Promoter Regulation of the Visinin-like Subfamily of Neuronal Calcium Sensor Proteins by Nuclear Respiratory Factor-1

Jian Fu,Jirong Zhang,Fang Jin,Jamie Patchefsky,Karl-Heinz Braunewell,Andres J Klein-Szanto

doi:10.1074/jbc.m109.049361

Jian Fu, Jirong Zhang + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.049361

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Abstract

VILIP-1 (gene name VSNL1), a member of the neuronal Ca(2+) 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/alpha-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 VSNL1 promoter activity. Our results further indicated that NRF-1 could be a regulatory factor for gene expression of the other visinin-like subfamily members including HPCAL4, HPCAL1, HPCA, and NCALD.

Highlights

Pocalcin (HPCA), VILIP-1, VILIP-2 (HPCAL4), and VILIP-3 (HPCAL1) [1,2,3]
To define the boundaries of the minimal promoter and to identify cis elements that govern the transcriptional activity of VSNL1, we prepared a series of truncation constructs (Fig. 1A) and tested them in a transient luciferase reporter system
To confirm essential requirement of nuclear respiratory factor (NRF)-1 for VILIP-1 transcription was these findings, we introduced point mutations to the sites and corroborated by the observation that Nuclear respiratory factor 1 (NRF-1) silencing led to a tested the mutant constructs in both cell lines (Fig. 3A)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Two non-small cell lung cancer cell lines, NCIH522 and NCI-H520, which express different levels of VILIP-1, and normal human bronchial epithelial cells were cultured as previously described [11]. DNA mixture containing 0.8 ␮g of VSNL1 promoter constructs and 8 ng of pGL4.73, a transfection efficiency control, was diluted in 50 ␮l of Opti-Mem I medium (Invitrogen) and mixed with 2 ␮l of Lipofectamine 2000 diluted in 50 ␮l of Opti-Mem I medium. The DNA binding was conducted at 4 °C for 30 min in a mixture containing 5 ␮g of nuclear extract, 1ϫ gel shift binding buffer, and 1 ␮l of 32P-labeled probe. Methylation of VSNL1 Promoter—The promoter-specific methylation of VSNL1 was performed according to a published protocol [11] In this case we used HhaI DNA methyltransferase (New England Biolabs, Ipswich, MA) in the methylationmodifying treatment. Construction of the Promoter Fragments of NCALD, HPCA, HPCAL4, and HPCAL1—The fragments containing the potential NRF-1-binding site were amplified by PCR using the following primers. For comparing the relative activity of different reporter constructs, unpaired Student’s t test for paired comparisons was performed, and a p value Ͻ0.05 was considered as significant

RESULTS

The Potential Regulation of the Promoter Activity of Other

DISCUSSION