In silico and functional studies of the regulation of the glucocerebrosidase gene

• Published in: Molecular genetics and metabolism Vol. 99; no. 3; pp. 275 - 282
• Main Authors: Blech-Hermoni, Yotam N., Ziegler, Shira G., Hruska, Kathleen S., Stubblefield, Barbara K., LaMarca, Mary E., Portnoy, Matthew E., Green, Eric D., Sidransky, Ellen
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2010
• Subjects: Animals; Base Sequence; Binding Sites; Cattle; Chlorocebus aethiops; Computational Biology - methods; Computer applications; Conserved Sequence; COS Cells; Dogs; Gaucher disease; Gaucher Disease - genetics; Gaucher Disease - physiopathology; Gene Expression Regulation, Enzymologic; Glucocerebrosidase; Glucosylceramidase - chemistry; Glucosylceramidase - genetics; Glucosylceramidase - metabolism; Humans; Luciferase assays; Luciferases, Firefly - genetics; Luciferases, Firefly - metabolism; Mice; Multi-species sequence comparisons; Mutagenesis, Site-Directed; Phenotype; Species Specificity; Transcription Factors - metabolism; Transcription, Genetic; Transcriptional regulation; Transfection; Vertebrates - classification; Vertebrates - genetics; Glucocerebrosidase; Gaucher disease; Multi-species sequence comparisons; Transcriptional regulation; Luciferase assays
• ISSN: 1096-7192; 1096-7206
• DOI: 10.1016/j.ymgme.2009.10.189

In Gaucher disease (GD), the inherited deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within lysosomes. Although almost 300 mutations in the glucocerebrosidase gene (GBA) have been identified, the ability to predict phenotype from genotype is quite limited. In this study, we sought to examine potential GBA transcriptional regulatory elements for variants that contribute to phenotypic diversity. Specifically, we generated the genomic sequence for the orthologous genomic region (∼39.4kb) encompassing GBA in eight non-human mammals. Computational comparisons of the resulting sequences, using human sequence as the reference, allowed the identification of multi-species conserved sequences (MCSs). Further analyses predicted the presence of two putative clusters of transcriptional regulatory elements upstream and downstream of GBA, containing five and three transcription factor-binding sites (TFBSs), respectively. A firefly luciferase (Fluc) reporter construct containing sequence flanking the GBA gene was used to test the functional consequences of altering these conserved sequences. The predicted TFBSs were individually altered by targeted mutagenesis, resulting in enhanced Fluc expression for one site and decreased expression for seven others sites. Gel-shift assays confirmed the loss of nuclear-protein binding for several of the mutated constructs. These identified conserved non-coding sequences flanking GBA could play a role in the transcriptional regulation of the gene contributing to the complexity underlying the phenotypic diversity seen in GD.