A Preliminary Study On Transcriptional Regulation Of Chicken NLRC5 By DNA Methylation And Key Transcription Factors

NLRC5,found in mice in 2010,is known as a new type of NOD-like receptor,,which is the largest NLR family member.It has been concluded from quantities of researches that NLRC5 plays an important role in innate immunity and adaptive immunity.However,the regulation mechanism remains unclear.In order to identify the regulation mechanism of NLRC5 promoter,the specificity of chicken NLRC5 gene expression in different tissues of chickens was tested in this research.The CpG islands in the promoter region of NLRC5 were predicted by software,and the methylation status of NLRC5 promoters in DF1 and HD11 cells was detected by BSP.Treating cells with methyltransferases inhibitors,we then compared the degree of methylation with gene expression,and finally verify transcription factors,genes expression of which is influenced by methylation,by promoter-binding transcription factor assays,site-directed mutagenesis,and EMSA.The 2108 bp promoter region of the NLRC5 start codon was then cloned to construct a series of promoter vector deletion vectors,which was then transfected into DF1 cells.Dual luciferase assays reflect the activity of promoters of different lengths to find the core region and predict and analyze cis-acting elements which may exist in the region of promoters.The cis-elements and their effect on NLRC5 promoter activity was subsequently verified by promoter-binding transcription factor experiments,site-directed mutagenesis experiments,and EMSA.Finally,HD11 cells were treated with LPS and IFN-?,RT-qPCR and ELISA used to determine the activation condition of NF-?B,and the NF-?B transcription factor in the promoter region of chicken NLRC5 gene was verified.The results showed that:1.The expression of NLRC5 in immune-related tissues,such as spleen and liver,of chickens detected by RT-qPCR was significantly higher than that in hearts,muscles,etc.,while the expression of NLRC5 in chicken macrophage cell line HD 11 detected by qPCR was significantly higher than that in chickens fibroblast cell line DF-1.2.A series of experiments were conducted to investigate the effect of methylation on the transcription level of NLRC5 promoter region,among which MethPrimer predicted and discovered two CpG islands;results of BSP experiment showed that DF1 is hyper-methylated,while the methylation level of HD 11 is low.Different concentrations of methylation transferase inhibitor 5-aza-cd were used to treat the two types of cells.CCK-8 was used to detect the cell survival rate,and the expression of related genes was detected by qPCR.The results showed that under the treatment of 40 ?mol/L 5-aza-cd,the survival rate of DF-1 cells was significantly different from that of the control group(P<0.01).In addition,methylation detection of the treated cells revealed specific methylation reduction sites,And methylation affects the transcriptional regulation of NLRC5 gene through E2F transcription factors was finally determined by dual luciferase assay,before which a deletion vector in the region was constructed,promoter-binding transcription factor assay,site-directed mutagenesis assay3.To find potential regulatory elements of the NLRC5 promoter region,JASPAR analysis was used and found that the NLRC5 promoter-2108 to-1 did not contain a typical ribonucleic polymerase II binding element.A deletion vector was then constructed to perform double luciferase assay,which confirmed that the three regions with higher activities.Promoter-binding transcription factor experiments revealed the presence of the STAT1 transcription factor in the-459 region.The point mutations of the cis-element STAT1 in this core region and EMSA experiments were targeted so that the presence of the cis-element STAT1 in this region and an important role in regulating the activity of NLRC5 initiation was further confirmed.4.To explore the NF-?B transcription factor binding site in the NLRC5 promoter region,the overexpressed binding deletion vector was used to determine the possible binding region of NF-?B.Stimulating chicken macrophages HD 11 with lipopolysaccharide(LPS)and interferon gamma(IFN-?),we found that NF-?B in cells could be activated under the 8-hour stimulation with lOng/ml LPS and 12-hour stimulation with lng/ml LPS+10ng/ml IFN-?.The presence of two NF-?B trans action elements in-2423 and-2342 were finally validated by EMSA.