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.
This grant proposal is to understand epigenetic regulations of myeloid lineage differentiation from the yolk sac derived erythroid-myeloid progenitors (EMPs). Particularly, we will delineate the role of BAF155, a subunit of the mammalian SWI/SNF chromatin-remodeling BAF (BRG1/BRM associated factor) complex, in chromatin remodeling at the myeloid gene loci, interplay with PU.1, a transcription factor critical for myeloid lineage differentiation, and cytokine mediated epigenetic changes contributing to myeloid lineage program activation.
