Promoter regulation of the visinin-like subfamily of neuronal calcium sensor proteins by nuclear respiratory factor-1

Abstract

VILIP-1 (gene name VSNL1), a member of the neuronal Ca2+ sensor protein family, acts as a tumor suppressor gene by inhibiting cell proliferation, adhesion, and invasiveness. VILIP-1 expression is down-regulated in several types of human cancer. In human non-small cell lung cancer, we found that down-regulation was due to epigenetic changes. Consequently, in this study we analyzed the VSNL1 promoter and its regulation. Serial truncation of the proximal 2-kb VSNL1 promoter (VP-1998) from its 5′ terminus disclosed that the last 3′ terminal 100-bp promoter fragment maintained similar promoter activity as compared with VP-1998 and therefore was referred to as VSNL1 minimal promoter. When the 5′ terminal 50 bp were deleted from the minimal promoter, the activity was dramatically decreased, suggesting that the deleted 50 bp contained a potential cis-acting element crucial for promoter activity. Deletion and site-directed mutagenesis combined with in silico transcription factor binding analysis of VSNL1 promoter identified nuclear respiratory factor (NRF)-1/α-PAL as a major player in regulating VSNL1 minimal promoter activity. The function of NRF-1 was further confirmed using dominant-negative NRF-1 overexpression and NRF-1 small interfering RNA knockdown. Electrophoretic mobility shift assay and chromatin immunoprecipitation provided evidence for direct NRF-1 binding to the VSNL1 promoter. Methylation of the NRF-1-binding site was found to be able to regulate VSNL1promoter activity.Ourresults further indicated that NRF-1 couldbea regulatory factor forgeneexpression of the other visininlike subfamily members including HPCAL4, HPCAL1, HPCA, and NCALD. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.