A functional mutation associated with piglet diarrhea partially by regulating the transcription of porcine STAT3

Abstract

The present study aimed to search for functional mutations within the promoter of porcine STAT3 and to provide causative genetic variants associated with piglet diarrhea. We firstly confirmed that STAT3 expressed higher in the small intestine than in the spleen, stomach and large intestine of SPF piglets, respectively (P < 0.05). Then, 10 genetic variations in the porcine STAT3 promoter region was identified by direct sequencing. Among them, three mutations SNP1: g.-870 G>A, SNP2: g.-584 A>C and a 6-bp Indel in the promoter region that displayed significant differential transcriptional activities were identified. Association analyses showed that SNP1: g.-870 G>A was significantly associated with piglet diarrhea (P < 0.05) and the GG animals had lower diarrhea score than AA piglets (P < 0.01) in both Min and Landrace population. Further functional analysis revealed that E2F6 repressed the transcriptional efficiency of STAT3 in vitro, by binding the G allele of SNP1. The present study suggested that SNP1: g.-870 G>A was a piglet diarrhea-associated variant that directly affected binding with E2F6, leading to changes in STAT3 transcription which might partially contribute to piglet diarrhea susceptibility or resistance.

Keywords: STAT3; piglet diarrhea; polymorphism; porcine; promoter.

Figure 1

The mRNA expression of porcine STAT3 in the tissues of the 28-day SPF piglets (n = 3). The tissues expression data was analyzed by one-way ANOVA. Different capital letter indicated significant difference (P < 0.01); different lowercase letter indicated significant difference (P < 0.05). Data are presented as means ± SD.

Figure 2

Sequence and genotyping results of SNP1 in porcine STAT3 promoter region. (A,B) show the sequencing results of SNP1 in the promoter region of porcine STAT3. (C) The enforced MspI PCR-RFLP assay for SNP1. Lane M molecular marker DL 2,000.

Figure 3

The effects of candidate genetic variations on porcine STAT3 transcriptional activity. (A) Schematic presentation of luciferase reporter construct for SNP1, SNP2, and SNP3. Genomic fragments carrying the SNPs were inserted the upstream of the SV40 promoter of the pGL3 vector. SNPs were named and numbered from the first nucleotide of the first Exon of porcine STAT3 (ENSSSCT00000018944.5) which was assumed as the putative transcriptional initial site and assigned as +1. (B) Luciferase assays of different SNPs luciferase reporter constructs in IPEC-J2 cells. (C) Schematic presentation of luciferase reporter constructs of the 6-bp Indel. (D) Luciferase assays of the 6-bp Indel in IPEC-J2 cells. Values are shown as the mean ± SD (n = 3). **P < 0.01.

Figure 4

Effect of E2F4 and E2F6 on transcriptional activity of STAT3 gene carrying SNP1. (A) IPEC-J2 cells were transfected with the luciferase reporter construct containing A or G at STAT3 SNP1 and E2F4 expression vector. (B) IPEC-J2 cells were transfected with the luciferase reporter construct containing A or G at STAT3 SNP1 and E2F46 expression vector. Relative luciferase activity was given as firefly activity over Renilla activity. Values are shown as the mean ± SD (n = 3). **P < 0.01.

Figure 5

In vitro analysis of the interaction between transcription factors and STAT3 gene carrying SNP1. (A) Over expression of porcine E2F4 in IPEC-J2. Lane M: marker; Lane 1: IPEC-J2 transfected with pCMA-HA-E2F4; Lane 2: IPEC-J2 transfected with pCMA-HA. (B) Over expression of porcine E2F6 in IPEC-J2. Lane M: marker; Lane 1: IPEC-J2 transfected with pCMA-HA-E2F6; Lane 2: IPEC-J2 transfected with pCMA-HA. (C) The binding of E2F4 on the promoter of STAT3 gene. (D) The binding of E2F6 on the promoter of STAT3 gene. Arrows represent the free probe and the specific band for the GG probe. Lane 1: Free probe containing the G allele is observed. Lane 2: Binding of nuclear proteins is observed with the probe containing the G allele. Lane 3: Binding was competed by unlabeled probe.