Expression Patterns Of Bcl6 Genes And Mutual Regulation Between Bcl6 And Prdm1 In Medaka

B cell lymphoma 6(Bcl6),a member of the BTB-ZF family,contains both of conservative BTB domain and zinc finger domain.As a transcription factor,it regulates the expressions of many genes by directly combining the promoters of target genes or recruiting some co-inhibitors.Bcl6 is an important regulator that participates in many of immune response,include the differentiation and activation of macrophages,proliferation of B lymphocytes,differentiation of follicular helper T cells.Similarly,beta-interferon positive regulatory domain binding factor 1(Prdm1)is also an important immunomodulatory factor that regulates the immune response by antagonizing each other with Bcl6.In addition,both Bcl6 and Prdml play an important role in the development of embryo,tissue formation,inflammatory response and the occurrence of tumor in many animals.The studies about Bcl6 and Prdml are relatively perfect in mammals,but there are relatively few reports in fish.Fish have large nonspecific immune system;their specific immune systems are less complex than that of mammals.We have previously reported that prdm1a participates the immune response in medaka(Oryzias latipes).In order to explore the possible molecular mechanism of pcl6 and prdm1 in fish,the expression profiles of bcl6 homologous genes were analyzed,and the mutual relationship between bcl6 and prdm1 was studied in medaka.BCL6(BCL6A)is homologous to BCL6B in human,and they are members of the BTB-ZF protein family.Due to genome duplication events occurred in the evolution of bony fish,bcl6a,direct homolog of human BCL6A,was duplicated.In order to avoid confusion,we named two bcl6a genes as bcl6aa and bcl6ab in fish.Medaka's bcl6b is homologous to human BCL6B.Bioinformatics analysis demonstrated that bcl6 proteins are very conservative among different species,they all contain a BTB domain and several zinc finger damains.Quantitative results showed that those three homologous genes are expressed in different tissues in medaka,and they are highly expressed in the embryos of 4 cells and morular stage and moderately expressed in the embryos after 3 days during embryonic development.In situ hybridization results showed that bcl6 homologous genes' mRNA can be detected in early stage of embryos.However,they have different expression patterns in the embryos of 5 days after fertilization,those results indicated that they may have different functions during embryonic development.Immune stimulation experiment showed that all of bcl6aa,bcl6ab and bcl6b participate in the immune response,and prdm1a and bcl6aa are reciprocally repressive in vivo.Luciferase reporter assay showed that the core promoter of bcl6aa is located in the upstream region of ATG,the length of this region is about 118 bp and it contains two putative prdm1 binding sites.Indeed,the fact that prdm1 directly repress the transcription of bcl6aa promoter was verified by luciferase reporter assay,because the luciferase activity was not affected after mutating the prdm1 binding sites.In addition,we found that bcl6 mRNA may have four alternative splicings:bcl6b.1,bcl6b.2,bcl6b.3 and bcl6b.4,and RT-PCR detection showed that bcl6b.3 may paly more important role in immune response.In short,the bcl6 genes,similar to mammals,are involved in the immune response in medaka,and bcl6aa and prdm1 may also have mutual inhibition in vivo.In addition,4 alternative bcl6b mRNAs may have different physiological functions in medaka.These findings will provide important evidences for better understanding of the immune mechanism of fish in the future.