Shouyong Sun

Correlation between BPI gene upstream CpG island methylation and mRNA expression in piglets.

Diarrhea and edematous disease are two major causes of mortality in postweaning piglets, and these conditions lead to huge economic losses in the swine industry. E. coli F18 is the primary causative agent of these two diseases. Bactericidal/permeability-increasing protein (BPI) plays an important role in the natural defense of the host. The aim of this study was to determine the correlation between BPI gene upstream CpG island methylation and mRNA expression. In this study, bisulfite sequencing PCR (BSP) was used to detect the methylation status of the BPI gene upstream CpG island and fluorescence quantitative PCR was used to detect BPI expression in the duodenum of piglets from birth to weaning age. BPI upstream CpG islands were shown to have many putative transcription factor binding sites, 10 CpG sites and every CpG site was methylated. The CpG island methylation level was lowest in 30-day piglets and was significantly lower than levels in 8-day piglets (p < 0.05). BPI mRNA expression was significantly higher in 30-day piglets than at any other age (p < 0.05). Pearson’s correlation analysis showed that the methylation status of the CpG island was negatively correlated with BPI mRNA expression. Statistical significances were found in CpG_1, CpG_3, CpG_4, CpG_7 and CpG_10 (p < 0.05). The data indicate that BPI expression is improved by demethylation of the BPI gene upstream CpG island. Furthermore, CpG_1, CpG_3, CpG_4, CpG_7 and CpG_10 may be critical sites in the regulation of BPI gene expression.

Identification of a 5-Methylcytosine Site that may Regulate C/EBPβ Binding and Determine Tissue-Specific Expression of the BPI Gene in Piglets

Bactericidal/permeability-increasing protein (BPI) plays an important role in innate immune defense in mammals. A previous study showed that BPI gene expression correlates to gram-negative bacteria resistance. However, this gene showed tissue-specific expression in piglets and strongly expressed only in the digestive tract. To investigate the mechanisms governing the tissue-specificity, bisulfite sequencing PCR and next generation sequencing were used for high accuracy methylation quantitation of CpG islands of BPI gene upstream in 11 different tissues from weaned Yorkshire piglets. Additionally, qPCR was used to examine mRNA levels of BPI gene as well as transcription factor. We additionally analyzed transcriptional regulation by studying key 5-methylcytosine sites and transcription factors. Results showed that BPI mRNA levels significantly correlated with the overall methylation as well as methylation at mC-15 which was non-CpG site, no significant correlation could be found between the BPI and transcription factor mRNA levels, EMSA test showed that C/EBPβ could interact with BPI wild-type promoter DNA, but not methylated DNA. So we confirmed that methylation of mC-15 residue could inhibit the ability of C/EBPβ binding to the BPI promoter and affect the expression, and this mechanism probably plays a role in the tissue specificity of BPI gene expression in weaned piglets.

Age-associated methylation change of TAP1 promoter in piglet.

Diarrhea and edematous disease are two major causes of mortality in postweaning piglets. These conditions lead to huge economic losses in the swine industry. Escherichia coli F18 is the primary causative agent of these two diseases. Transported associated with antigen processing (TAP) plays an important role in the immune response and the TAP1 gene could be an effective anti-E. coli F18 molecular marker in pigs. The aim of this study was to determine the correlation between TAP1 gene promoter CpG island methylation status and mRNA expression in piglets. In this study, bisulfite sequencing PCR (BSP) was used to detect the methylation status of the TAP1 gene promoter CpG islands and fluorescence quantitative PCR was used to detect TAP1 expression in the jejunum of Sutai piglets from birth to weaning age. The fragment of the TAP1 gene promoter region under investigation has no mutation, has 13 putative transcription factor binding sites containing 19 CpG sites, and may be important for regulation of gene expression. With increasing age, the overall methylation levels decreased, while the TAP1 expression levels increased, indicating a negative correlation between TAP1 expression and promoter methylation levels. Variance analysis showed significant differences in the methylation status of CpG_4, CpG_13 and CpG_15 among the different age groups (P<0.05). Our data indicate that TAP1 expression is increased by demethylation of promoter CpG islands, with CpG_4, CpG_13 and CpG_15 implicated as the critical regulatory sites.

New insights into the codon usage patterns of the bactericidal/permeability-increasing (BPI) gene across nine species

Bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins known as super-antibiotics that are implicated as endotoxin neutralising agents. Non-uniform usage of synonymous codons for a specific amino acid during translation of a protein is known as codon usage bias (CUB). Analysis of CUB and compositional dynamics of coding sequences could contribute to a better understanding of the molecular mechanism and the evolution of a particular gene. In this study, we performed CUB analysis of the complete coding sequences of the BPI gene from nine different species. The codon usage patterns of BPI across different species were found to be influenced by GC bias, particularly GC3s, with a moderate bias in the codon usage of BPI. We found significant similarities in the codon usage patterns in BPI gene among closely related species, such as Sus_scrofa and Bos_taurus. Moreover, we observed evolutionary conservation of the most over-represented codon CUG for the amino acid leucine in the BPI gene across all species. In conclusion, our analysis provides a novel insight into the codon usage patterns of BPI. This information facilitates an improved understanding of the structural, functional and evolutionary significance of BPI gene among species, and provides a theoretical reference for developing antiseptic drug proteins with high efficiency across species.

DIFFERENTIAL EXPRESSION OF PORCINE TAP1 GENE IN THE POPULATIONS OF PIGS

Transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum (ER) for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. TAP is consisted of two subunits: TAP1 and TAP2. Using Real-time PCR technology, this study detected tissue expression profile and analyzed the differential expression of TAP1 gene in Sutai Escherichia coli-resistant group, Yorkshire and Meishan pigs. Tissue expression profile revealed that TAP1 gene expressed in all tissues we detected, and the expression levels were high in lung, immune tissues and intestines. Through the comparation of gene expression differention in different populations, TAP1 expression level of Sutai E. coli-resistant group was significantly higher than that of Yorkshire and Meishan populations in liver, spleen, lung, kidney, thymus, lymph, duodenum and jejunum (P < 0.05). Meanwhile TAP1 gene was more highly expressed in Sutai E. coli-resistant group than that of Meishan population in stomach (P < 0.05). In conclusion, the upregulation of TAP1 expression level in E. coli resistant group could be related to E. coli F18 infection. In addition, Chinese local pigs may have special immune response and genetic mechanism in resisting E. coli F18 infection which is differing from MHC I moleculars.

Differential expression of genes BPI , TAP1 , SLA-1 and SLA-3 in Escherichia coli F18-resistant and sensitive Meishan post-weaning piglets

The genetic basis and mechanism of sensitivity or resistance to the significant pathogen Escherichia coli F18 is not clear in native Chinese pig breeds and may differ from that in non-native breeds. Our previous research showed that the genes BPI, TAP1, SLA-1 and SLA-3 may play important roles in resistance to E. coli F18 in post-weaning piglets. This study was based on success in selecting and identifying full sib Chinese native Meishan breed post-weaning E. coli F18-resistant and sensitive piglets. Real-time PCR was used to detect expression levels of the genes BPI, TAP1, SLA-1 and SLA-3 in liver, spleen, thymus, lymph node, duodenum, and jejunum tissues between E. coli F18-resistant and -sensitive Meishan piglets. Only the BPI gene was obviously tissue specific; it was highly expressed in the duodenum and jejunum, but expressed at a low level in other tissues. The other three genes were expressed in all the studied tissues, and particularly highly in immune organs and intestinal tissues. The expression level of BPI in the duodenum of the resistant group was significantly higher than that in the sensitive group (P < 0.01). The SLA-3 expression level in the thymus of the resistant group was significantly higher than that in the sensitive group (P < 0.05). TAP1 and SLA-1 gene expression levels were generally higher in the resistant group than the sensitive group, but there were no significant differences. Genes BPI and SLA-3 play an important role in the processes of resistance to E. coli F18 in Meishan weaned piglets. We speculate that BPI protein may have a direct killing effect on Gram-negative bacteria such as E. coli strain F18 in the intestine; the resistance of Meishan weaned piglets to E. coli F18 is likely to be related to the upregulation of intestinal BPI. The upregulation of SLA-3 may increase the resistance of weaned piglets to E. coli F18 by regulation of the immune response.