title: NonO binds to the CpG island of oct4 promoter and functions as a transcriptional activator of oct4 gene expression.
abstract: We investigated the relationship between oct4 gene expression patterns and CpG sites methylation profiles during ES cell differentiation into neurons, and identified relevant binding factor. The oct4 gene expression level gradually declined as ES cell differentiation progressed, and the CpG sites in the oct4 proximal enhancer (PE) and promoter regions were methylated in concert with ES cell differentiation. An electro-mobility shift assay (EMSA) showed that putative proteins bind to CpG sites in the oct4 PE/promoter. We purified CpG binding proteins with DNAbinding purification method, and NonO was identified by liquid chromatography-mass spectrometry. EMSA with specific competitors revealed that NonO specifically binds to the conserved CCGGTGAC sequence in the oct4 promoter. Methylation at a specific cytosine residue (CC* GGTGAC) reduced the binding affinity of NonO for the recognition sequence. Chromatin immunoprecipitation analysis confirmed that NonO binds to the unmethylated oct4 promoter. There were no changes in the NonO mRNA and protein levels between ES cells and differentiated cells. The transcriptional role of NonO in oct4 gene expression was evaluated by luciferase assays and knockdown experiments. The luciferase activity significantly increased threefold when the NonO expression vector was cotransfected with the NonO recognition sequence, indicating that NonO has a transcription activator effect on oct4 gene expression. In accordance with this effect, when NonO expression was inhibited by siRNA treatment, oct4 expression was also significantly reduced. In summary, we purified NonO, a novel protein that binds to the CpG island of oct4 promoter, and positively regulates oct4 gene expression in ES cells.
authors.affiliation: Graduate School of Medicine, Korea University, Seoul, Korea.
authors.forename: Yoojin
authors.initials: Y
authors.lastname: Park
authors.forename: Ja-Myong
authors.initials: JM
authors.lastname: Lee
authors.forename: Min-Young
authors.initials: MY
authors.lastname: Hwang
authors.forename: Gi-hoon
authors.initials: GH
authors.lastname: Son
authors.forename: Dongho
authors.initials: D
authors.lastname: Geum
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D007124:Immunoenzyme Techniques; D008156:Luciferases; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins; D060888:Real-Time Polymerase Chain Reaction; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; C500331:Nono protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D008156:Luciferases