实验室表明，炎症诱导的骨髓分化是表观遗传驱动的， DNA甲基转移酶DNMT 3A的敲除导致骨髓分化反应的抑制。基于 根据我们小组以前的研究数据，我假设HSPCs经历了一个可塑性的表观遗传过程， 对IFNγ应答的重编程促进骨髓分化并影响下游免疫 应答通过定义表观遗传重编程的程度，我试图确定保存HSC的策略 尽管感染应激，但仍能发挥功能，从而改善HSCT结果。 而我们的实验室表明，IFNγ依赖的DNA甲基化变化有助于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髓样分化。 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。接下来我会 通过移植M.鸟， 将HSPC亚群刺激成幼稚受体，并在3个月后用M. avium最后，我将研究这些表观遗传修饰的可塑性，通过暴露M。鸟， 刺激的HSPCs对随后的高剂量LPS。我将执行RNA-seq、CUT&RUN-seq和ATAC-seq， 移植后免疫攻击实验以确定在M.鸟 与暴露于M相比，刺激显示改变的分化反应。只有avium。总体看 这项工作将揭示IFNγ促进髓系分化和HSC的机制 慢性炎症期间的衰竭，并使治疗方法的发展，以防止移植 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.