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.

卵黄囊（YS）衍生的红系-髓系祖细胞（EMP）提供具有定形红系和髓系祖细胞的胚胎。 在造血干细胞形成之前形成组织驻留巨噬细胞的髓样细胞 （HSC）。调节EMP髓系分化的分子决定因素知之甚少。 哺乳动物SWI/SNF染色质重塑BAF（BRG 1/BRM相关因子）复合物在哺乳动物中起作用。 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 Baf 155（aka Srg 3或Smarcc 1），BAF复合物的亚基，在Tie 2-Cre谱系中（Tie 2-Cre; Baf 155 f/f，Baf 155 CKO）导致胚胎死亡，卵黄囊巨噬细胞分化缺陷。流式细胞术显示 在E9.5和E10.5，卵黄囊中几乎没有成熟的髓样细胞。然而，在Baf 155中存在EMP CKO卵黄囊。通过单细胞RNA测序分析野生型和Baf 155缺陷型卵黄囊细胞， 虽然髓系主调节因子Pu.1和Myb在EMP群体中表达， BAF 155缺失时，髓系成熟基因如Irf 8、Csf 1 r和Cx 3cr 1的表达， 表明PU.1和BAF介导的BAF之间可能存在层次关系 染色质重塑激活髓系成熟程序。出乎意料的是，骨髓细胞因子 可以挽救培养中Baf 155缺陷型EMPs的分化缺陷。染色质检查 通过使用针对Baf 155 CKO骨髓细胞的骨髓基因位点的可及性的测定， 转座酶可降解染色质与高通量测序（ATAC-seq）也表明常见 作为独特的表观遗传变化可能有助于骨髓谱系拯救。从这些基本的 研究结果，总体目标是证明BAF 155介导的染色质重塑是必需的， 骨髓谱系分化发生的EMP阶段，细胞因子可以绕过BAF 155的要求， 髓系发育特别是，我们将研究髓系细胞和非髓系细胞之间的层次关系。 谱系转录因子PU.1和BAF 155染色质重塑因子在髓系发育中的作用。 我们还将确定由PU.1-BAF 155途径调节的髓系程序。我们将 研究BAF 155在允许骨髓谱系的染色质可及性和染色质状态中的作用 基因计划我们还将研究细胞因子介导的表观遗传变化，证实BAF 155 在骨髓谱系程序激活中起作用。工作的顺利完成将确立以下方面的作用： 染色质重塑，染色质结构和转录因子之间的相互作用对髓样细胞的关键 谱系分化该结果也将有助于产生组织巨噬细胞， 多能干细胞或体细胞重编程以及这些细胞在广泛应用中的功能 在再生医学中，以及操纵组织修复所需的组织巨噬细胞功能， 再生或疾病。