Epigenetic control of vascular niche capacity to support hematopoiesis.

表观遗传控制血管生态位能力以支持造血。

• 批准号: 10403595
• 负责人: Bradley Wayne Blaser
• 金额: $ 31.35万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10403595
• 关键词: ANGPT1 gene; ATAC-seq; Adult; Animals; Bar Codes; Binding; Binding Sites; Blood; Blood Vessels; Bone Marrow; CRISPR screen; CXCL12 gene; Cell Culture Techniques; Cell Line; Cells; Chromatin; Clonal Hematopoietic Stem Cell; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Cytotoxic Chemotherapy; DNA; Data; Development; Developmental Process; Elements; Embryo; Endothelial Cells; Enhancers; Epigenetic Process; Erythroid; Erythroid Progenitor Cells; Fertilization; Gene Expression; Genes; Genetic Enhancer Element; Genetic Screening; Genetic Transcription; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Homologous Gene; Human; In Vitro; KITLG gene; Kidney; Lead; Link; Longevity; Marrow; Molecular; Morbidity - disease rate; NF-kappa B; Natural regeneration; Organ; Outcome; Patients; Phenotype; Phylogenetic Analysis; Population; Population Heterogeneity; Process; Promoter Regions; Recovery; Regulation; Role; Signal Transduction; Signaling Molecule; Source; Stem cell transplant; System; Tissues; Toxin; Transplantation; Work; Zebrafish; cell type; chemotherapy; cytopenia; hematopoietic stem cell fate; hematopoietic stem cell niche; improved; in vitro Assay; in vivo; mortality; multimodality; mutant; neutrophil; novel; novel therapeutics; overexpression; prevent; programs; promoter; reconstitution; single-cell RNA sequencing; stem cell biology; stem cell population; therapy development; transcription factor; unpublished works

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.

项目摘要/摘要 造血是一种细胞发育过程，由微环境中的细胞内部发育程序和细胞超级因素控制。骨髓和其他造血器官中存在的血管生态市场提供了适当调节此过程所需的物理空间和信号。造血干细胞（HSC）系统发育和功能多样性受到接受化疗或受到造血干细胞移植（HSCT）的接受者的限制，导致克隆造血和细胞质减少症，这是这些患者的重要来源。目前，关于微环境在调节HSC多样性中的作用知之甚少。我们假设在血管生态层中存在机制，可以调节其在系统发育和功能上多样化的HSC种群。我们在条形码格式斑马鱼中进行了遗传筛选，发现斑马鱼PKC-DELTA同源物PrkCDA的表达失调增加，增加了对造血的HSC克隆的数量，增加了50％以上。使用单细胞RNA-seq进行表型分析证明了新的未成熟嗜中性粒细胞的存在和红斑前体细胞的扩展。单细胞ATAC-SEQ分析表明，在血管生态位细胞种群中专门降低了天然PRKCDA基因座的启动子的染色质可及性。这些数据导致我们假设在血管生态位中存在特定表观遗传程序的存在，该计划调节了支持造血的因素的表达，从而维持利基市场支持HSC系统发育和功能多样性的能力。在AIM 1中，我们将对特定表观遗传因素进行剪切和运行分析，以表征人内皮细胞培养物中的血管生态位。多模式单细胞RNA-seq/atac-seq将用于成人和胚胎斑马鱼，以鉴定基因附近的小众细胞群体和顺式作用DNA元素，对支撑造血的基因很重要。在AIM 2中，将使用CRISPR屏幕来识别靠近属于CXCL12，ANG1和KITLG在内的关键基因的人血管生态位细胞中特定的转录因子结合位点。将制作一组斑马鱼CRISPR突变体，以研究体内选定的增强子元件的功能。利基功能的变化将通过与HSPC共同培养和在体内通过Gestalt Barcoding和SCRNA-Seq在体外分析。这项工作将提供新的机械理解，了解如何调节造血生态位以支持多样化的HSC的能力，并可能导致新的疗法改善化学疗法或移植后造血恢复。