THAP11 Maintains Hematopoietic Stem Cells Function By Regulating Valine Metabolism

Substance metabolism is crucial for proliferation and differentiation of stem cells.The roles of glucose and lipid in hematopoietic stem cells（HSCs）have been widely reported.However,little is known about the roles of amino acids on HSCs.Previous study suggests that specific amino acids were required for maintaining HSCs function such as valine,leucine,and aspartate.It remains unknown how these specific amino acids are regulated in HSCs.THAP11 is a member of the THAP family of transcription factors which control a series of important biological processes.THAP11 can regulate embryo implantation,early development of heart,and retinal development in mice and craniofacial development in zebrafish embryos.Our previous study found that knockdown of Thap11 in human cord blood CD34⁺cells reduced the ability of colony formation in vitro and the ability of transplanted reconstitution in vivo,indicating that THAP11 regulates the function of human hematopoietic stem cells.Ronin is the homologous gene of THAP11 in mice.We generated Vav-i Cre⁺;Thap11^fl/fl mice that specifically knocked out the Thap11 gene in the mouse hematopoietic system and found that Thap11 knockout in hematopoietic cells led to embryonic death due to anemia,erythroid developmental disorders,and HSCs dysfunction,indicating that THAP11 is essential for hematopoietic system development.To further reveal the role of THAP11 in adult HSCs,we generated a inducible knockout mouse model Mx1-Cre⁺;Thap11^fl/fl to delete Thap11 gene in adult hematopoietic system.In the present study,using this mice model,we investigated the effects of THAP11 on mouse adult HSCs function and its possible mechanism.The results showed that in hematopoietic stem/progenitor cells of Mx1-Cre⁺;Thap11^fl/flmice Thap11 was effectively deleted after five injections of poly（I:C）,and the knockout mice showed increased mortality,decreased numbers of peripheral blood cells and bone marrow cells,and impaired HSCs function with reduced colony formation in vitro and loss of hematopoietic reconstitution activity in vivo.Mx1-Cre⁺;Thap11^fl/fl mice bone marrow cells were transplanted into recipients to generate chimeric mice,and 8 weeks after transplantation poly（I:C）were injected in to the chimeric mice.The Mx1-Cre⁺;Thap11^fl/fl chimeric mice displayed reduced numbers of peripheral blood cells and donor cells in bone marrow.Furthermore,Mx1-Cre⁺;Thap11^fl/fl mouse c-Kit⁺cells were sorted and stimulated by IFN-αto knock out Thap11 in vitro.Consistently,the colony forming ability was reduced and the ability to reconstitute in vivo after transplantation is lost.These results suggest deletion of THAP11 causes HSCs dysfunction.To investigate the mechanism of THAP11 regulating HSCs function,RNA-Seq was performed and the result showed that the expression of mitochondrial function-related genes,autophagy-related genes and amino acid metabolism-related genes in HSCs was down-regulated after Thap11 deletion.Using small-molecule compounds targeting mitochondria and autophagy,as well as specific amino acids and metabolites,we evaluated the colony forming ability.The results showed that the valine metabolic pathway products can be partially rescue the defects of colony formation in Thap11knockout hematopoietic cells.To further evaluate the rescue effect of valine metabolic pathway products,we treated chimeric mice transplanted with bone marrow cells from Mx1-Cre⁺;Thap11^fl/fl mice with valine metabolites,and the results showed that the combination of valine metabolites can increase the number of bone marrow HSCs in chimeric mice,and enhance the ability of HSCs to constitute hematopoietic system in secondary transplantation.The above results show that THAP11 affects HSCs function partially through regulating the valine metabolic pathway.