Identifying necessary chromatin states and targets for hemogenic specification and reprogramming

确定造血规范和重编程所需的染色质状态和目标

• 批准号: 9907211
• 负责人: Elizabeth Howell
• 金额: $ 4.55万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907211
• 关键词: ATAC-seq; Adult; Affect; Binding; Blood; Blood Cells; Cell Nucleus; ChIP-seq; Chromatin; Chromatin Structure; Competence; DNA; Data; Derivation procedure; Development; Ectopic Expression; Embryo; Endothelial Cells; Endothelium; Fetus; Fluorescent in Situ Hybridization; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genome; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heterochromatin; Histones; Lamin B1; Methods; Microscopy; Molecular; Mus; Nuclear; Nuclear Structure; Nucleosomes; Optics; Patients; Peripheral; Positioning Attribute; Process; RUNX1 gene; Resolution; Role; Route; Specific qualifier value; Testing; Therapeutic; Therapeutic Uses; To specify; Transitional Cell; Visualization; XCL1 gene; clinical application; dosage; endothelial stem cell; experimental study; fetal; hematopoietic stem cell formation; hemogenic endothelium; improved; in vivo; insight; mouse development; prevent; reconstruction; stem cell therapy; transcription factor; transcriptome sequencing

Project Summary Many patients with hematological disorders rely on hematopoietic stem cell (HSC) transplants for treatment. However, for numerous patients, this option is dramatically impacted by the lack of optimally matched donors. Generation of HSCs ex vivo would greatly improve treatment for these patients. Understanding how HSCs form in vivo is crucial for the efficient derivation of HSCs ex vivo. During development, HSCs originate from a subset of endothelium, known as hemogenic endothelium. HSCs form through a process known as endothelial-to- hematopoietic transition (EHT). The complete genetic and molecular mechanism underlying EHT is still not fully understood, but it is known that RUNX1 is absolutely necessary for EHT. Ectopic expression of RUNX1 in embryonic non-hemogenic endothelium at E8.5 is able to induce EHT and blood cell formation. However, four days later in mouse development, RUNX1 is not as effective at reprogramming fetal endothelium to undergo EHT. Efficient hemogenic specification of fetal endothelium requires twice as much RUNX1, and proceeds via a different route than that of embryonic ECs. We hypothesize that the differential hemogenic competency observed between fetal and embryonic endothelium is due, at least in part, to changes in chromatin organization that prevent RUNX1 from binding key target genes. In order to understand how RUNX1 can efficiently specify embryonic endothelium as hemogenic, we will identify direct targets of RUNX1 and determine if these targets are marked by repressive heterochromatin in fetal endothelium. Additionally, we will determine if there are underlying baseline differences in chromatin structure between fetal and embryonic endothelium by profiling the genes in lamina associated domains (LADs) and using stochastic optical reconstruction microscopy (STORM) to determine global chromatin and nucleosome organization. Additionally, we will use DNA fluorescent in situ hybridization (FISH) to determine if important RUNX1 targets are localized to the nuclear periphery in fetal endothelium, which is what is preventing these genes from getting activated. These experiments will provide insight on the precise chromatin landscape and gene expression necessary for efficient hemogenic specification from endothelium. These studies will provide potential avenues for ex vivo generation of HSCs for therapeutic uses.

项目摘要 许多血液病患者依赖造血干细胞（HSC）移植进行治疗。 然而，对于许多患者来说，这种选择受到缺乏最佳匹配供体的严重影响。 离体产生HSC将极大地改善对这些患者的治疗。了解HSC如何形成 在体内对于离体HSC的有效衍生至关重要。在发育过程中，HSC起源于一个亚群， 也就是生血内皮。HSC的形成是通过一个称为内皮细胞-内皮细胞转化的过程。 造血转变（EHT）。EHT的完整遗传和分子机制尚不完全清楚， 理解，但众所周知，RUNX 1对于EHT是绝对必要的。RUNX 1在大肠杆菌中的异位表达 E8.5的胚胎非生血内皮细胞能够诱导EHT和血细胞形成。不过四 在小鼠发育的几天后，RUNX 1在重新编程胎儿内皮细胞以进行 EHT。胎儿内皮细胞的有效生血特化需要两倍的RUNX 1，并通过一个 与胚胎内皮细胞不同的途径。我们假设，观察到的不同生血能力 胎儿和胚胎内皮细胞之间的差异至少部分是由于染色质组织的变化， 阻止RUNX 1结合关键靶基因。为了理解RUNX 1如何有效地指定 胚胎内皮作为生血细胞，我们将确定RUNX 1的直接靶点，并确定这些靶点是否 以胎儿内皮细胞的抑制性异染色质为标志。此外，我们将确定是否有 胎儿和胚胎内皮细胞之间染色质结构的基础差异， 基因在纤层相关结构域（LAD）和使用随机光学重建显微镜（STORM） 以确定整体染色质和核小体组织。此外，我们将使用DNA荧光原位 用荧光原位杂交（FISH）确定重要的RUNX 1靶点是否定位于胎儿细胞核周围。 内皮细胞，这是什么阻止这些基因被激活。这些实验将提供 深入了解高效造血特化所需的精确染色质景观和基因表达 内皮细胞。这些研究将为离体产生用于治疗的HSC提供潜在途径。 使用.