Establishing the role and mechanisms of LSD1 during megakaryocytic and erythroid fate commitment

建立LSD1在巨核细胞和红细胞命运决定过程中的作用和机制

• 批准号: 10605993
• 负责人: Evrett Thompson
• 金额: $ 5.02万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605993
• 关键词: ATAC-seq; Adult; Anemia; Benign; Biological Models; Blood; Blood Cells; Blood Platelets; Bone Marrow; CD34 gene; Candidate Disease Gene; Cell Culture Techniques; Cell Differentiation process; Cell Lineage; Cell Therapy; Cells; Chimeric Proteins; Chromatin; Computer Analysis; Data; Data Set; Disease; Enhancers; Epigenetic Process; Erythrocytes; Erythroid; Erythroid Progenitor Cells; Erythropoiesis; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Guide RNA; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Individual; Investigation; KDM1A gene; Lead; Lysine; Maintenance; Malignant - descriptor; Mediating; Megakaryocytes; Megakaryocytopoieses; Messenger RNA; Modification; Mus; Myelogenous; Pathology; Pathway interactions; Play; Population; Process; Production; Publishing; Research; Research Personnel; Role; Series; Site; Specific qualifier value; Testing; Training; candidate identification; career; cell fate specification; cell type; demethylation; endonuclease; granulocyte; hematopoietic differentiation; histone modification; inhibitor; insight; monocyte; peripheral blood; progenitor; programs; promoter; stem; stem cells; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Hematopoiesis is responsible for producing the varied cell types found in blood. In adults, this process primarily takes place in the bone marrow and is characterized by successive rounds of differentiation beginning with the hematopoietic stem cell through to lineage commitment. This process involves progressive narrowing of lineage potency as progenitors eventually commit to the production of a single cell lineage. Disruption of hematopoiesis can lead to benign and malignant pathologies. We focus on the bipotent megakaryocytic-erythroid progenitor (MEP), which has the potential to differentiate into a lineage committed erythroid progenitor (ErP) or a lineage committed megakaryocytic progenitor (MkP). MEP fate specification has been studied to a limited extent, uncovering only a small number of influences that contribute to this process. One understudied aspect of human MEP fate specification is the role of epigenetics. KDM1A (LSD1) is known to be important for erythroid maturation, but its role in MEP fate specification is unknown. Preliminary data using primary human cells show that inhibition of LSD1 in primary human ErP results in the ability of erythroid committed progenitors to undergo granulocytic-monocytic and megakaryocytic commitment. In contrast, LSD1 inhibition does not affect MkP commitment to the megakaryocytic fate. This suggests that LSD1 only antagonizes alternative lineage potential during erythropoiesis, and not megakaryopoiesis, even though LSD1 mRNA is expressed at similar levels in MEP, ErP, and MkP. The goal of this proposal is to establish the mechanism by which LSD1 promotes erythroid commitment while silencing alternative myeloid and megakaryocytic lineage potential. I will achieve these goals utilizing a mix of functional (cell culture), genetic and epigenetic approaches to determine where and how LSD1 regulates gene expression and fate specification in MEP, ErP, and MkP. Computational analyses will determine candidate gene targets, and potential genomic sites to force epigenetic modifications using different fusion proteins based on the catalytically inactive Cas9 endonuclease (dCas9). These results will establish the epigenetic mechanisms that govern erythroid lineage commitment mediated by LSD1. They will also shed light on the distinct lineage-specific epigenetic mechanisms that mediate and maintain fate specification in hematopoiesis.

项目总结/摘要 造血是负责生产血液中发现的各种细胞类型。在成年人中，这一过程主要是 发生在骨髓中，其特征是从骨髓开始的连续几轮分化 造血干细胞通过谱系定型。这一过程涉及血统的逐渐缩小 作为祖细胞的潜能最终致力于单细胞谱系的产生。造血功能障碍 会导致良性和恶性病变。我们关注的是双能巨核-红系祖细胞 (MEP)，其具有分化为谱系定向红系祖细胞（ErP）或谱系定向红系祖细胞（ErP）的潜力。 定向巨核细胞祖细胞（MkP）。MEP结局规格已在有限程度上进行了研究， 只揭示了一小部分影响这一过程的因素。人类的一个未被充分研究的方面 MEP命运特化是表观遗传学的作用。已知KDM 1A（LSD 1）对红系细胞重要， 成熟，但其在MEP命运规范的作用是未知的。使用原代人类细胞的初步数据显示， 在原代人ErP中LSD 1的抑制导致红系定向祖细胞经历 粒细胞-单核细胞和巨核细胞定型。相反，LSD 1抑制不影响MkP 巨核细胞的命运。这表明LSD 1仅拮抗替代谱系潜力， 在红细胞生成过程中，而不是在巨核细胞生成过程中，尽管LSD 1 mRNA在 MEP、ErP和MkP。该提案的目标是建立LSD 1促进红系造血细胞增殖的机制。 同时沉默替代性髓系和巨核细胞谱系潜力。我将实现这些目标 利用功能（细胞培养），遗传和表观遗传方法的混合来确定LSD 1在哪里以及如何 调节MEP、ErP和MkP中的基因表达和命运特化。计算分析将确定 候选基因靶点和潜在的基因组位点，以使用不同的融合蛋白迫使表观遗传修饰， 基于无催化活性的Cas9内切核酸酶（dCas 9）的蛋白质。这些结果将建立 LSD 1介导的控制红系定型的表观遗传机制。他们也将揭示 不同的谱系特异性表观遗传机制，介导和维持命运的规范， 造血