Atf7ip And Setdb1 Interaction Orchestrates The Hematopoietic Stem And Progenitor Cell State With Diverse Lineage Differentiation

As a heterogeneous group of stem cells with proliferation,differentiation and regeneration abilities,the balance of proliferation and differentiation of hematopoietic stem and progenitor cells is crucial for the maintenance of homeostasis,and disruption of this balance often leads to anemia or malignant blood diseases.However,the cellular and molecular mechanisms and key factors by which hematopoietic stem and progenitor cells coordinate stemness and differentiation into various blood cells are still poorly understood.ATF7IP,an epigenetic regulator known to mediate immune evasion and T-cell function,plays a pivotal role in controlling the activity of the H3K9methyltransferase SETDB1.Nevertheless,the functional roles and mechanisms of ATF7IP/SETDB1 in the proliferation and differentiation of hematopoietic stem and progenitor cells are currently poorly understood.Zebrafish,due to its highly conserved hematopoietic processes and regulatory mechanisms compared to mammals,along with its unique advantages such as external fertilization,transparency during early embryonic stages,and the availability of rich transgenic strains,has emerged as a commonly utilized model organism in the study of hematopoietic development and human disease.Therefore,this study uses zebrafish as the main animal model,combined with mouse and human cell lines,to explore the role and mechanism of epigenetic factor ATF7IP/SETDB1 in regulating hematopoietic stem and progenitor cell proliferation and differentiation.First,we used CRISPR-Cas9 technology to construct atf7ip and setdb1 zebrafish mutants,and found that atf7ip or setdb1 deficiency leads to impaired hematopoietic stem and progenitor cell expansion and myeloid differentiation bias,accompanied by reduced erythrocytes and lymphocytes,by in situ hybridization and flow cytometry analysis.Using fluorescence confocal microscopy and lineage tracing techniques,we found that atf7ip-deficient hematopoietic stem and progenitor cells have reduced proliferation capacity while increasing their myeloid differentiation potential.Given that atf7ip and setdb1 are widely expressed epigenetic factors,we used cell transplantation and tissue-specific overexpression experiments to demonstrate that atf7ip/setdb1 regulates hematopoietic stem and progenitor cell expansion and differentiation in a cell-autonomous manner.To explore the mechanism of atf7ip/setdb1regulation of hematopoietic stem and progenitor cell proliferation and differentiation,we performed H3K9me3 Ch IP-seq,ATAC-seq and RNA-seq analysis on atf7ip/setdb1mutant zebrafish hematopoietic stem and progenitor cells.The results showed that knockout of atf7ip or setdb1 led to H3K9me3 deposition decreased and chromatin openness increased in the promoter regions of cell cycle inhibitor cdkn1a and myeloid differentiation key factor cebpβ,resulting in impaired hematopoietic stem and progenitor cell expansion,thereby promoting myeloid monocyte and neutrophil differentiation.In addition,we also found that Atf7ip/Setdb1-mediated H3K9me3 modification is mainly enriched in hematopoietic stem and progenitor cell DNA transposons and retrotransposons(LTR,LINE,SINE)regions.Atf7ip interacts with Setdb1 to catalyze H3K9me3 modification in the regulatory regions of LTR and LINE,reducing chromatin openness,silencing LTR and LINE expression,thereby inhibiting Mda5/Rig-I receptor-mediated innate immune response,blocking stress-driven excessive myeloid differentiation,to maintain normal development and differentiation of the blood system.To explore the role of histone methylation modification in human hematopoietic stem and progenitor cell development and differentiation and disease,we used mouse bone marrow transplantation and colony formation experiments to demonstrate that ATF7IP deficiency significantly reduces the hematopoietic reconstitution ability of human CD34~+cells,but retains their myeloid differentiation potential.Meanwhile,we found that ATF7IP is highly expressed in acute myeloid leukemia cells,and knocking out ATF7IP/SETDB1 can inhibit the growth of acute myeloid leukemia cells,promote myeloid differentiation and innate immune response.In summary,this study reveals an important regulatory mechanism of ATF7IP/SETDB1-mediated H3K9me3 deposition and chromatin remodeling in controlling hematopoietic stem and progenitor cell expansion and differentiation into various blood cells,providing new ideas and strategies for the intervention of human blood diseases.