Epigenetic regulations of macrophage development

卵黄囊(YS)来源的红系-髓系祖细胞(EMPS)为胚胎提供明确的红系和 在造血干细胞建立之前形成组织驻留巨噬细胞的髓系细胞 (HSCs)。调控髓系向EMPs分化的分子决定因素目前知之甚少。 哺乳动物SWI/SNF染色质重塑BAF(BRG1/BRM相关因子)复合体在 在发育和病理过程中发挥重要作用。我们的初步数据表明， Baf155(又名SRG3或Smarcc1)，BAF复合体的一个亚单位，位于Tie2-Cre谱系(Tie2-Cre；Baf155f/f，Baf155 CKO)导致胚胎死亡，卵黄囊巨噬细胞分化缺陷。流式细胞仪显示 E9.5和E10.5的卵黄囊内几乎没有成熟的髓样细胞。然而，在BAF155中存在EMP CKO卵黄囊。野生型和Baf155缺陷卵黄囊细胞的单细胞RNA测序分析 虽然髓系主调制子PU.1和Myb在EMP人群中表达 缺乏BAF155，髓系成熟基因如IRF8、CSF1R和CX3CR1的表达 大大减少，表明PU.1和BAF之间可能存在一种等级关系 染色质重塑在激活髓系成熟程序中的作用。出乎意料的是，髓系细胞因子 可挽救Baf155缺陷的EMPs在培养中的分化缺陷。染色质的检查 解救的Baf155 CKO髓系细胞髓系基因位点的可及性 转座酶可及染色质高通量测序(atac-seq)也很常见 作为独特的表观遗传学变化，可能有助于髓系谱系的挽救。从这些基本原则出发 发现，首要目标是证明BAF155介导的染色质重塑在 髓系分化发生的EMP阶段，细胞因子可以绕过BAF155的要求 骨髓系发育。特别是，我们将调查髓系之间的等级关系 谱系转录因子PU1和BAF155染色质重塑因子与髓系发育 我们还将确定由PU.1-BAF155途径调节的髓系谱系程序。我们会 探讨BAF155在允许髓系染色质可及性和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，这些变化证实了BAF155 在髓系谱系程序激活中的作用。工作的成功完成将为以下工作奠定基础 染色质重塑、染色质结构与髓系关键转录因子之间的相互作用 世系分化。这一结果也将有助于从 多能干细胞或体细胞重新编程及其在广泛应用中的功能 在再生医学中，组织修复和组织修复所需的机动组织巨噬细胞功能 再生或疾病。

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.