Characterization of regulatory elements in the 5 '-flanking region of the GM2 activator gene

Lysosomal degradation of the ganglioside GM2 by human beta-hexosaminidase A requires the presence of the GM2 activator protein as an essential cofacto r. Here we demonstrate that GM2 activator mRNA is differentially expressed and mainly localized to the apical part of the epithelial cells of distal r enal tubules and the collecting duct. In order to understand the mechanism underlying the regulation of the GM2 a ctivator gene, we analyzed the genomic organization upstream exon 2 as well as the 5'-flanking region. The GM2 activator gene spans about 16.8 kb with a first intron of 6.5 kb, and the transcription start is located at positi on -96 upstream from the ATG. DNA elements responsible for GM2 activator ex pression were identified in a PCR-based method of long-distance DNA walking . Sequence analysis revealed a 2.9 kb region upstream of the ATG that conta ined regulatory elements like CAAT boxes, Spl binding sites as well as AP1, and AP2 sites. Transfection experiments in COS-1 cells with a series of ch imeras of 5'-stepwise deletion mutants of the GM2 activator gene 5'-flankin g region and the secretory alkaline phosphatase (SEAP)-reporter gene indica ted that a genomic fragment encompassing -323 to +1bp had significant promo ter activity. EMSA experiments showed that Spl and other transcription fact ors like AP1, AP2 and CCAAT-Box binding proteins are involved in GM2 activa tor gene regulation.