Study On The Molecular Mechanism Of EDAG/THAP11 Interaction In Regulating HSC Function

HSC?Hematopoietic stem cell?is the initial source of various mature cells in the blood system,which maintain the HSC population by the ability of self-renewal and differentiate into hematopoietic progenitor cells and mature functional blood cells by the ability of determination.The balance of self-renewal and differentiation is necessary for long-term maintenance of hematopoietic function,and its regulation is a complex process.Transcription factors are the driving factors regulating hematopoietic stem cell lineage commitment,and generally,transcription factors forms dynamic complexes through protein-protien interaction and selectively regulate target genes,determing the fact-decision of HSC.EDAG?Erythroid differentiation-associated gene?is a hematopoietic specific transcriptional regulator with both transcripitonal activationa activity and repression activity,and our previous study suggest that EDAG plays critical roles in regulating the proliferation and differentiation of hematopoietic stem cells.Bioinformation analysis reveals that although EDAG reuglates the expression of multiple clusters of genes,it does not contain any known DNA-binding domain,suggesting that EDAG regulates target genes expression through interacting with other transcription factors.THAP11?Thanatos-associated protein11?is a member of the THAP family,which is a transcription factor that contains a C2-CH zinc finger DNA binding domain that recognizes a 15bp?ACTACNNTCCCAG?double-stranded DNA sequence.Previous studies suggest that THAP11 plays an important role in the survival and maintenance of pluripotent in embryonic stem cells,however,the role of THAP11 in hematopoietic stem cells remains largely unknown.Our previous experiments demonstrate that EDAG is a binding partner of THAP11.Therefore,we propose that EDAG might form a transcriptional regulatory complex with THAP11 and affect the function of HSC by selectively regulating the expression of hematopoietic related genes.In this study,Ch IP-Seq was used to analyze the binding profile of EDAG and THAP11 on chromosome in K562 cells.Gene microarray were used to determine the target genes regulated by EDAG and THAP11.Intergration of the Chip-Seq data and microarray data reveals the direct target genes regulated by EDAG and THAP11.Data analysis found 71 genes were regulated by EDAG and binding by EDAG and THAP11.GO analysis found that those genes were enriched in the translation,cell senescence,histone modification,protein deubiquitination,amino acid metabolism and other processes.The results also suggest that mitochondrial function-related genes were highly enriched in EDAG and THAP11 regulated target genes.By immunofluorescence,we found that knockdown of EDAG affects CD34 + mitochondrial morphology,mitochondrial fusion,and mitochondrial mass reduction.These results suggest that EDAG plays an important role in maintaining the mitochondrial stability of hematopoietic stem cells,but whether or not to interact with THAP11 requires further experimental confirmation.In order to further study the role of THAP11 in hematopoiesis,we constructed Ronin?murine THAP11?conditional knockout mice.Ronin was specifically knocked out in hematopoietic cells by hybridizated with Vav-Cre tool mice.First,we explored the phenotype of Ronin^flox/+Vav-Cre?9Tg? mice.The results showed that the mRNA and protein levels of Ronin in bone marrow and thymus were down-regulated.Blood routine examination showed that red blood cells and hemoglobin content decreased.Bone marrow erythrocyte development retradated,early development of erythrocyte?EryA?ratio increased,the late EryC ratio decreased.Spleen and bone marrow LSK ratio decreased.Thymus T cell development reduced.The proportion of B cells was decreased in peripheral blood,spleen and bone marrow.These results suggest that THAP11 is likely to affect the development and differentiation of hematopoietic cells from multiple lineage.