Mishra Dipti Ranjan, Chaudhary Sanjib, Krishna B Madhu, Mishra Sandip K
Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.
PLoS One. 2015 Apr 29;10(4):e0124864. doi: 10.1371/journal.pone.0124864. eCollection 2015.
Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.
胞质无机焦磷酸酶通过水解各种代谢反应副产物形成的无机焦磷酸（PPi）在细胞代谢中发挥重要作用。已知无机焦磷酸酶与各种生物体生长发育相关的重要功能有关。在人类中，无机焦磷酸酶（PPA1）的表达在不同类型的癌症中失调，并参与胃癌细胞的迁移和侵袭以及卵巢癌细胞的增殖。然而，编码PPA1的基因的转录调控知之甚少。为了深入了解PPA1基因调控，克隆并分析了其5'侧翼区域的1217 bp。启动子的5'缺失分析显示，一个266 bp的近端启动子区域表现出大部分转录活性，经序列分析发现该区域存在三个推定的Sp1结合位点。通过电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）分析证实了Sp1与PPA1启动子的结合。通过定点诱变验证了这些结合位点的重要性，Sp1的过表达激活PPA1启动子活性，上调蛋白表达并增加染色质可及性。p300与PPA1启动子结合并刺激Sp1诱导的启动子活性。曲古抑菌素A（TSA），一种组蛋白脱乙酰酶（HDAC）抑制剂，诱导PPA1启动子活性和蛋白表达，并且p300的组蛋白乙酰转移酶（HAT）活性在PPA1表达的调控中很重要。这些结果表明，PPA1受到Sp1的正调控，p300共激活Sp1诱导的PPA1启动子活性，组蛋白乙酰化/去乙酰化可能有助于PPA1启动子上的局部染色质重塑。此外，PPA1的敲低降低了MCF7细胞的集落形成和活力。
