NonO Binds to the CpG Island of oct4 Promoter and Functions as a Transcriptional Activator of oct4 Gene Expression|RISS 상세보기

다국어 초록 (Multilingual Abstract)

We investigated the relationship between oct4 gene ex-pression 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 DNA-binding 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 co-transfected 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.

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참고문헌 (Reference)

1 Dong, X, "p54nrb is a transcriptional corepressor of the progesterone receptor that modulates transcription of the laborassociated gene, connexin 43 (Gja1)" 23 : 1147-1160, 2009

2 Ishitani, K, "p54nrb acts as a transcriptional coactivator for activation function 1 of the human androgen receptor" 306 : 660-665, 2003

3 Kameoka, S, "p54(nrb) associates with the 5′ splice site within large transcription/splicing complexes" 23 : 1782-1791, 2004

4 Yu, H.B, "Zfp206, Oct4, and Sox2 are integrated components of a transcriptional regulatory network in embryonic stem cells" 284 : 31327-31335, 2009

5 Yang, H.M, "Transcriptional regulation of human Oct4 by steroidogenic factor-1" 101 : 1198-1209, 2007

6 Sewer, M.B, "Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription" 143 : 1280-1290, 2002

7 Hwang, M, "The neuronal differentiation potential of Ldb1-null mutant embryonic stem cells is dependent on extrinsic influences" 26 : 1490-1495, 2008

8 Basu, A, "The intracisternal A-particle proximal enhancer-binding protein activates transcription and is identical to the RNA- and DNA-binding protein p54nrb/NonO" 17 : 677-686, 1997

9 Zhang, Z, "The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs" 106 : 465-475, 2001

10 Straub, T, "The RNA-splicing factor PSF/p54 controls DNA-topoisomerase I activity by a direct interaction" 273 : 26261-26264, 1998