Study On The Function And Mechanism Of Ronin In Regulating Hematopoietic Stem Cells

THAP11 is a member of THAP transcription factors family and is widely involved in a variety of biological processes.Ronin is homologue of THAP11 in mice,with DNA sequence homology up to 83%.It has been reported that Ronin affects physiological processes such as stemness maintenance,multipotential stem cell proliferation and differentiation,mitochondrial electron transport chain,and cell metabolism through binding with transcription cofactors to regulate specific genes expression.Previous study show that the global ablation of Ronin leads to development arrest at E7.0 day,Ronin knockout in heart causes heart hypoplasia and dysfunction.and knockout of Ronin in mouse retina cells leads to retina development disorder.Moreover,Zebrafish Ronin deletion causes the craniofacial development deformity,indicating that Ronin is crucial for the development of multiple organs.However,the role of Ronin in the hematopoietic system has not been reported.Our previous study found that THAP11regulates the erythroid and megakaryocyte differentiation balance,and knockdown of THAP11 in CD34~+cells led to a reduction of hematopoietic reconstitution capacity in vivo.Mice show the fetal death after Ronin conditional knockout in the hematopoietic system(Ronin CKO),suggesting that Ronin plays an critical role in regulating the mouse embryonic hematopoietic system.In this research,Ronin CKO mice model was used to assess the regulation of fetal hematopoiesis by Ronin.First,the erythropoiesis in fetal liver was determined by analyzing the number of erythrocyte peripheral blood and hemoglobin content of E13.5-E15.5 days of embryos,erythroid development marker,erythrocyte enucleated status and detecting the expression of erythroid related genes in fetal liver.Secondly,the impact of Ronin on hematopoietic stem progenitor cells(HSPC)development was analyzed by the flow cytometry.Fetal liver HSPC function was evaluated using colony formation assay,competitive and non-competitive transplantation experiments and treatment of acute radiation injury.In addition,we detected the cell cycle,apoptosis,mitochondrial energy supply capacity and ROS level of HSPCs by flow cytometry and Seahorse XF Analyzers.Finally,RNA-Seq was used to detect gene expression changes in Ronin CKO fetal liver Lin-sca1c-kit(LSK)cells.Molecular mechanism of Ronin was investigated using RNA-Seq and ChIP-seq in human CD34~+cells.The results of this study showed that the number of erythrocyte and the hemoglobin content in peripheral blood of Ronin CKO fetal were reduced,the proportion of erythrocytes unucleated and the expression of erythroid-related genes were significantly down-regulated,indicating that severe anemia might be the direct cause of embryo death.Simultaneously,Ronin CKO fetal showed decreased R3 phase during erythrocyte development,indicating that the erythroid development was arrested at R3 stage.Furthermore,analysis of fetal liver HSPC reveal that the number of LSK and MPP cells in fetal liver of Ronin CKO mice were increased,in contrast,the number of HPC,commited progenitor cells(CMP,GMP,and MEP)and multi-Lineage cells were decreased,indicating that Ronin regulates the development of HSPC in mice.Functional analysis of fetal liver HSPC in Ronin CKO mice reveal that Ronin deletion led to decreased colony formation activity in vitro,failure to reconstitute hematopoiesis in vivo after competitive and non-competitive transplantation,as well as with reduced activity to treat acute radiation injury,indicating that Ronin ablation causes HSPC malfunction.In addition,analysis of HSPC cell cycle showed that the percentage of S phase of LSK cells increased significantly in Ronin CKO fetal liver,indicating that Ronin was involved in regulating the cell cycle of fetal liver HSPC in mice.Analysis of HSPC mitochonrial function suggested that the respiratory mode of fetal liver HSPC cells in Ronin CKO mice was biased to glycolysis with reduced maximum respiratory capacity and full lost of spare respiratory capacity.This data provides evidence that Ronin regulates HSPC mitochondrial function.ROS assay showed that the amount of ROS in LSK cells in fetal liver increased significantly after the absence of Ronin.The gene expression alteration analysis suggest that in Ronin CKO LSK cells the differentiated genes are mainly enriched on cell cycle,mitochondria,methylation regulation,acetylation and hematopoietic related genes.Integrating analysis of mouse LSK cell RNA-seq data with ChIP-seq data in human CD34~+cells and mouse ES cells suggested that Ronin is highly conserved as a transcription factor binding DNA sequence with preferred binding sequence is-CTGGGAYKTGTAGT-(Y is G or A,K is T or A).In summary,Ronin is an essential transcription factor in mouse embryonic hematopoietic development through regulating various genes expression with specific binding sites on chromosome.