Epigenetic control of vascular niche capacity to support hematopoiesis.

项目摘要/摘要 造血是一个细胞发育过程，它受细胞内在发育程序和微环境中细胞外在因素的控制。骨髓和其他造血器官中存在的血管壁龛提供了适当调节这一过程所需的物理空间和信号。在接受化疗或接受造血干细胞移植(HSCT)的患者中，造血干细胞(HSC)的系统发育和功能多样性受到限制，导致克隆性造血和细胞减少，这是这些患者发病率和死亡率的重要来源。目前，对微环境在调节HSC多样性中的作用知之甚少。我们假设血管壁龛内存在的机制可以被调节，使其能够支持更具系统发育和功能多样性的造血干细胞群体。我们对条形码格式塔斑马鱼进行了遗传筛选，发现斑马鱼PKC-Delta同源物prkcda的异常表达使有助于造血的HSC克隆数量增加了50%以上。使用单细胞RNA-SEQ的表型分析表明，新的未成熟中性粒细胞群体的存在和红系前体细胞的扩张。单细胞ATAC-SEQ分析表明，天然prkcda基因启动子的染色质可及性在血管壁细胞群体中尤其减少。这些数据使我们假设在血管生态位中存在特定的表观遗传程序，该程序调节支持造血的因子的表达，从而维持生态位支持HSC系统发育和功能多样性的能力。在目标1中，我们将使用特定表观遗传因子的切割和运行分析来表征人内皮细胞培养中的血管生态位。多模式单细胞RNA-seq/ATAC-seq将用于成年斑马鱼和胚胎斑马鱼，以确定支持造血的重要基因附近的利基细胞群和顺式作用DNA元件。在目标2中，CRISPR筛选将用于识别位于人血管壁龛细胞中的特定转录因子结合位点，这些细胞位于壁龛功能关键基因附近并控制其转录，包括CXCL12、Ang1和KITLG。一组斑马鱼CRISPR突变体将被用来研究选定的增强子元件在体内的功能。利基功能的变化将通过体外与HSPC共培养和体内格式塔条形码和scRNA-seq来检测。这项工作将提供新的机制上的理解，以支持不同的造血干细胞群体的能力是如何调节的，并可能导致新的治疗方法，以改善化疗或移植后的造血恢复。

PROJECT SUMMARY/ABSTRACT Hematopoiesis is a cellular developmental process that is controlled by a cell-intrinsic developmental program and cell-extrinsic factors from the microenvironment. The vascular niche present in the bone marrow and other hematopoietic organs provides the physical space and signals necessary for the proper regulation of this process. Hematopoietic stem cell (HSC) phylogenetic and functional diversity are restricted in patients who receive chemotherapy or are recipients of hematopoietic stem cell transplantation (HSCT), leading to clonal hematopoiesis and cytopenias which are significant sources of morbidity and mortality in these patients. At present, very little is known about the role of the microenvironment in regulating HSC diversity. We hypothesized that mechanisms exist within the vascular niche that can be modulated to allow it to support a more phylogenetically and functionally diverse population of HSCs. We performed a genetic screen in barcoded GESTALT zebrafish and found that dysregulated expression of prkcda, the zebrafish PKC-delta homolog, increased the number of HSC clones contributing to hematopoiesis by more than 50%. Phenotypic analysis using single cell RNA-seq demonstrated the presence of a novel population of immature neutrophils and expansion of erythroid precursor cells. Single cell ATAC-seq analysis showed that chromatin accessibility at the promoter of the native prkcda locus is reduced specifically in vascular niche cell populations. These data led us to hypothesize the existence of a specific epigenetic program within the vascular niche that regulates expression of factors supporting hematopoiesis and thereby maintains the capacity of the niche to support HSC phylogenetic and functional diversity. In Aim 1, we will use Cut and Run analysis for specific epigenetic factors to characterize the vascular niche in human endothelial cell cultures. Multimodal single cell RNA-seq/ATAC-seq will be used in adult and embryonic zebrafish to identify niche cell populations and cis-acting DNA elements near genes important for supporting hematopoiesis. In Aim 2, a CRISPR screen will be used to identify specific transcription factor binding sites in human vascular niche cells lying proximal to and controlling transcription of key genes involved in niche function, including CXCL12, ANG1, and KITLG. A panel of zebrafish CRISPR mutants will be made to study the function of selected enhancer elements in vivo. Changes in niche function will be assayed in vitro by co-culture with HSPCs and in vivo by GESTALT barcoding and scRNA-seq. This work will provide new mechanistic understanding of how the capacity of the hematopoietic niche to support a diverse population of HSCs is regulated and may lead to new therapies that improve hematopoietic recovery after chemotherapy or transplantation.