A Study On The Mechanism Of Gene Setd2 In Mouse Hemopoietic Stem Cell And Lymphocyte Development

The adaptive immune system,mainly composed of T and B lymphocytes,is the key guarantee for humans to live healthy and longer by recognizing multiple antigens from in vivo and in vitro.The repertoire of diverse T cell receptors(TCRs)and immunoglobulins(Igs)is generated through the somatic rearrangement of respective V,D and J gene segments,termed V(D)J recombination during early T or B cell development,which determinate the ability of the immune system to recognize antigens.The RAG1/2 proteins mediate V(D)J recombination at antigen receptor gene loci,and the histone modifications might be involved in the recombination process and lymphocyte development due to the connatural histone-DNA interactions.SETD2,a histone methyltransferase,catalyzes lysine 36 trimethylation on histone 3(H3K36me3)that conserved between species.In this study,we systematically explored the role of SETD2 and its catalytic product H3K36me3 in hematopoietic system development especially in lymphocyte development.We generate the transgenic mouse model to knock out the Setd2 in the hematopoietic system and result in the removing of the Setd2 and H3K36me3.We find the developmental disorder both in hematopoietic stem cells and common lymphoid progenitors,as well as the T and B cell development are blocked at CD4~-CD8~-double negative 3(DN3)stage and pro-B cell,respectively.We find the consistent phenotype in T and B cell lineage specific knock-out mouse model.The V(D)J rearrangement assay reveal that the blockage of lymphocytes development was caused by impaired V(D)J rearrangement in DN3 stage and pro-B cell,respectively.We further employ the Chromatin immunoprecipitation,RNA-seq,immunoblotting to uncover the mechanism of how Setd2 regulates the V(D)J rearrangement and find that the loss of Setd2 impairs the binding of Rag1 to the antigen receptor gene loci,meanwhile the deficient of Setd2 also causes decreased phosphorylation of the ATM,the sensor of DNA double strand break(DSB),that result in the insufficient DNA repair during the recombination in RSS.We identify new SETD2 mutations by analyzing whole exome sequencing data from the primary immunodeficiency patient cohort,and find these mutations failed to restore the H3K36me3 in vitro.In summary,our study expounds the function of Setd2 in lymphocyte development and provides a new clinical diagnostic candidate for the primary immunodeficiency.