Search for the regulation of human D-amino acid oxidase gene, a potential risk factor of schizophrenia

Introduction: D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes certain D-amino acids, proposed to play a main role in the degradation of D-serine, an allosteric modulator of the N -methyl-D-aspartate-type glutamate receptor. DAO is also suggested to be one of potential candidate genes involved in known biological pathways implicated in the pathogenesis of schizophrenia. Until now, the DAO expression mechanism is still unclear. Aim: Investigating the regulation of gene expression of DAO. Results & Discussion: Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (-237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4126/+4929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1163/+1940) in intron1; that would act in concert with P1 and P2. Bioinformatics analyses elucidated the conservation of transcription factor PAX5 family binding sites among species. These sites (-60/-31) and (+4464/+4493), locate in P1 and P2 of hDAO, respectively. Gel shift assays demonstrated P1 contains a site (-60/-31) for PAX5 binding while P2 has three sites for both PAX2 and PAX5 binding. The dual roles of PAX5 family in regulating hDAO transcription by modulating promoter activity of P1 and activating promoter activity of P2 were implicated based on the site-directed mutagenesis experiment. Our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5. Furthermore, by using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5’-flanking region of the hDAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). Altogether, our findings will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.