Identification of DNA-binding proteins that interact with the 5′-flanking region of the human d-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry

• Pull-down assay coupled with 2-dimensional gel electrophoresis and MS was conducted.
• Six DNA-binding proteins of the 5′-flanking region of the hDAO gene were identified.
• Potential targets for studies on gene regulatory mechanism of hDAO are presented.

d-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5′-flanking region of this gene (−4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. 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 human DAO 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). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.

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