Study On The Role Of THAP11 In Regulating Embryonic Hematopoiesis And Its Molecular Mechanism

THAP11 is a newly discovered member of the THAP transcription factor family,which is involved in various biological processes and plays a role in cell proliferation,differentiation and apoptosis.THAP11 controls the maintenance of stemness of some stem cells,mitochondria function,and cell metabolism through combining other transcription cofactors to specifically regulate the expression of various target genes.The function of THAP11 on individual organisms is multiple.Embryos fail to implant or die after implantation after systemic knockout of THAP11,and embryonic stem cells cannot survive after THAP11 knockout.Knockout of THAP11 also increases the sensitivity of embryonic stem cells to DNA damage.Deletion of THAP11 in the heart leads to growth defects of cardiomyocytes,deletion of THAP11 in zebrafish leads to craniofacial developmental abnormalities.Multiple studies have shown that THAP11 is essential for organ development.However,the role of THAP11 in the hematopoietic system remains unknown.Our group previously generated THAP11 hematopoietic system-specific knockout mice(Vav-i Cre;THAP11^fl/fl)and found that deletion of THAP11 in hematopoietic cells led to embryonic lethality with severe anemia and down-regulation of erythroid differentiation-related genes.In this study,we used the Vav-i Cre;THAP11^fl/fl conditional knockout mouse model to illustrate the role of THAP11 in the fetal hematopoiesis.According to the developmental characteristics of mouse embryonic hematopoiesis,E14.5 days fetal liver were selected for investigation.First,the erythroid differentiation stage of THAP11-knockout fetal liver was analyzed by using specific surface markers to clarify that the erythroid development was blocked.Then,the distribution of hematopoietic stem and progenitor cells（HSPCs）in the fetal liver was detected by flow cytometry analysis,and the cells number of multlineage was counted.Colony formation assay was performed to detect the effect of THAP11 on hematopoietic stem cells function in vitro.Competitive transplantation experiments were performed to exclude the effect of THAP11 on the hematopoietic reconstitution capacity in vivo.Cell cycle was analyzed by Brd U incorporation assay,the apoptosis was detected with Annexin V staining,and ROS production was analyzed by ROS Fluorescent Probe Kit.In this study we showed that in THAP11-knockout fetal liver,the cells number of fetal liver and red blood cells were significantly decreased,the appearance of the embryo was pale,and the fetal liver,the main hematopoietic organ,was much smaller than wild type fetal liver.Analysis of erythroid development showed that in THAP11-knockout fetal liver,the erythroid development was blocked at R3 stage.Combined with our previous research results,it is clear that severe anemia occurred in THAP11-knockout embryos,which led to embryo death.Analysis of the lineage differentiation suggested that the proportion and number of B cells,neutrophils,and macrophages were decreased in THAP11-knockout fetal liver,indicating that THAP11 deletion caused multilineage differentiation disorder.Statistical analysis of HSPCs suggested that the number of LSK cells was increased and hematopoietic progenitor cells（HPC）was decreased.According to the stem cell surface markers,LSK cells were divided into three types of cells:long-term hematopoietic stem cells(Long-time hematopoietic stem cells（LT-HSC）,short-term hematopoietic stem cells（Long-time hematopoietic stem cells,ST-HSC）,and multipotent progenitors（MPP）.No significant difference was observed in cell number of LT-HSC,while the numbers of MPP and ST-HSC were increased significantly in THAP11-knockout fetal liver,indicating that THAP11 regulates the development of HSPCs in fetal liver.Analysis of cell cycle showed that in THAP11-knockout fetal liver the proportion of S phase increases and the proportion of G0 phase decreases in LSK and LT-HSC,while no difference was observed in HPC,indicating that THAP11 might be involved in the regulation of the cell cycle of HSPC.Apoptosis and ROS assays suggested that the apoptosis and ROS levels of HPC,LSK,and LT-HSC in THAP11-knockout fetal liver were significantly increased.To rule out effect of microenvironment on HSPCs in THAP11-knockout fetal liver,an inducible knockout mouse model Mx1-Cre;THAP11^fl/flwas used.Wild type or Mx1-Cre;THAP11^fl/fl feta liver cells were transpanted into recipients with competitor cells and after8 weeks,the chimeric recipients were injected with poly（I:C）.The results showed that in Mx1-Cre;THAP11^fl/fl chimeric recipients the number of peripheral blood cells were decreased,the numbers of bone marrow LSK cells,LT-HSC,ST-HSC,and MPP were all increased dramatically,while the number of HPC cells were decreased after poly（I:C）induction.analysis of lineage differentiation indicated that the proportion and numbers of donor-derived B cells,T cells,neutrophils,and macrophages were decreased in Mx1-Cre;THAP11^fl/fl chimeric recipients,which was consistent with the results in Vav-i Cre;THAP11^fl/fl mice model.The fluorescence quantitative PCR results showed that multiple genes expression were altered after THAP11 knockout,which related to epigenetics,cell proliferation and differentiation,and activation of the interferon signaling pathway,suggesting that THAP11 regulates HSPC function through various mechanisms acting together.In summary,THAP11 is essential for maintaining the fetal liver hematopoietic homeostasis and controlling cell cycle,apoptosis and ROS production in HSPCs.