Pig FUT3 Methylation Regulates E. coli F18 Susceptibility

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Shenglong Wu	+ 5212 word(s)	5212	2021-10-15 05:34:03	\|
2	update layout and reference	Rita Xu	-3193 word(s)	2019	2021-10-27 04:53:45	\|

This entry is adapted from the peer-reviewed paper 10.3390/genes12101586

Post-weaning diarrhea (PWD) is frequently associated with E. coli F18 infections in piglets. However, the underlying molecular mechanism concerning the resistance of E. coli F18 in local weaned piglets in China is not clearly understood. In the present study, our findings indicated that the methylation of mC-3 and mC-5 sites has certain inhibitory effect on FUT3 expression and promotes the resistance of E. coli F18 in piglets. The underlined study may explore FUT3 as a new candidate target in E. coli F18 infection in Chinese local weaned piglets.

pig FUT3 DNA methylation E. coli F18

1. Introduction

DNA methylation widely existing in mammals can regulate gene expression and maintain genetic stability with transcription and cell division. Under the catalysis of DNA methyltransferase (DNMTs), S-adenosyl-L-methionine (SAM) as methyl group is added to DNA segments and is converted to 5-methylcytosine (5-mC), N⁶-methyladenine (m⁶A), and 7-Methylguanine (7-mG). Among these, 5-mC is the most important methylation modification in mammals. DNA methylation is the main epigenetic mechanism in promoter regions of the eukaryotic genomes that regulate the expression of a gene by enhancing or attenuating the interaction of TF with sequences of DNA in the promoter region ^[1]. It has been reported that DNA methylation plays a crucial role in maintaining cell function, regulating individual development and disease ^[2]^[3]^[4]. DNA methylation is considered to be a significant research hotspot in the current field of pig genetics and breeding, relevant studies mainly focus on tissue-specific expression ^[5]^[6], cell apoptosis ^[7], variety differences ^[8], growth and development ^[9]^[10]^[11], immune response ^[12]^[13]. However, DNA methylation has been rarely reported in expression regulation of pig resistance to pathogenic microorganism infection.

Piglets’ bacterial diarrhea is one of the most common intestinal inflammatory diseases that leads to severe financial damages to pig farming on large scale. Enterotoxigenic Escherichia coli F18 (E. coli F18) is the main pathogenic microbe that results in post-weaning diarrhea ^[14]. However, it is evident from the study that receptors for E. coli F18 in the small intestine of a piglet at the brush border of the epithelial cell, are potentially associated with the pathogenicity of E. coli F18 in terms of their expression levels. In addition, the mechanism being involved in E. coli F18 resistance in Chinese local weaned piglets still remains undiscovered. In this study, based on previously obtained Sutai (a new crossbred between Meishan and the Duroc strains) and Meishan piglets, i.e., resistant and sensitive to E. coli F18 ^[15]^[16], we conducted a comparative mRNA sequencing of duodenum tissues and identified a functioning gene, designated a-1,3-fucosyltransferase (FUT3), that may have critically contributed in anti-E. coli F18 infection. FUT3, belonging to the fucosyltransferase family, regulates the formation of ABH and Lewis antigens and resistance to pathogen infection ^[17]^[18]. To further investigate the mechanism of FUT3 expression in regulating E. coli F18 resistance, this study systematically verified the linkage between the expression of FUT3 and infection of E. coli F18 at the tissue and cellular levels. Then, the FUT3 core promoter was determined by bioinformatics analysis and dual-luciferase assay, meanwhile, bisulfite amplicon sequencing (BSAS) was used to detect the level of methylation in FUT3 core promoter in intestinal tissues of piglets, i.e., sensitive and resistant to E. coli F18. Then we determined the impact of the methylation sites on the expression of FUT3 mRNA and evaluated considerable transcription factor in the core promoter region.

2. FUT3 Was Evaluated as a New Target to Combat E. coli F18 Infection on the Basis of Comparative Transcriptome Analysis

To determine the molecular mechanism of regulating E. coli F18 resistance in local pig breeds in Chinese indigenous pigs, we conducted a comparative RNA-seq analysis of duodenal tissues from Sutai and Meishan piglets exhibiting sensitivity or resistance to E. coli F18. In Sutai piglets, 238 differentially expressed genes (DEGs) were screened out between piglets that were sensitive or resistant to E. coli F18 (p-value <0.05 and |fold change)| <2 (Figure 1a). Likewise, 310 DEGs were detected in Meishan piglets (Figure 1b). Interestingly, we identified 46 common DEGs between two pig breeds (Figure 1c,d). Among these DEGs, α-1,3-fucosyltransferase (FUT3) is involved in glycosphingolipid biosynthesis (KO pathway: ko00601) correlated with the generation of E. coli F18 receptor ^[19]^[20]^[21], which is probably considered as a novel target to combat E. coli F18 infection in piglets.

Figure 1. Pig FUT3 is found as a host gene involved in the infection of E. coli F18. (a) DGEs between Sutai F18-resistant (SR) and -sensitive (SS) piglets. (b) DGEs between Meishan F18-resistant (MR) and -sensitive (MS) piglets. (c) Venny screening of common DGEs between Sutai and Meishan piglets. (d) Gene list of common DGEs has been associated with infection caused via E. coli (F18).

3. Knockdown of Pig FUT3 Enhances E. coli F18 Resistance

To analyze the correlation between the expression of FUT3 and E. coli F18 susceptibility in pigs, we analyzed the differential mRNA and protein expression of FUT3 between piglets that were sensitive and resistant to E. coli F18. qPCR evaluation revealed that the FUT3 gene’s expression level was considerably elevated in the duodenum and jejunum of piglets that were sensitive to E. coli F18 relative to those that showed resistance (p < 0.01), as depicted in Figure 2a. The results were further confirmed by the immunoblotting which revealed that the protein expression of FUT3 was significantly upregulated in a sensitive group, as depicted in Figure 2b.

Figure 2. Differential expression analysis of pig FUT3 gene in intestinal tissues between Sutai E. coli F18-resistant and -sensitive piglets. (a) qRT-PCR detection, n = 3 biological replicates, ** p < 0.01. (b) Immunoblot analysis, β-actin, internal reference.

To further understand how the expression of FUT3 regulated E. coli F18 invasion, the lentivirus-activated RNAi was used to facilitate the attenuation of FUT3 in the intestinal porcine epithelial cells (IPEC-J2). The expression of a GFP was determined in more than 90% of cells treated with shRNA vector. As indicated in Figure 3a, The knockdown potential of FUT3 in shFUT3-n (n = 1, 2, 3, 4) treated IPEC-J2 cells were 39.9%, 71.6%, 23.1%, and 86.9%, accordingly, relative to non-treated cells (control) and shFUT3-4 treated IPEC-J2 cells were considered for the successive evaluation. The level of Protein expression showed consistency with transcription levels (Figure 3b). Therefore, we successfully established the IPEC-J2 cell line with the silencing of FUT3. Besides, we further investigated the effects of FUT3 expression on the interaction of E. coli F18 to IPEC-J2 cells. After FUT3 knockdown, bacteria enumeration (Figure 3c) and relative quantification (Figure 3d) revealed that the adhesion levels of E. coli F18ab/ac-expressing fimbriae to IPEC-J2 cells in the shFUT3 group were considerably reduced than that in control groups (p < 0.01). Immunofluorescence assay (Figure 3e), scanning electron microscopy (Figure 3f), and gram staining (Figure 3g) also showed that the distribution of E. coli F18 in the shFUT3 group was markedly higher than that in the control group. These results suggested that FUT3 contributed to regulating E. coli F18 infection and lowered expression level enhances E. coli F18 resistance.

Figure 3. Knockdown of FUT3 enhances E. coli F18 resistance. (a) Interference efficiency evaluation of FUT3 in RNAi-n (n = 1, 2, 3, 4) by qRT-PCR. (b) Interference efficiency of FUT3 for RNAi-4 was assessed by immunoblot validation. (c,d) Adhesion of the F18 fimbria to IPEC-J2 cells were evaluated via bacteria enumeration and relative quantification detection, results are represented as mean± SEM, n = 3 biological replicates, ** p < 0.01. (e) Immunofluorescence assay. Blue fluorescence indicates nuclear staining via DAPI, red fluorescence indicates E. coli antibody. (f) Scanning electron microscopy (SEM) assay, cells were found under a scanning electron microscope (2500×). (g) Gram staining assay. An optical microscope (400×) was used to observe cells.

4. Impact of Pig FUT3 Promoter Methylation on Gene Expression

To further investigate the molecular mechanisms of FUT3 expression, we focused on the DNA methylation modification analysis of pig FUT3 promoter. Firstly, we carried out the determination of the FUT3 core promoter region. According to the IGV analysis of transcriptome sequencing, we achieved a 2000 bp sequence upstream of the FUT3 gene. The prediction of three promoter regions of pig FUT3 was carried out which were present at –400–(–350)bp, –1297 –(–1247)bp, –1848–(–1798)bp upstream of the transcription start site. Then, the 2000-bp upstream sequence was divided into four fragments, namely, –200–0 bp (control), –500–0 bp (P1), –1500–0 bp (P2), and –2000–0 bp (P3) (Figure 4a). Agarose gel electrophoresis was employed for evaluating the PCR products, followed by sequencing. PCR amplification products were evaluated via agarose gel electrophoresis (1%), as depicted in Figure 4b. As shown in Figure 4c, luciferase assay revealed that the luciferase intensity of the pRL-P1 was considerably elevated (unpaired t-test, p < 0.01) than that of the other transfected groups. We identified that the core promoter region of FUT3 was found at –500–(–200) bp (chr. 2: g.73171117–g.73171616). Based on sequence, we designed the methylation amplification region (Figure 4d) using the MethPrimer software.

Figure 4. Identification of pig FUT3 gene core promoter region. (a) prediction of FUT3 core promoter region and truncation of detection fragment. (b) Agarose gel electrophoresis of PCR products that were digested by restriction enzymes. Lane 1: Plasmid digested by KpnI and HindIII, Lane 2: Plasmid DNA, Lane M: DL5000 Marker. (c) Luciferase assay of different vectors. Basic: negative control. Results are indicated as mean± SEM, n = 3 biological replicates, ** p < 0.01. (d) Methylation amplification fragment was designed using the MethPrimer software. Box labeled means the methylated CpG sites, below box means the predicted transcription factors.

Furthermore, we conducted the methylation analysis of the FUT3 promoter in intestinal tissues of individuals that were resistant and sensitive to E. coli F18 via bisulfite amplicon sequencing (BSAS). As shown in Figure 5a, we detected nine CpG sites methylated in pig FUT3 core promoter. On the whole, the average methylation levels in the duodenum (78.25%) and jejunum (86.63%) tissues from E. coli F18-resistant individuals were considerably elevated than that in the duodenum (72.20%) and jejunum (75.15%) tissues from E. coli F18-sensitive individuals (p < 0.05). From single CpG sites, the methylation levels of mC-5 and mC-6 sites in intestinal tissues from individuals that were resistant to E. coli F18 have considerably elevated than that from E. coli F18-sensitive individuals (p < 0.05 or p < 0.01, Figure 5b,c). Interestingly, Pearson correlation analysis revealed that the methylation level of the CpG sites remained in a negative correlation with the mRNA expression of FUT3 (Figure 5d, R = −0.643, p < 0.05), with considerable correlation coefficients being attained only for the mC-3 (R = −0.53, p < 0.05), mC-5 (R = −0.818, p < 0.01) and mC-6 sites (R = −0.83, p < 0.01), indicating the methylation of CpG-(3, 5, 6) sites probably inhibit FUT3 mRNA expression.

Figure 5. Effect of pig FUT3 promoter methylation level on gene expression. (a) The methylation level of the identified CpG sites. 1~9 mean the different CpG sites, accordingly. CpG sites are observed with pie charts in which the black region shows the level of methylation. AV: the average degree of methylation. (b) Differential methylation analysis in jejunum tissues from E. coli F18-sensitive and -resistant individuals. (c) Differential methylation analysis in duodenum tissues from E. coli F18-sensitive and -resistant individuals. Results are indicated as mean± SEM, * p < 0.05, ** p < 0.01. (d) Correlation analysis between the FUT3 methylation and mRNA expression level.

5. Evaluation of Key Transcription Factors in Pig FUT3 Core Promoter

To further identify the important transcription factors in regulating FUT3 expression, we presented the potential transcription factor binding sites (TFBS) found within CpG sites in the core promoter region of pig FUT3 genes, such as SP1, HIF1A, AP2, USF, C/EBPα, and CREB (Figure 4d). In the above study, we found a significant correlation between the methylation level of CpG-(3, 5, 6) sites and FUT3 mRNA expression (Figure 5d). Furthermore, mC-3, mC-5, and mC-6 were located in the HIF1A (hypoxia-inducible factor 1-α), SP1 (specificity protein 1), and USF (upstream stimulatory factor) transcription factor binding site, which reveals their significant contribution in facilitating expression level of FUT3 by influencing the interaction of TFs with a sequence of the promoter. Herein, we performed dual luciferase activity assay to investigate the effects of HIF1A, SP1, and USF on transcriptional activity in the FUT3 promoter. As shown in Figure 6, HIF1A and SP1 could promote FUT3 transcriptional activity, while USF led to the inhibition of transcriptional activity.

Figure 6. Effects of HIF1A, SP1, and USF on FUT3 transcriptional activity by dual-luciferase activity assay. The obtained results are indicated as mean± SEM, *** p < 0.001.

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