The Impact Of Brg1 On Hematopoietic Stem Cells Differentiation And Self-renewal

Part ⅠTHE EXPRESSION OF BRG1 IN THE HEMATOPOIETIC SYSTEM AND CONSTRUCTION OF A SPECIFIC BRG1 TRANSGENIC KNOCKOUT MOUSE MODELOBJECTIVE:To observe the expression of Brg1 gene in hematopoietic stem/progenitor cells and mature cells of C57BL/6 mice.To explore the appropriate Poly（I:C）induced Cre/Lox P recombinant system for specific deletion of Brg1 gene in hematopoietic system of mice.METHODS:Cell surface molecular staining and flow cytometry were used to sort out hematopoietic stem/progenitor cells,myeloid cells and red line cells in bone marrow,immature B cells and mature B cells in spleen,immature T cells and mature T cells in thymus.RT-PCR was used to detect the expression of Brg1 in cell subgroup.Cross Lox P transgenic mice(Brg1^fl/fl)with Cre recombinase transgenic mice（Mx1-Cre）to obtain Brg1^fl/fl and Brg1^fl/flMx1-Cre mice,and the expression of Cre recombinase was induced by injecting Poly（I:C）to knock out Brg1 gene.Poly（I:C）was intraperitoneally injected at 10μg/g or 6μg/g（dose/body weight）,once every 48 hours for three times in total.After 10～15 days,the survival of mice was observed,and the knockout rate of Brg1 m RNA and protein levels in mouse bone marrow cells was detected by RT-PCR and WB.RESULTS:The m RNA levels of Brg1 were higher in surface molecular labeled hematopoietic stem/progenitor cells and lower in mature cells.Poly（I:C）was injected with 10μg/g,the mortality rate of Brg1^fl/flMx1-Cre（Brg1-CKO）mice that specifically knock out the Brg1 gene in the hematopoietic system was about 90%,and the expression of Brg1 was completely knocked out.Poly（I:C）was injected with 6μg/g,the mortality rate of Brg1-CKO mice was approximately 50%,and the expression of Brg1 was completely knocked out.CONCLUSION:The m RNA levels of Brg1 was higher in hematopoietic stem/progenitor cells than in other cells,suggesting that Brg1 may play an important role in hematopoietic stem/progenitor cells.Using Poly（I:C）to inject mice at 6μg/g,the mortality rate of the mice specifically knocking out the Brg1 gene was low,ensuring that Brg1 was completely knocked out,which is conducive to the implementation of the next experiment.After Poly（I:C）was reduced to 6μg/g,the mortality of Brg1-CKO mice was reduced and the expression of Brg1 was completely knocked out,which was conducive to the implementation of the next experiment.Part Ⅱ KNOCKOUT OF BRG1 RESULTS IN THREELINEAGES DIFFERENTIATION ARREST IN THE HEMATOPOIETIC SYSTEM IN MICEOBJECTIVE: To observe the effect of knockout of Brg1 gene in hematopoietic system on peripheral blood,thymus and spleen of mice;and to observe the effect of knockout of Brg1 gene on the development of Bline cells in bone marrow,T-line cells in the thymus,and myeloid cells in the bone marrow and spleen.METHODS: After knocking out the Brg1 gene of hematopoietic system,the peripheral blood of Brg1fl/fl and Brg1-CKO mice was detected,and the changes of the thymus and spleen of the mice were observed and weighed.H&E staining was used to observe the changes of sternum,thymus and spleen.Cell surface molecular staining and flow cytometry were used to detect the changes of mouse B-line cells,T-line cells and myeloid cells.RESULTS: Compared with Brg1fl/fl mice,the number of white blood cells（WBC）,neutrophils（NE）,lymphocytes（LY）,red blood cells（RBC）,hemoglobin（Hb）and platelets（PLT）in Brg1-CKO mice were significantly decreased.In Brg1-CKO mice,the thymus and spleen were significantly reduced in size and weight.H&E staining showed that Brg1-CKO mice had fewer cells in the sternum,thinner thymus cortex,and smaller and fewer germinal centers in the spleen.Flow cytometry showed that compared with Brg1fl/fl mice,the common lymphoid progenitor（CLP）cells in the bone marrow of Brg1-CKO mice did not change significantly.During the B cell differentiation process,the total number of Pro-B cells were increased,while the total number of Pre-B cells,immature B cells and mature B cells was decreased in the bone marrow of Brg1-CKO mice.At the stage of T cell differentiation process,the counts of DN1-DN4 cells,immature T cells,CD4+ T cells and CD8+ T cells were significantly reduced in the thymocytes of Brg1-CKO mice.The total number of common myeloid progenitor（CMP）and granulocyte-macrophage progenitor（GMP）cells was significantly increased in bone marrow of Brg1-CKO mice,while the total number of the megakaryocyte-erythrocyte progenitor（MEP）cells were not significantly changed.The total number of myeloid cells was significantly decreased in bone marrow and spleen of Brg1-CKO mice,while macrophages were significantly increased in bone marrow.In Brg1-CKO mice,the total number of R1,R2 and R3 cells decreased in bone marrow and spleen,the total number of mature R4 cells did not change significantly in bone marrow but decreased significantly in spleen,while the total number of megakaryocytes increased significantly in bone marrow.CONCLUSION: The number of WBC,NE,LY,RBC,Hb and PLT in peripheral blood of Brg1-CKO mice were significantly decreased,suggesting that knockout of Brg1 lead to the arrest of three-lineages differentiation.After Brg1 was knockout,the total number of CLP cells did not change significantly,the total number of CMP and GMP cells increased significantly,the total number of MEP cells did not change significantly,while the number of the mature B cells,mature T cells,myeloid cells and mature red blood cells was decreased.These results indicate that the differentiation of lymphoid progenitor cells into B cells and T cells were arrest,and the differentiation of myeloid progenitor cells into myeloid and red blood cells were arrest.Part Ⅲ KNOCKOUT OF BRG1 DAMAGES THE SELFRENEWAL ABILITY OF HEMATOPOIETIC STEM CELLSOBJECTIVE: To observe the effect of Brg1 on hematopoietic stem cells and whether it is affected by the microenvironment;and to observe the effect of Brg1 on the cycle and self-renewal ability of hematopoietic stem cells.METHODS: The changes of hematopoietic stem cells were detected by flow cytometry staining.Bone marrow cells from Brg1fl/fl and Brg1fl/flMx1-Cre（CD45.2）mice were transplanted into CD45.1 recipient mice by ordinary transplantation technique.Poly（I:C）was injected after transplantation to induce Brg1 gene knockout,and observe whether the changes of Brg1 knockout on hematopoietic stem cells were affected by the endothelial microenvironment.Ki67 staining and Brd U incorporation experiments were used to detect changes in the hematopoietic stem cell cycle.Continuous competitive transplantation technology was to mix the bone marrow cells of Brg1fl/fl and Brg1fl/flMx1-Cre mice with CD45.1-CD45.2 mouse bone marrow cells at a ratio of 1:1 and then transplanted into CD45.1 recipient mice.After 6 weeks of transplantation,Poly（I:C）was injected to induce Brg1 gene knockout,and the second round of transplantation was performed 4 months later.The CD45.1-45.2/CD45.2 ratio in peripheral blood monocytes,T cells,B cells and myeloid cells was detected by flow cytometry to observe the self-renewal ability of hematopoietic stem cells..RESULTS: Compared with Brg1fl/fl mice,the total number of LSK cell population and HSC cells were increased significantly in the bone marrow of Brg1-CKO mice,and further analysis showed that the total number of LT-HSC were increased in the bone marrow of Brg1-CKO mice,while the total number of ST-HSC did not change significantly.Poly（I:C）was injected after transplantation to induce Brg1 gene knockout,and the changes of LSK,HSC,LT-HSC and ST-HSC were consistent with the phenotypic results of primary mice.Ki67 staining showed that 80%-90% of HSC were in the quiescent phase in the Brg1fl/fl mice,while only 60%-70% in the Brg1-CKO mice.The results of Brd U incorporation showed that the HSC of Brg1fl/fl mice in G0-G1 phase accounted for about 80%,and that of Brg1-CKO mice accounted for about 70%.Compared with Brg1fl/fl mice,the proportion of peripheral blood monocytes,myeloid cells,B and T cells generated from bone marrow of Brg1-CKO mice in peripheral blood cells of recipient mice decreased with time extension and the second round of transplantation.At 4 months after the second round of transplantation,the bone marrow of Brg1-CKO mice produced few peripheral blood monocytes,myeloid cells,B and T cells in recipient mice.CONCLUSION: The total number of HSC cells in bone marrow of Brg1-CKO mice increased,which promoted the proliferation of HSC from quiescent phase into cell cycle.Donor bone marrow formation was reduced in Brg1-CKO mice,suggesting impaired self-renewal ability after HSC knockout of Brg1.Part Ⅳ INVESTIGATION THE MECHANISMS OF BRG1 IN HEMATOPOIETIC STEM CELL DIFFERENTIATION AND SELF-RENEWALOBJECTIVE: To investigate the possible mechanisms of Brg1 in hematopoietic stem cell differentiation and self-renewal.METHODS: RNA-Seq was used to detect the changes of HSC expression profile after knockout of Brg1 in mice HSC.CUT&Tag was used to detect the changes of H3K27 ac,H3K4me3 and H3K27me3 histone modification levels in HSC of Brg1 knockout mice.ATAC-seq sequencing was used to detect changes in chromatin accessibility of HSC after Brg1 was knockout.Brg1-CUT&Tag sequencing was used to detect gene clusters directly or indirectly regulated by Brg1 in normal HSC.RT-PCR was used to verify the downstream target genes of Brg1 in HSC.RNA-Seq data and the differentially peaks of H3K27ac-CUT&Tag were analyzed by GSEA Hallmark gene set enrichment.RNA-Seq data and the differentially peaks of H3K27ac-CUT&Tag were analyzed by KEGG enrichment with GSEA gene set.RNA-Seq data and Brg1-CUT&Tag data were used to analyze the gene set of Brg1 regulating the differentiation and self-renewal of hematopoietic stem cells.RESULTS: RNA-Seq sequencing results showed that 128 differentially expressed genes were up-regulated and 705 differentially expressed genes were down-regulated in HSC after Brg1 was knocked out.The sequencing results of ATAC-Seq showed that there were 8103 peaks only in Brg1-CKO mice,537 peaks only in Brg1fl/fl mice,and 7321 peaks in two groups.Differential peak analysis revealed that 573 genes were upregulated and 799 genes were down-regulated in HSC of Brg1-CKO mice.The results of H3K27 ac,H3K4me3 and H3K27me3 histone modification showed that the H3K27 ac histone modification near 679/768 genes in HSC of Brg1fl/fl mice was increased compared with that of Brg1-CKO;in Brg1fl/fl mice,the H3K4me3 histone modification near 244/414 genes in HSC increased compared with Brg1-CKO;in Brg1-CKO mice,H3K27me3 histone modifications near 159/189 genes were enriched in HSC compared with Brg1fl/fl.GSEA enrichment analysis of RNA-Seq data and H3K27 ac CUT&Tag difference peaks in HSC of Brg1fl/fl and Brg1-CKO mice showed that estrogen receptor pathway was enriched.RNA-Seq data for HSC in Brg1fl/fl and Brg1-CKO mice were overlapped with Brg1-CUT&Tag data,and the results showed that Egr1/2/3,Nr4a1/2,Klf2/4/6 and other related genes were detected.RT-PCR showed that the expression of Egr1,Egr2,Egr3 and Nr4a1 were significantly down-regulated in HSC of BRG1-CKO mice,among which Egr1,Egr3 and Nr4a1 were downstream molecules of estrogen receptor,while Nr4a2 and Klf2 were not significantly changed.KEGG enrichment analysis of GSEA gene set was performed on RNA-Seq data and H3K27ac-CUT&Tag difference peaks in HSC of Brg1fl/fl and Brg1-CKO mice,and it was found that both were enriched in PPAR signaling pathway.RT-PCR showed the expression of PPARδ in HSC was slightly decreased in BRG1-CKO mice,but the downstream genes RXRa and Fabp4 were not significantly changed.The expression of PPARγ and its downstream genes Fbp1 and Acsl6 decreased significantly,while other downstream genes Me1,Scd,Src1 and Src2 did not change significantly.CONCLUSION: HSC chromatin openness was increased and more down-regulated genes were found in Brg1 knockout mice,suggesting that Brg1 plays a transcriptional activation role in HSC.The differential genes are mainly enriched in estrogen and PPAR signaling pathways,and their downstream molecules are down-regulated,suggesting that Brg1 may regulate HSC through nuclear receptor complex.