In vivo footprinting analysis of the Glypican 3 (GPC3) promoter region in neuroblastoma cells

Abstract

Glypican 3 (GPC3) is an X-linked gene that has its peak expression during development and is down-regulated in all studied tissues after birth. We have shown that GPC3 was expressed in neuroblastoma and Wilms' tumor. To understand the mechanisms regulating the transcription of this gene in neuroblastoma cells, we have focused our study on the identification of putative transcription factors binding the promoter. In this report we performed in vivo dimethylsulfate, UV type C irradiation and DNaseI footprinting analyses coupled with ligation-mediated PCR on nearly 1000 bp of promoter in two neuroblastoma cell lines, SJNB-7 (expressing GPC3) and SK-N-FI (not expressing GPC3). Nucleosome signature footprints were observed in the most distal part of the studied region in both cell lines. We detected eight large differentially protected regions, suggesting the presence of binding proteins in both cell lines but more DNA–protein interactions in GPC3-expressing cells. Sp1 was previously shown to be able to bind some of these regions. Here by combining electromobility shift assays and chromatin immunoprecipitations we showed that the transcription factor NFY was part of the DNA–protein complex found in footprinted regions upstream of the described minimal promoter. These studies performed on chromatin in situ suggest that NFY and yet unknown cell type-specific factors may play an important role in the regulation of GPC3.