title: Two c-Myc binding sites are crucial in upregulating the expression of human phospholipid scramblase 1 gene.
abstract: Human phospholipid scramblase 1 (hPLSCR1) is a type II endofacial membrane protein which mediates bi-directional transport of phospholipids across the plasma membrane. hPLSCR1, a multifunctional protein with variety of roles in apoptosis, tumor progression, cell signaling and anti-viral defense. The expression of such a multifunctional protein should be under tight regulation. Apart from a single report showing snail mediated down regulation of hPLSCR1, the molecular mechanisms regulating the expression of scramblases are not well elucidated. In this study we identified c-Myc as a transcriptional regulator of hPLSCR1. Transcription factor prediction tool ConSite predicted three binding sites for c-Myc. Reporter gene assays and western blot analysis revealed c-Myc mediated up regulation of hPLSCR1 expression. Deletion construct -790 lacking one c-Myc binding site showed a 27% decrease in promoter activity while deletion construct -469 lacking two c-Myc binding sites showed a 62% decrease in promoter activity. Site directed mutagenesis revealed the importance of c-Myc binding sites from -751 to -756 and -548 to -553 on the promoter of hPLSCR1in transcriptionally regulating the expression of hPLSCR1. The results were further confirmed by shRNA mediated knock down of endogenous c-Myc and in vivo interactions by ChIP assay.
authors.affiliation: Applied and Industrial Microbiology Lab, Bhupat and Jyoti Mehta School of Biosciences, Department of Biotechnology, IIT Madras, Chennai, 600036, India.
authors.forename: Janaki Manoja
authors.initials: JM
authors.lastname: Vinnakota
authors.affiliation: Applied and Industrial Microbiology Lab, Bhupat and Jyoti Mehta School of Biosciences, Department of Biotechnology, IIT Madras, Chennai, 600036, India. Electronic address: gummadi@iitm.ac.in.
authors.forename: Sathyanarayana N
authors.initials: SN
authors.lastname: Gummadi
mesh_terms: D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; C116115:MYCBP protein, human; C493422:PLSCR1 protein, human; D049294:Phospholipid Transfer Proteins; D014157:Transcription Factors
keywords: Apoptosis; Human phospholipid scramblase 1; Transcriptional regulation; c-Myc