Transcription Factor Blimp1 Involves In The B-cell Differentiation Of Common Carp(Cyprinus Carpio) By The Recruitment Of HDAC3 And Histone Deacetylation Modification

Transcription factor Blimp1 is a key regulator of plasma cell differentiation,which can promote the B lymphocytes differentiation into the plasma cell with antigen stimulation,and then regulate the antibody secretion and cell proliferation.Blimp1 inhibit the transcription of different downstream target genes including Pax5 and Bcl6 by recruitment of histone deacetylase(HDAC)and methyltransferase G9a forming transcriptional inhibition complexes.Histone acetylation and deacetylation is a dynamic,reversible process catalyzed by two classes of enzymes,histone acetyltransferase(HAT)and histone deacetylase(HDAC),at lysine residues along histone tails.In general,histone acetylation correlates with transcriptional activation,and histone deacetylation correlates with transcriptional repression.Until now,Studies on HDAC,especially the role of HD AC in B-cell differentiation and plasma in bony fish cell has been limited.In this research,the HD AC genes in common carp(Cyprinus carpio.)were cloned and the expression profiles of hdacl,hdac3 and hdac8 were analyzed.The transcriptional repression of Blimp1,the role of HDAC3 and the acetylation of histone in stimulated lymphocytes with LPS were analyzed as following:The experiment mainly includes the following three aspects:1)the gene sequences of hdacl,hdac3 and hdac8 of carp were cloned,all of which contained complete ORF region,highly conserved histone deacetylase domains,conserved catalytic sites and partial phosphorylation sites.Sequence analysis predicted that all of them had multiple glycosylation sites and ubiquitination sites.2)the results of real-time quantitative PCR showed that hdacl,hdac3 and hdac8 were highly expressed in the gonads,brain and spleen,but were low in the gills.Common carp stimulated by intraperitoneal injection of TNP-LPS(TI antigen)and TNP-KLH(TD antigen)were used for the following experiments.Lymphocytes from common carp abdominal cavity,peripheral blood and spleen were isolated by Histopaque density gradient centrifugation on day 1,3,7 and 15 after stimulation.The expression of hdacl,hdac3 and hdac8,the transcription factors blimp1,pax5,bcl6aa,bcl6ab,the expression of sIgM and IgZ3,and the expression of mIgM and cd79a were detected and expression correlation analysis was performed.The results show that hdacl expression was the highest,followed by hdac3 and hdac8 expression was the lowest.TNP-LPS stimulation promoted HDAC3 expression in immune response.On the other hand,the correlation analysis of all the genes showed that blimp1 was negatively correlated with pax5,mIgM and cd79a.However,in lymphocytes from spleen,blimp1 showed negative correlation with bcl6aa and bcl6ab,among which bcl6aa had significant difference statistically.Except that,the expression of blimp1 showed significant positive correlation with hdac3.The spleen is the main site for mature B lymphocytes to differentiate and form plasma cells after contacting with antigens.Plasma cells formed in the spleen can migrate into peripheral blood and abdominal cavity,or migrate to the head kidney to form long-lived plasma cells.Therefore,the above expression profiles in spleen may suggest a functional correlation between blimp1 and hdac3.3)HDAC3 expression vector was constructed,HDAC3 protein was purified,and commercial HDAC3 antibody was confirmed and validated.The co-expression of IgM and HDAC3 as well as Blimpl and HDAC3 were detected in carp spleen tissues stimulated by TI and TD antigens by immunofluorescence labeling.The results showed that HDAC3 was expressed in IgM+B lymphocytes,and HDAC3 and Blimp1 were colocalized in the nucleus of spleen cells,which suggested that Blimpl and HDAC3 might both play a role in IgM+B cells.In order to further clarify the transcriptional regulation effect of Blimp1 on target genes and the influence of histone acetylation modification on Blimpl transcriptional regulation,the following experiments were performed.1)the carp spleen lymphocytes and peripheral blood lymphocytes were stimulated with LPS in vitro,and the expressions of 12 genes were detected and the correlation analysis was performed.The results showed that blimp1 was positively correlated with the expression of hdac3 and aicda in splenic lymphocytes.2)the inhibitory effect of Blimp1 on the transcriptional activity of several target gene promoters was detected by Double-luciferase assay.Promoter reporter vectors of bcl6aa,pax5 and aicda were constructed respectively,and were co-transfected with the eukaryotic expression vector of Blimp1 into 293T cells respectively.Combined with the treatment of histone deacetylation inhibitor TSA and histone deacetylase HDAC3 specific inhibitor RGFP966,the effects of these two inhibitor treatments on transcriptional regulation were analyzed.The results showed that Blimp1 could significantly inhibit the transcriptional activity of bcl6aa,pax5 and aicda promoter,while TSA and RGFP966 treatment could significantly eliminate the transcriptional inhibition of Blimp 1.3)In order to further study the effect of HDAC3 on the regulation of Blimp1,on the basis of the above experiments,the expression vector of HDAC3 was co-transfected with Blimp1 expression vector.The results showed that the overexpression of hdac3 further enhanced the transcriptional inhibition of Blimp1 on the promoter of bcl6 and pax5.4)Co-Immunoprecipitation results showed that HDAC3 and Blimp1 interacted with each other in co-transfected 293T cells.These results suggest that Blimp1 and HDAC3 have synergistic effect on Blimp1 transcriptional repression,which can be performed in two different ways.On one hand,the HDAC3 itself can directly bind to the specific recognition sequence of the target gene promoter region and keep interaction with Blimp1;on the other hand,HDAC3 involved in the transcriptional inhibition complex by recruitment of Blimp 1.This complex exerts transcriptional inhibition through histone acetylation modification affecting chromatin opening or closing conformation in the promoter region of specific target genes.In order to analyze the interaction between Blimp1 and HDAC3 and acetylation of histone in carp lymphocytes in the immune response stimulated by LPS.Two experiments were carried out.1)HDAC3 antibody was used to perform immunoprecipitation experiment in peripheral blood lymphocytes and spleen lymphocytes stimulated by LPS.The results showed that HDAC3-bound Blimpl was enriched and increased in the activation of lymphocytes.The binding Blimp1 to HDAC3 decreased after TSA and RGFP966 treatment.Therefore,TSA can influence the recruitment of HDAC3 by Blimp1.2)Histone acetylation was detected on LPSactivated lymphocytes,and Western blotting hybridization was performed using specific histone acetylation antibodies.The results showed that the overall level of multisite acetylation of histones in peripheral blood lymphocytes and spleen lymphocytes decreased at day 1 and day 3 after LPS stimulation and TSA treatment resulted in significant increase of histone acetylation,especially at H3K4 and H3K9 sites.These results preliminarily suggest that histone acetylation modification is an important process in the differentiation of LPS-activated lymphocytes.At the same time,this study provides a basis for further identifying the role of histone acetylation modification in the co-regulation of Blimp1 and HDAC3 and its regulatory mechanism on target genes by ChIP-qPCR or ChIP-seq.In summary,HDAC3 plays an important role in transcriptional regulation of common carp Blimp1,which is involved in cell differentiation and immune response of mature B lymphocytes.The research of HD AC and the histone acetylation modification will provide basic data for understanding the molecular regulation mechanism of B-cell immune response in bony fish and will be helpful to elucidate the importance of epigenetic regulation mechanism in B-cellmediated specific immunity in bony fish.