Tissue Expression Characteristics Of Chicken BRD2/3/4 Genes And Their Role In NDV Replication

The Bromodomain and extra terminal domain(BET)family includes four members,BRD2,BRD3,BRD4 and BRDT.Among them,BRD2,BRD3 and BRD4 are widely expressed in human and mammalian tissues and are major intracellular transcriptional regulators with various biological functions such as gene transcriptional regulation,cell cycle,inflammatory response,natural immunity and viral replication,while BRDT is specifically expressed only in testicular tissues.Our group found that BRD2,BRD3 and BRD4 genes were expressed in chicken visceral and muscle tissues,but their expression characteristics in chicken immune organs were not clear.Also their relationship with the replication of the infectious agent Newcastle disease virus(NDV)in poultry has not been clarified.Therefore,in this study,we used Real-time quantitative PCR(RT-qPCR)to detect the expression characteristics of BRD2,BRD3 and BRD4 genes in immune organs of chickens at different months of age and in NDV-infected chickens.Meanwhile,the expression levels of BRD2,BRD3 and BRD4 genes were detected by RT-qPCR after NDV inoculation of chicken embryonic fibroblasts(DF-1).The BRD2 gene with significant expression differences was selected and its role in NDV replication was explored by RNA interference and gene overexpression assays.The main findings are as follows.1.RT-qPCR results showed that BRD2,BRD3 and BRD4 genes were expressed in the immune organs of 1-6-month-old chickens,but there were significant differences in their expression characteristics and expression trends.The highest expression of BRD2 gene was found in the spleen and thymus of 3-month-old chickens(P < 0.01),while the highest expression was found in the bursa of fabricius of 4-month-old chickens(P < 0.01),and the expression changes in chicken spleen,thymus and bursa of fabricius showed an "inverted V" trend.BRD3 gene expression was highest in the spleen of 1-month-old chickens and in the thymus and bursa of fabricius of 6-month-old chickens(P < 0.01),and the expression changes in the chicken spleen,thymus and bursa of fabricius showed "inverted N" and "Uptrend" trends,respectively.BRD4 expression was highest in the spleen of 3-month-old chickens and in the thymus and bursa of fabricius of 6-month-old chickens(P < 0.01),and the expression changes in chicken spleen,thymus and bursa of fabricius showed three trends of "inverted V","inverted N" and "Uptrend",respectively.2.The RT-qPCR results showed that the expression levels of BRD2,BRD3 and BRD4 genes were significantly different in the immune organs of chickens at d 3-5 after NDV infection.The expression of BRD2 gene was highest in chicken spleen at d 3 and lowest at d 5(P < 0.01),and in the thymus and bursa of fabricius at d 4(P < 0.01),and it showed two trends of " Declining" and "inverted V" in the spleen,thymus and bursa of fabricius,respectively.BRD3 gene expression was highest in the spleen and bursa of fabricius of chickens at d 3 and in the thymus of chickens at d 5(P< 0.01),and showed both a " Declining" and a " Uptrend" in the spleen,thymus and bursa of fabricius.BRD4 gene expression was highest in both spleen,thymus and bursa of fabricius of chickens at d 3,with highly significant differences in expression in thymus and bursa of fabricius(P < 0.01).changes in expression of BRD4 gene in spleen,thymus and bursa of fabricius of chickens showed "V" and "Declining " trends,respectively.3.After DF-1 cells were infected with NDV,BRD2,BRD3 and BRD4 were expressed at all time points,with the highest expression at the 6th and 12 th h,which were 16,9.8 and 4.3 times higher than those of the control group,respectively(P < 0.05).The BRD2 gene with the most significant expression differences was selected for RNA interference and gene overexpression experiments.Tthe results showed that the highest interference efficiency(P < 0.01)was found for BRD2 sh RNA1 among the four BRD2 sh RNA interference vectors,at 79.8%.DF-1 cells after NDV infection with BRD2 sh RNA1 interfered with BRD2 gene expression showed significant syncytia at 24 h,and the syncytia formed at 36 h-72 h were significantly larger than those in the Control sh RNA group and Normal group.Meanwhile,the viral titers of NDV at 36 h and 48 h were significantly higher than those of Control sh RNA and Normal groups(P < 0.05).However,in NDV-infected DF-1 cells with BRD2 gene overexpression,the overexpression group formed significantly smaller syncytia at 36 h-72 h than the EGFP-C1 group and the Normal group,and the viral titer at 48 h was significantly lower than that of the EGFP-C1 group and the Normal group(P < 0.01).The above findings showed that BRD2,BRD3 and BRD4 genes were expressed in the spleen,thymus and bursa of fabricius of NDV-infected SPF chickens in immune organs and NDV-infected chickens at different months of age,but the expression characteristics and expression trends were significantly different.Meanwhile,interference with BRD2 genes expression promoted NDV replication and cellular pathogenicity,while overexpression of BRD2 showed the opposite result.This lays the foundation for subsequent work to investigate the mechanism of action of chicken BRD2 protein in regulating NDV replication and as a candidate gene for chicken disease resistance breeding.