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

Chen Zi,Wenbin Bao,Jing Wang,Shouyong Sun,Xuemei Yin,Shenglong Wu,Guoqiang Zhu,Li Sun

doi:10.1038/srep28506

Abstract

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.

Highlights

Bactericidal/permeability-increasing protein (BPI), an endogenous cationic protein found in humans and other mammals, plays an important role in the innate immune defense
We examined whether there was a correlation between expression of the transcription factors of the C/EBPβand that of BPI; C/EBPβwere only highly expressed in the lungs (Fig. 4a), and no significant correlation could be found when examining the BPI and C/EBPβmRNA levels (R =−0.13)
This bacterium relies on its fimbriae to adhere to the surface of epithelial cells in the small intestine and to bind to the F18 receptor on porcine small intestinal epithelial brush cells

Summary

Introduction

Bactericidal/permeability-increasing protein (BPI), an endogenous cationic protein found in humans and other mammals, plays an important role in the innate immune defense This protein, which is primarily located in the aniline blue particles of polymorphonuclear leukocytes (PMNs), participates in killing gram-negative bacteria and in neutralizing endotoxin and lipopolysaccharide (LPS)[1]. To investigate the genetic mechanisms underlying the tissue-specific expression of the BPI gene, we used BSAS to examine the BPI gene promoter CpG island methylation, and analyzed the regulation of mRNA expression by examining key 5-mC sites and key transcription factors in 11 different tissues from weaned Yorkshire weaned piglets

Methods

Results

Conclusion