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J. Gao, T. Li, L. Lu; Role of DNA Methylation in CTCF–Regulated Pax6 Transcription . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3129.
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© ARVO (1962-2015); The Authors (2016-present)
Homeobox gene Pax6 plays a predominant role in the development of the eye. We found previously that CTCF (CCCTC binding factor) controls Pax6 transcription by binding to a repressor element upstream from Pax6 P0 promoter. The present study is to further investigate the effect of DNA methylation on CTCF–regulated Pax6 transcription.
Mouse J1 embryonic stem (ES) cells were cultured in serum–free medium without feeder cells. Microarray and Western analysis were performed to compare ES cells with/without treatment of 5–AzadCyd (5–Aza–2’–deoxycytidine), a demethylation reagent. On the other hand, ES cells were transfected by electroporation with cDNA encoding mouse Dnmt 3a (DNA methyltransferase). Expression levels of CTCF, Dnmt 3a and Pax6 proteins were detected by Western blot with specific antibodies.
Demethylation induced by 5–AzadCyd treatment in ES cells resulted in significant changes in imprinting genes including: H19 and IGF–2. Demethylation treatment in ES cells did not affect CTCF mRNA expression, however, it caused a 2.5–fold decrease in Pax6 mRNA level. Over–expression of CTCF in ES cells markedly suppressed Pax6 expression. Pax6 expression in CTCF–transfected ES cells was further suppressed by demethlation treatment. In contrast, over–expression of Dnmt 3a in ES cells enhanced Pax6 expression and the effect of Dnmt 3a on Pax6 expression was diminished by treatment of 5–AzadCyd. In addition, co–expression of CTCF and Dnmt 3a in ES cells resulted in further inhibition of Pax6 expression, indicating that CTCF regulates Pax6 expression is through a methylation–sensitive mechanism.
CTCF–controlled Pax6 transcription in ES cells may play an important role in directing differentiation of these cells toward to the eye–related cells. The genetic control of CTCF in Pax6 expression is through a DNA methylation mechanism similar to those imprinting genes.
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