Epigenetic regulations of macrophage development

巨噬细胞发育的表观遗传调控

• 批准号: 10083228
• 负责人: KYUNGHEE CHOI
• 金额: $ 51.67万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-20 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10083228
• 关键词: ATAC-seq; Acetylation; Applications Grants; Architecture; Biological Assay; Blood Cells; Bypass; Cells; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Complex; Cytokine Signaling; Data; Defect; Development; Developmental Process; Disease; EP300 gene; Embryo; Epigenetic Process; Erythroid; Erythroid Cells; Flow Cytometry; Gene Expression; Genes; Genetic Transcription; Goals; Hematopoietic stem cells; High-Throughput Nucleotide Sequencing; Histones; In Vitro; Knock-out; Maps; Mediating; Molecular; Myelogenous; Myeloid Cells; Myeloid Progenitor Cells; Outcome; Output; Pathologic Processes; Pathway interactions; Phenotype; Play; Pluripotent Stem Cells; Population; Regenerative Medicine; Regulatory Element; Role; SMARCA4 gene; SMARCC1 gene; Somatic Cell; TREM2 gene; Testing; Tissues; Transferase; Transposase; Work; XCL1 gene; Yolk Sac; base; chromatin remodeling; cytokine; demethylation; embryonic stem cell; epigenetic regulation; erythroid differentiation; genetic effector; genome-wide; genomic locus; insight; knock-down; macrophage; novel; progenitor; programs; recruit; self-renewal; single-cell RNA sequencing; tissue regeneration; tissue repair; transcription factor; virtual

Yolk sac (YS) derived erythroid-myeloid progenitors (EMPs) provide embryos with definitive erythroid and myeloid cells that form tissue resident macrophages prior to the establishment of hematopoietic stem cell (HSCs). Molecular determinants that regulate myeloid lineage differentiation from EMPs are poorly understood. The mammalian SWI/SNF chromatin-remodeling BAF (BRG1/BRM associated factor) complex plays an important role in developmental and pathological processes. Our preliminary data indicates that deletion of Baf155 (aka Srg3 or Smarcc1), a subunit of BAF complex, in the Tie2-Cre lineage (Tie2-Cre; Baf155f/f, Baf155 CKO) leads to embryonic lethality, with defects in yolk sac macrophage differentiation. Flow cytometry showed virtually no mature myeloid cells in the yolk sac at E9.5 and E10.5. However, EMPs were present in the Baf155 CKO yolk sac. Analysis of wild type and Baf155 deficient yolk sac cells by single cell RNA sequencing revealed that while myeloid lineage master regulators, Pu.1 and Myb, were expressed in the EMP population in the absence of BAF155, expression of myeloid lineage maturation genes, such as Irf8, Csf1r, and Cx3cr1 was greatly reduced, suggesting that there might be a hierarchical relationship between PU.1 and BAF mediated chromatin remodeling in activating myeloid lineage maturation program. Unexpectedly, myeloid cytokines could rescue the differentiation defect of Baf155 deficient EMPs in culture. Examination of the chromatin accessibility at the myeloid gene loci of the rescued Baf155 CKO myeloid cells by using Assay for Transposase Accessible Chromatin with high throughput sequencing (ATAC-seq) suggested common as well as unique epigenetic changes potentially contributing to myeloid lineage rescue. From these fundamental findings, the overarching goal is to demonstrate that BAF155 mediated chromatin remodeling is required at the EMP stage for myeloid lineage differentiation to occur and that cytokines can bypass BAF155 requirements in myeloid lineage development. Particularly, we will investigate the hierarchical relationship between myeloid lineage transcription factor PU.1 and BAF155 chromatin-remodeling factor in myeloid lineage development. We will also determine the myeloid lineage program that is regulated by the PU.1-BAF155 pathway. We will investigate the role of BAF155 in permitting chromatin accessibility and chromatin states of myeloid lineage gene program. We will also investigate cytokine mediated epigenetic changes that corroborate with BAF155 function in the myeloid lineage program activation. Successful completion of the work will establish the role for chromatin remodeling, interplay between chromatin architecture and transcription factors critical for myeloid lineage differentiation. The outcome will also be instrumental for generating tissue macrophages from pluripotent stem cells or somatic cell reprogramming and function of such cells in a wide range of applications in regenerative medicine, and maneuvering tissue macrophage function required for tissue repair and regeneration or diseases.

蛋黄囊（Ys）衍生的红细胞乳突祖细胞（EMP）提供具有确定性红细胞和的胚胎 在建立造血干细胞之前形成组织常驻巨噬细胞的髓样细胞 （HSC）。对EMP的调节分化的分子决定因素知之甚少。 哺乳动物SWI/SNF染色质复制BAF（BRG1/BRM相关因子）配合物 在发育和病理过程中的重要作用。我们的初步数据表明删除 BAF155（又名SRG3或SMARCC1），BAF综合体的亚基，在TIE2-CRE谱系中（TIE2-CRE； BAF155F/F，BAF155 CKO）导致胚胎致死性，在蛋黄囊巨噬细胞分化中存在缺陷。流式细胞仪显示 在E9.5和E10.5处的蛋黄囊中几乎没有成熟的髓样细胞。但是，BAF155中存在EMP CKO蛋黄囊。通过单细胞RNA测序分析野生型和BAF155缺乏蛋黄囊细胞 虽然髓样谱系主要调节剂PU.1和MYB在EMP人群中表达 没有BAF155，髓样谱系成熟基因的表达，例如IRF8，CSF1R和CX3CR1 大大减少了 激活髓样谱系成熟程序中的染色质重塑。出乎意料的是，髓样细胞因子 可以挽救BAF155培养中BAF155缺乏的分化缺陷。染色质检查 通过使用测定法 带有高吞吐量测序（ATAC-SEQ）的转座酶可访问的染色质也建议常见 随着独特的表观遗传变化可能导致髓样谱系救援。来自这些基本 调查结果，总体目标是证明BAF155介导的染色质重塑是需要在 EMP阶段的发生分化发生，细胞因子可以绕过BAF155的要求 髓样谱系的发展。特别是，我们将研究髓样的分层关系 谱系转录因子PU.1和BAF155色胶谱系因子在髓样谱系发育中。 我们还将确定由PU.1-BAF155途径调节的髓样谱系程序。我们将 研究BAF155在允许染色质访问性和髓样谱系染色质状态中的作用 基因程序。我们还将研究细胞因子介导的表观遗传学变化，以证实BAF155 在髓样谱系程序激活中的功能。成功完成工作将确立 染色质重塑，染色质结构和转录因子之间的相互作用至关重要 谱系分化。结果也将对从 多能干细胞或在广泛应用中的这些细胞的体细胞重编程和功能 在再生医学和组织修复和机动组织巨噬细胞功能和 再生或疾病。