Transcriptional Regulation of HMOX1 Gene in Hezuo Tibetan Pigs: Roles of WT1, Sp1, and C/EBPα

Abstract

Heme oxygenase 1 (HMOX1) is a stress-inducing enzyme with multiple cardiovascular protective functions, especially in hypoxia stress. However, transcriptional regulation of swine HMOX1 gene remains unclear. In the present study, we first detected tissue expression profiles of HMOX1 gene in adult Hezuo Tibetan pig and analyzed the gene structure. We found that the expression level of HMOX1 gene was highest in the spleen of the Hezuo Tibetan pig, followed by liver, lung, and kidney. A series of 5' deletion promoter plasmids in pGL3-basic vector were used to identify the core promoter region and confirmed that the minimum core promoter region of swine HMOX1 gene was located at -387 bp to -158 bp region. Then we used bioinformatics analysis to predict transcription factors in this region. Combined with site-directed mutagenesis and RNA interference assays, it was demonstrated that the three transcription factors WT1, Sp1 and C/EBPα were important transcription regulators of HMOX1 gene. In summary, our study may lay the groundwork for further functional study of HMOX1 gene.

Keywords: HMOX1 gene; Hezuo Tibetan pig; promoter; transcriptional regulation.

Figure 1

Relative expression patterns of Hezuo Tibetan pig HMOX1 gene in different tissues. The result was normalized with β-actin gene and relative to gene expression in the duodenum group. ** indicates p < 0.01.

Figure 2

Promoter region cloning and bioinformatics analysis. (A) PCR amplification product of HMOX1 gene promoter region. M, DL2000 DNA marker; 1, PCR product. (B) Genomic structure of HMOX1 gene. (C) The predict results of CpG island in HMOX1 gene. (D) The promoter sequence information of HMOX1 gene. The box represents the transcription factor binding sites, and the 4 bp core binding sites are shown in red. The underline represents the amplification primer and the arrow represents the transcription start site (TSS).

Figure 3

Construction of different deletion vectors of HMOX1 gene promoter region and luciferase activity analysis. (A) Luciferase activity analysis of promoter with different deletion vectors (pGL3-1878/+115, pGL3-1617/+115, pGL3-919/+115, pGL3-387/+115, pGL3-158/+115), pGL3-Basic as a negative control. (B) Double enzyme digestion identification of different deletion vectors. M, DL5000 DNA marker, lanes 1–5 are different deletion fragments. (C) Analysis of luciferase activity in the core promoter region. All ** represents p < 0.01.

Figure 4

Transcription factor prediction and multi-species homology analysis in the core promoter region. (A) Transcription factor prediction of core promoter region. Four essential transcription factors WT1, Sp1, C/EBPα, and c-Ets-1 were predicted in −387/−158, the location of the 4 bp core binding is indicated in red. (B) Sequence alignment and phylogenetic construction of core promoter regions in six species. The box represents the 4 bp core binding site.

Figure 5

Role of WT1, Sp1, and C/EBPα in the transcription regulation of HMOX1 gene. (A) Luciferase activity analysis with site-directed mutagenesis of WT1, Sp1, C/EBPα, and c-Ets-1 sites. Solid and hollow represent wild-type and mutant-type, respectively. (B) Interference efficiency of WT1, Sp1, and C/EBPα. (C) The mRNA expression level of HMOX1 gene by inhibition with si-WT1, si-Sp1, and si-C/EBPα. (D) Luciferase activity after co- transfection of si-WT1, si-Sp1, and si-C/EBPα with pGL3-387/−158. (* indicates p < 0.05, ** indicates p < 0.01).