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

Yotam N. Blech-Hermoni, Shira G. Ziegler, Kathleen S. Hruska, Barbara K. Stubblefield, Mary E. LaMarca, Matthew E. Portnoy, Eric D. Green, Ellen Sidransky

Research output: Contribution to journalArticlepeer-review

Abstract

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 (∼39.4 kb) 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.

Original languageEnglish (US)
Pages (from-to)275-282
Number of pages8
JournalMolecular genetics and metabolism
Volume99
Issue number3
DOIs
StatePublished - Mar 2010
Externally publishedYes

Keywords

  • Gaucher disease
  • Glucocerebrosidase
  • Luciferase assays
  • Multi-species sequence comparisons
  • Transcriptional regulation

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Endocrinology

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