Identification of Critical Elements for Regulation of Inorganic Pyrophosphatase (PPA1) in MCF7 Breast Cancer Cells.
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.
Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.
Dipti Ranjan
DR
Mishra
Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.
Sanjib
S
Chaudhary
Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.
B Madhu
BM
Krishna
Cancer Biology Laboratory, Gene function and regulation Group, Institute of Life Sciences, Bhubaneswar, Odisha, India.
Sandip K
SK
Mishra
D024506:5' Flanking Region; D000107:Acetylation; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D042002:Chromatin Assembly and Disassembly; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D006657:Histones / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D043564:Inorganic Pyrophosphatase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D015854:Up-Regulation; D050880:p300-CBP Transcription Factors / Q000378:metabolism
D006657:Histones; D006877:Hydroxamic Acids; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; C012589:trichostatin A; D050880:p300-CBP Transcription Factors; D043564:Inorganic Pyrophosphatase