关键词: 猪, Sall4, 启动子, 多能性, 转录调控

Abstract: 【Objective】Sall4 (spalt-like transcription factor 4), as a zinc finger transcription factor, plays an important role in establishing and maintaining the pluripotency of embryonic stem cells. To investigate the expression pattern and regulatory mechanism of Sall4, we cloned porcine Sall4 promoter and functionally analyzed the core regulatory domains of this promoter. 【Method】A 2.1 kb fragment of porcine Sall4 promoter was cloned from pig genomic DNA by PCR and was used to construct reporter vectors. The pE2.1 vector was transfected into different kinds of cells to determine the expression of fluorescent protein for cellular specificity. The Sall4 expression in different tissues was detected by real-time RT-PCR. The core regulatory elements of Sall4 promoter, which share homology to known transcription factor binding sites, were analyzed by GPminer, Methprimer and JASPER program. Sall4 promoter and pluripotent factors, including Oct4, Sox2, Klf4, c-Myc, Esrra/b and Smad were co-transfected into 293T cells to determine the activation of Sall4 promoter. A series of deletion fragments with different sizes of Sall4 promoter were produced by PCR and inserted into pGL3-Basic vector. After transient transfection into 293T, dual luciferase assay was used to measure promoter activities. 【Result】 Sall4 has specific expression in pluripotent cell type P19, reproductive cell type CHO and cancerization cell types 293T and Hela. The real-time fluorescent quantitative RT-PCR results showed that the expression of porcine Sall4 was significantly higher in ovary and testis tissues and porcine iPS cells, but there was low expression in brain, heart, liver and muscle tissues, suggesting that Sall4 is a key factor maintaining pluripotency of stem cells. Bioinformatics analysis of Sall4 promoter by JASPER and GPminer programs indicated that cis-acting elements like TATA box, GC box and CAAT box, and the potential binding sites for Oct4, Sox2, Klf4, Myc, Esrra/b, Stat3 and Smad were found within the promoter sequence. We found that Oct4, Sox2 and TGF-beta signal pathway could significantly activate Sall4 promoter, in which Sall4 promoter activity increased three times compared with the control. However, Klf4 could repress Sall4 promoter activity, showing a negative regulatory role. Deletion constructs of pL2.1, pL1.0 and pL0.5 were constructed based on PCR method. The result of dual luciferase assay indicated there was no significant differences in promoter activity between pL2.1 and pL1.0 though pL2.1 retains more than a 1 kb fragment. However, the promoter activity of pL1.0 which is 486 bp larger than pL0.5 increased two times compared to pL0.5. Additionally, the promoter activity in pL0.5 was 8 times over that of the control. These results demonstrate that two core regulatory elements of Sall4 promoter are located within -367 bp to -852 bp region and -1 bp to -366 bp region. 【Conclusion】Sall4 promoter has been cloned and showed tissue-specific expression. Numerous potential transcription factor binding sites and core promoter regulatory region on Sall4 promoter regulated the expression of Sall4 gene. The interaction between these transcription factor and Sall4 gene would be essential for transcriptional regulation of porcine Sall4 gene in pluripotency stem cells.

Key words: porcine, Sall4, promoter, pluripotency, transcriptional regulation