Identification of two promoters for human D-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2

• Published in: Journal of biochemistry (Tokyo) Vol. 157; no. 5; pp. 377 - 387
• Main Authors: Tran, Diem Hong, Shishido, Yuji, Chung, Seong Pil, Trinh, Huong Thi Thanh, Yorita, Kazuko, Sakai, Takashi, Fukui, Kiyoshi
• Format: Journal Article
• Language: English
• Published: England 01.05.2015
• Subjects: Animals; Base Sequence; D-Amino-Acid Oxidase - genetics; Humans; LLC-PK1 Cells; Molecular Sequence Data; PAX2 Transcription Factor - genetics; PAX5 Transcription Factor - genetics; Promoter Regions, Genetic; Sequence Homology, Nucleic Acid; Swine; transcription factor; promoter; PAX5; d-amino acid oxidase; PAX2
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvu084

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids. Until now, the DAO expression mechanism is still unclear. 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 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) 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 (+4,464/+4,493), 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 paired box gene 2 (PAX2) and paired box gene 5 (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. Altogether, our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5.