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.

Keywords: CpG sites, ES cells, methylation, NonO, Oct4