Transcription Factor Bcl11a Regulates Lymphocyte Delopment And Proliferation

The body’s immune system plays a protective role by resisting pathogens.The health immune system will have a good disease resistance.The key part of immune system is lymphocyte.Lymphopoiesis is tightly regulated by interplay of intrinsic and extrinsic factors,the transcription factors and cytokines.The lymphoid transcription factors activating lymphoid genes and/or suppressing non-lymphoid genes to regulate the lymphocyte development.The lineage determining transcription factors always express in lineage specific cells.When the lymphoid determining factors express and what regulate its expression?What’s the connection among these transcription factors?How the gene regulatory networks in directing lymphoid lineage choice and development?These are the recent interesting questions to answer.Bcl11a is a C2H2 transcription factor.The previous study demonstrated that Bcl11a play an essential role in fetal liver lymphocyte development.Complete absence of B cells and abnormal T cell development in Bcl11a germline null embryo.Since Bcl11a germline null alleles caused neonatal lethality,we generate Bcl11a condition knockout(cko)mice to elucidate Bcl11a in adult lymphopoiesis.The results are as follows:(1)Bcl11a is highly enriched in HSCs,CLPs,ETPs,B cells and dendritic cells,which eGFP expression was detected in Bcl11aeGF eGFP reporter mice by flow cytometry and transcripts were detected by a qRT-PCR.GFP+ cells were detected in almost all Lin-BM cells,including HSCs,MPPs,LMPPs,CLPs,common myeloid progenitors(CMPs),granulocyte-monocyte progenitors(GMPs),MEPs,monocyte-dendritic precursors(MDPs)and common dendritic precursors(CDPs).Bcl11a expression was also detected in erythroid progenitors(EPs),differentiated macrophages,granulocytes and megakaryocytes.All B cells expressed Bcl11a,Pro-B cells,Pre-B cells,Immature B cells and Mature B cells.In T cells compartments,Bcl11a only expressed in uncommitted ETPs and DN2 cells.Some of NKPs expressed Bcl11a.qRT-PCR analysis further demonstrated that the expression of Bcl11a in HSCs,CLPs,and B cells were higher than in CMPs,GMPs,MEPs,macrophages,granulocytes and EPs.(2)Deletion of Bcl11a in adult mice caused loss of early B cells and depletion of lymphoid progenitors.5 days after Bcl11a deletion,the total BM B cell numbers in the flox/flox mice being reduced to 30%of those in the wild type control mice.Pro-B cells and pre-B cells were affected the most with few presenting in the flox/flox BM.Immature B cells reduced to approximately one fifth of the wild type number.However,within the experiment time window,B cells in the spleen,lymph node and peripheral blood were not obviously reduced.the flox/flox thymus had few ETP cells and DN2 thymocytes.The flox/flox mice had no Ly6d+ CLPs and a 7-fold reduction of total CLPs.(3)Bcl11a regulates lymphopoiesis in a dosage manner.Compared to the wild type control,the heterozygous mutants(50%Bcl11a expression)had about 50%of LMPPs,CLPs,ETPs and DN2 thymocytes.(4)Complete loss of lymphoid development caused by Bcl11a deficiency is cell autonomous.After specific deletion of Bcl11a hematopoietic cells,the analysis showed that no donor pro-B cells,pre-B cells,CLPs in bone marrow and no donor ETPs and DN2 cells in thymus.(5)Bcl11a is required for survival of early B cells.2-3 days after Bcl11a deletion,pro-B,pre-B and immature B cells are apoptosis in the mouse bone marrow.The pro-B cells were also apoptosis in vitro once Bcl11a deletion.Pre-B cell colony assay show that Bcl11a has a essential function in pre-B cells.(6)Apoptosis in Bcl11a-deficient pro-B cells is rescued by exogenous Bcl2.(7)Co-expression of Bcl2 and Mdm2 rescues both apoptotic and proliferative defects in Bcl11a-deficient pro-B cells.Although over-expression of Bcl2,Bcl-xL or Mcl1 efficiently rescued Bcl11a-deficient B cells from apoptosis,these B cells appeared to be in senescence since no cells were in S phase.Consistent with a cell cycle defect,expression of the cyclin-dependent kinase inhibitor p21,was up-regulated in Bcl2-rescued B cells.P21 promotes cell cycle arrest in response to many stimuli,and is induced by p53.Mdm2 inhibits p53 transcriptional activity.Therefore,over-expressing Mdm2 together with Bcl2 should relieve the cell cycle block.(8)The in vivo and in vitro experiments show that deletion of p53 can rescue Bcllla-deficent early B cells development and rescue Bcl11a-deficient pro-B cells apoptosis and proliferation defects.Bcl11a and p53 double deficient pro-B cells can survival and grow in B cells culture condition in vitro.Furthermore,Bcl11a and p53 double deficient CLPs can differentiate to B cells in vitro.(9)Complete loss of lymphoid development potential in Bc11a-deficient HSCs.Some of HSCs are apoptosis once Bcl11a deleted.Bcl11a-deficient LSKs can’t differentiate to B cells in vitro.Bcl11a-deficient can’t generate CLPs,ETPs,B cells,T cells and NK cells.(10)No lymphocytes were generated from Bcl11a and p53 double deficient HSCs.Deletion of p53 can rescued the Bell la-deficient HSCs from apoptosis,however,it can’t rescue the lymphoid development potential in Bcl11a-deficient HSCs.Consistent with early B cells,p21 was up regulated in HSCs once Bcl11a deleted.P53 deficiency indeed reduced p21 expression to the wild type level suggesting that cell cycle defects would unlikely be a reason for lymphoid development defects in Bell la-deficient LSKs or HSCs and that Bell la functions by another mechanism(s).(11)QRT-PCR analysis shows that the lymphoid genes,myeloid genes and erythroid genes were altered in Bcl11a-deficient HSCs and LMPPs.In Bcl11a-deficient and Bcllla/p53 double-deficient HSCs,the lymphoid transcription factor Pbx1 was significantly decreased,foxol and Runxl were also down regulated,Ikzfl,Pu.l and E2A were not altered.The myeloid gene Mpo and lymphoid genes Gatal and Klf1 were significantly up regulated.In Bcl11a-deficient and Bcl11a/p53 double-deficient LMPPs,The lymphoid genes Runxl,Flt3,I17r,Foxol and Notch1 were down regulated,Ikzfl,Pu.1,E2A and Mef2c were not altered.The myeloid genes Mpo and Cebpa were significantly up regulated.The inability of p53 deficiency to restore lymphoid potential in Bcl11a-deficient LMPPs is therefore reflected on the fact that deletion of p53 did not substantially change the expression patterns of the key lymphoid genes in these progenitors.(12)ChIP-PCR demonstrated that Bcl11a directly binds to Bcl2,Mdm4 and Mdm2 locus to regulate their expression.Bcl11a binds the intronic region of Bcl2,binds promoter region of Mdm4 and binds exon region of Mdm2.(13)By ChIP-Seq analysis,we identified 4,517 binding loci of Bcl11a in pro-B cells,including E2A,Pax5,Bcl11b,BclxL and p21.Bcl11a binding sites in E2A,PaX5,BclxL and p21 were confirmed by ChIP-PCR.