Transcriptional Regulation Of MDia2 Gene

The final stages of mammalian erythrocyte production include enucleation,membrane and protein remodeling,and organelle clearance.mDia2 belongs to the mammalian Formin protein family,is involved in linear actin polymerization.mDia2 is highly expressed in the late stages of erythropoiesis^[1].Studies have shown that the deletion of mDia2 affects the production of erythrocytes at different stages,manifesting as cytoplasmic division and enucleation defects,and embryonic lethality as observed in mDia2 whole body knockout mice^[2].Gene expression regulation is critical for normal development^[3],however,the expression regulation of mDia2 gene is currently unclear.In this study,the transcriptional regulation mechanism of mDia2was investigated at the cellular level,and the main research contents included:（1）According to the structure of the mDia2 gene and the peak sequence of GATA1Ch IP-Seq,the 5’flanking sequence（-1765bp～+227bp）of the mDia2 gene is selected,and the 5’flanking sequence and one intron region of the human mDia2 gene are selected（-1245bp～+495bp and 660bp intron region）.All the fragments are inserted into luciferase construct.The luciferase reporter plasmid for the deletion of murine and human mDia2 genes are further constructed.We find the core promoter region of murine mDia2 gene is located at-320bp～+227bp,and the core promoter region of human mDia2 gene is located in the intron region of 660bp.（2）By using the transcription factor binding prediction website（TF Bind）,we find the potential GATA1 binding sites in the core promoter regions of both mouse and human mDia2 genes.The mouse mDia2 luciferase reporter constructs with mutated GATA1 binding site are constructed.We find the luciferase activity was significantly reduced,and the dose-dependent induction of luciferase expression upon GATA1transfection was largely attenuated,indicating that GATA1 regulates the transcription of mouse mDia2 gene.Specifically,we identify two potential GATA1 binding sites in mouse mDia2 gene,mutation of both sites leads to 50%decrease of luciferase activity.We further reveal four potential GATA1 binding sites in the intron region of human mDia2 gene,and the luciferase activity was completely blocked after combined mutation of all four sites,indicating these binding sites are essential elements to maintain the activity of mDia2 gene promoter regulated by GATA1.（3）To explore the role of mDia2 in human erythropoiesis,the in vitro differentiation of human CD34⁺stem/progenitor cells into erythroid cells were established.The downregulation of mDia2 expression mediated by sh RNA knock down attenuated the erythroid differentiation and enucleation during human erythropoiesis in vitro.In summary,we identified the GATA1 binding sites are necessary cis-acting elements for mDia2 gene expression and mDia2 plays an important role in the differentiation of human CD34⁺cells into the erythrocytes.