Research paperAge-associated methylation change of TAP1 promoter in piglet
Introduction
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. Escherichia coli F18 (E. coli F18), which is the primary causative agent of these two diseases (Imberechts et al., 1992), relies on its fimbriae to adhere to the surface of epithelial cells in the porcine small intestine and to bind to the F18 receptor on porcine small intestinal epithelial brush cells. The bacterium then reproduces, and produces enterotoxin, causing disease in piglets (Benin and Ducher-Suchaux, 1991). Transporter associated with antigen processing (TAP) is a heterodimer composed of TAP1 and TAP2 subunits. The major function of TAP is endogenous antigen presentation (Paulsson, 2004). Many studies have reported that the TAP1 gene is related to virus infection and cancer, with viruses thought to provide evasion from the host immune system, specifically by preventing the recognition of virally infected cells by cytotoxic T (CD8 +) cells (Zeidler et al., 1997, Ambagala et al., 2000, Ambagala et al., 2003, Bauer and Tampe, 2002). Therefore, as a protein antigen peptide transport carrier, TAP plays an important role in the immune response (Suh et al., 1994). Some reports have indicated that TAP1 could be an effective anti-E. coli F18 molecular marker in pigs (Witkowska-Toboła et al., 2004, Sun et al., 2012). Based on the paired full-sib individuals selected from an established resource of Sutai pigs (Duroc × Meishan) that were characterized as resistant or sensitive to E. coli F18, we investigated the expression profiles of some genes, including TAP1 (Bao et al., 2012). The results showed that TAP1 expression was increased following viral and bacterial infection, thus indicating a significant role in immune responses.
DNA methylation of 5-cytosine to 5-methylcytosine in guanine and cytosine-rich regions (CpG islands) is catalyzed by methyltransferases and is one of the most common mechanisms of epigenetic regulation. DNA methylation occurs mainly in the CpG island-rich promoter region, where it can hinder the binding of transcription factors to the promoter, thereby inhibiting gene transcription (Wang and Xu, 2014). In view of the importance of the promoter in the regulation of gene transcription regulation and the close relationship between TAP1 gene expression and E. coli F18-resistance, we conducted the present study to determine the correlation between promoter CpG island methylation status and TAP1 mRNA expression. Bisulfite sequencing PCR (BSP) was used to investigate the methylation status of TAP1 gene promoter CpG islands and fluorescence quantitative PCR was used to analyze TAP1 expression in the jejunum of Sutai piglets from birth to weaning age. This information will provide help to elucidate the mechanism responsible for resistance to E. coli F18 infection in piglets.
Section snippets
Experimental animals
The Sutai pig [Duroc (50%) × Meishan (50%) cross] is a new breed of high quality lean-meat pig breed by the Sutai Pig Breeding Center in Suzhou City (Jiangsu Province, China). It was approved by the National Committee of Livestock and Poultry Species as a new variety in 1999. All pigs (Sutai pigs, 8-, 18-, 30- and 35-days old, n = 4 per age group) included in the study were healthy, raised under the same conditions, with similar birth weights, weaning weights, and body sizes. The pigs were
Bioinformatic analysis
The result showed that the porcine TAP1 gene core promoter region is located in the region from − 700 to − 400 bp and upstream-2 kb region contains three CpG islands (Fig. 1): − 925 to − 808, − 595 to − 450 and − 273 to − 78. So we speculated that the second CpG island, which contains 19 CpG sites, regulates gene expression. Therefore, primers were designed for amplification of a fragment containing the whole CpG island-containing region. TFSEARCH and Promoter Scan were used to find identify putative
Discussion
A large study showed that the methylation of CpG islands in the promoter region could suppress gene expression (Antequera, 2003, Laird, 2010, Wang et al., 2012). There are three ways to explain the mechanism by which DNA methylation inhibits transcription. First, DNA methylation interferes directly with the binding of transcription factors (TF) to TFBS. Second, DNA methylation can influence chromatin organization to block the binding of TF and TFBS. Third, the methylation sequence can combine
Conclusions
In this study, we demonstrated that TAP1 expression is negatively related to promoter CpG island methylation and CpG_4, CpG_13 and CpG_15 may be the critical regulatory sites.
The following are the Supplementary data to this article. . 1% agarose gel electrophoresis for PCR products of TAP1 gene. (a) Bisulfite-treated DNA PCR products of TAP1 gene (b) RT-PCR products of TAP1 gene; (c) RT-PCR products of GAPDH gene; (d) Normal DNA PCR products of TAP1 gene; M means DL2000 Marker.
Acknowledgments
This research was supported by the National Natural Science Funds (31372285, 31172183), the Genetically Modified Organisms Technology Major Project (2014ZX08006-001B), the Science and Technology Support Program of Jiangsu Province (BE2014357, BE2013345), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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