Department of Animal Science, School of Agriculture and Biology, Shanghai Jiao Tong University, China.
J Appl Genet. 2011 Nov;52(4):451-8. doi: 10.1007/s13353-011-0050-4. Epub 2011 May 17.
Soluble glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays important roles in the synthesis of triacylglycerol and in the glycerol-3-phosphate shutter. Though GPD1 is expressed in most adult tissues, little is known about the regulation of its expression. In this study, we analyzed the characters, organization and core region of the promoter of pig GPD1 gene by in silico analysis and activity detection of deletion mutants. We also identified and testified the negative regulation effect of C/EBP β on pig GPD1 gene by Chromatin immunoprecipitation (ChIP) assay and over-expression experiments in cultured pig kidney cells. Compared to that of human, pig GPD1 gene promoter has three conserved regions and one deletion region. In silico analysis indicated that pig GPD1 promoter was TATA-less with at least 3 CpG islands of over 200 bp in length and over 60% in GC content. The activity detection of deletion mutants suggested that the essential elements required for the optimal promoter activity scatter in the promoter region, while the core promoter region was from -422 bp to -1 bp. Chromatin immunoprecipitation (ChIP) assay results indicated that C/EBP β had plenty of binding sites in pig GPD1 promoter with the common cis-element (5'- TKNNGCAAK -3'). The over-expression examination of C/EBP β showed that the expression of GPD1 was negatively regulated by C/EBP β in pig kidney cells. Overall, our study revealed that the pig GPD1 promoter is a TATA-less promoter, and in promoter region, the binding sites of C/EBP β share common motif of (5'-TKNNGCAAK -3'). We also showed that pig GPD1 gene is regulated negatively by C/EBP β in cultured kidney cells.
可溶性甘油-3-磷酸脱氢酶 1（GPD1，EC 1.1.1.8）在三酰基甘油的合成和甘油-3-磷酸门控中发挥重要作用。尽管 GPD1 在大多数成年组织中表达，但对其表达的调控知之甚少。在这项研究中，我们通过计算机分析和缺失突变体的活性检测，分析了猪 GPD1 基因启动子的特征、组织和核心区。我们还通过染色质免疫沉淀（ChIP）检测和在培养的猪肾细胞中的过表达实验，鉴定并验证了 C/EBPβ对猪 GPD1 基因的负调控作用。与人类相比，猪 GPD1 基因启动子有三个保守区和一个缺失区。计算机分析表明，猪 GPD1 启动子是 TATA 缺失的，至少有 3 个长度超过 200bp、GC 含量超过 60%的 CpG 岛。缺失突变体的活性检测表明，最佳启动子活性所需的基本元件分散在启动子区域，而核心启动子区域位于-422bp 到-1bp。染色质免疫沉淀（ChIP）检测结果表明，C/EBPβ在猪 GPD1 启动子上有大量结合位点，共同的顺式元件为（5'-TKNNGCAAK-3'）。C/EBPβ的过表达检测表明，在猪肾细胞中，C/EBPβ负调控 GPD1 的表达。总的来说，我们的研究表明，猪 GPD1 启动子是一个 TATA 缺失的启动子，在启动子区域，C/EBPβ的结合位点共享共同的基序（5'-TKNNGCAAK-3'）。我们还表明，在培养的肾细胞中，猪 GPD1 基因受 C/EBPβ的负调控。
