Role Of Mouse Gata1-3' Untranslated Region(UTR) And Its Regulatory Mechanism In Normal And Stress Erythropoiesis

Gatal,an essential transcription factor whose level varies during erythroid development,is precisely regulated.However,how the level of Gatal is controlled has not been fully understood.MicroRNAs(miRNAs)are small noncoding RNAs that can regulate the expression of specific target genes and play important roles in hematopoiesis.Our microarray data reveals that in the early erythroid developmental stages,the expression levels of 11 miRNAs are much higher compared to the levels of miR-144/451,the most abundantly expressed miRNAs in lata stage red biood cells.Further bioinformatics analysis indicates that two of those miRNAs(miR-709 and miR-706)can potentially bind to multiple sites of the 3' untranslated region(3' UTR)of Gatal mRNA.Therefore,we knocked out part of the Gatal 3' UTR sequence that contains potential miR-709 and miR-706 binding sites.Our preliminary data indicate that Gatal 3' UTR knockout mice(both heterozygous and homozygous)are anemic and their Gatal levels are higher than that in the normal littermates.We thus hypothesize that Gatal-3' UTR is an important functional element for Gatal expression and loss of binding between Gatal-3' UTR and miR-709/miR-706 increases the level of Gatal,which leads to disrupted erythropoiesis.This proposal describes plans for the extensiive study on 1)the role of Gatal-3' UTR in erythropoiesis under both baseline and stress conditions;2)the role of miR-709 and miR-706 in erythropoiesis;and 3)the direct regulatory relationship between miR-709/miR-706 and Gatal expression.We will analyze Gatal 3' UTR knockout mice we have generated as an in vivo model and utilize in vitro systems inclouding fetal liver-derived hematopoietic stem cell culture to examine what the function of Gatal-3' UTR and miR-709/miR-706 are,and wherther both miR-709/miR-706 affect erythroid development through the inhibition of Gatal.Our goal is to map a new molecular pathway through which Gatal is regulated in normal and stress erythropoiesis and to demonstrate a proof of principle that minRNAs can target major transcription factors for homeostasis during development.Methods:1.The Gatal-3' UTR knockout mice were constructed by CRISPR/cas9 technology.The erythroid phenotypic changes were observed in vivo under non stress state and acute anemia induced by multiple stresses;2.The number and proportion of various cells and platelets in peripheral blood were analyzed by CBC,the cell morphology was observed by blood smear,and the number,proportion and morphology of various cells in bone marrow and spleen were observed by histology and immunohistochemistry;3.Observe whether the maturation process of precursor erythrocytes of Gatal-31 UTR knockout mice is delayed and whether apoptosis is increased in vitro;4.Erythroid cells in different developmental stages of mice were sorted by flow cytometry,the proportion of various cells in peripheral blood,14.5-day embryonic liver,bone marrow and spleen were analyzed,and the expression of miR-709,miR-706 and miR-451 were detected by real-time fluorescence quantitative PCR(q-RT PCR);5.Embryonic hepatocytes,MEL cells and g1ejc4 cells were cultured in different media for 14.5 days in vitro.The dynamic expression of miR-709,miR-706 and miR-451 were detected by q-RT PCR;6.Based on the overexpression of miR-709 in MEL cells by mscv-pig retroviral vector,the changes of erythrocyte proliferation,differentiation and apoptosis were analyzed;7.Through the query results of biological database,Gata1 was predicted to be the target gene of miR-709,and then pGL3-BS-Gata1-3' UTR plasmid was constructed.The double Luciferase Report verified the direct targeting effect of both;8.The mRNA and protein expression of Gata1 were detected by q-RTPCR and Western blotting.Results:1.Gata1-3' UTR knockout mice were successfully constructed and bred normally.The mutant mice showed anemia symptoms;2.The total number of erythrocytes,hemoglobin and other erythroid indexes in peripheral blood of knockout mice,whether homozygous or heterozygous,were abnormal,and megakaryocytes and platelets were also changed;3.miR-709 and miR-706 were low expressed in mature erythrocytes of peripheral blood and reticulocytes of bone marrow of mice,which were much lower than that of miR-451;The expression of miR-709 and miR-706 was higher than that of miR-451 in immature erythrocytes and bone marrow nucleated erythrocytes of mouse embryonic liver at 14.5 days;4.In erythroid-like G1EJC4 cells,MEL cells and 14.5-day-old embryonic liver hematopoietic stem cells,the expression of miR-709 and miR-706 increased first and then decreased;5.miR-709 plasmid was constructed.MEL cells were infected with 293T cell packaging virus and screened by puromycin.Compared with MSCV,the miR-709 was successfully overexpressed,cell differentiation was blocked and proliferation was accelerated;6.Through the double Luciferase Report experiment,it was found that the luciferase activity decreased significantly after the co expression of Gata1-3' UTR and miR-709 in 293T cells,which verified the binding relationship between Gata1-3' UTR and miR-709;7.After overexpression of miR-709 in MEL cells,the mRNA and protein levels of Gata1 decreased.High lights:Gata1-3' UTR is a functional fragment that can regulate the level of Gata1.miR-709 and miR-706 can fine tune the expression level of Gata1 through this sequence to ensure the normal development of erythroid and may play an important role in maintaining the steady state of erythroid formation.