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Regulation of Pax6 by CTCF during induction of mouse ES cell differentiation.

Gao J, Wang J, Wang Y, Dai W, Lu L - PLoS ONE (2011)

Bottom Line: Instead, the interaction of CTCF with Pax6 gene was regulated by decreased CTCF occupancy in its binding motifs upstream from Pax6 P0 promoter following the course of ES cell differentiation.Furthermore, changes in DNA methylation levels in vitro and in vivo effectively altered methylation status of these identified CpG sites, which affected ability of CTCF to interact with the P0 promoter, resulting in increases in Pax6 expression.We conclude that there is an epigenetic mechanism involving regulations of Pax6 gene during ES cell differentiation to neural stem cells, which is through increases or decreases in methylation levels of Pax6 gene to effectively alter the ability of CTCF in control of Pax6 expression, respectively.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Torrance, California, United States of America.

ABSTRACT
Pax6 plays an important role in embryonic cell (ES) differentiation during embryonic development. Expression of Pax6 undergoes from a low level to high levels following ES cell differentiation to neural stem cells, and then fades away in most of the differentiated cell types. There is a limited knowledge concerning how Pax6 is regulated in ES cell differentiation. We report that Pax6 expression in mouse ES cells was controlled by CCCTC binding factor (CTCF) through a promoter repression mechanism. Pax6 expression was significantly enhanced while CTCF activity was kept in the constant during ES cell differentiation to radial glial cells. Instead, the interaction of CTCF with Pax6 gene was regulated by decreased CTCF occupancy in its binding motifs upstream from Pax6 P0 promoter following the course of ES cell differentiation. Reduced occupancy of CTCF in the binding motif region upstream from the P0 promoter was due to increased DNA methylations in the CpG sites identified in the region. Furthermore, changes in DNA methylation levels in vitro and in vivo effectively altered methylation status of these identified CpG sites, which affected ability of CTCF to interact with the P0 promoter, resulting in increases in Pax6 expression. We conclude that there is an epigenetic mechanism involving regulations of Pax6 gene during ES cell differentiation to neural stem cells, which is through increases or decreases in methylation levels of Pax6 gene to effectively alter the ability of CTCF in control of Pax6 expression, respectively.

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Effect of DNA methylation or de-methylation on Pax6 gene expression.(A) Effect of decreasing DNA methylation on Pax6 gene expression in ES cells. ES cells were treated with 1 µM 5-azadCyd for 24 and 48 h and subjected to Western analysis to compare the Pax6 expression w/wo 5-azadCyd-mediated de-methylation treatment. (B) Effect of increasing DNA methylation on Pax6 gene expression in ES cells. ES cells were transfected with full-length cDNA encoding Dmnt3a for 24–72 h and protein levels of Pax6, CTCF, Dmnt3a and β-actin were determined by Western analysis. (C) Data was shown as mean ±S.E. obtained from three independent experiments. Symbol “*” indicates significant differences between treated and un-treated groups.
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pone-0020954-g007: Effect of DNA methylation or de-methylation on Pax6 gene expression.(A) Effect of decreasing DNA methylation on Pax6 gene expression in ES cells. ES cells were treated with 1 µM 5-azadCyd for 24 and 48 h and subjected to Western analysis to compare the Pax6 expression w/wo 5-azadCyd-mediated de-methylation treatment. (B) Effect of increasing DNA methylation on Pax6 gene expression in ES cells. ES cells were transfected with full-length cDNA encoding Dmnt3a for 24–72 h and protein levels of Pax6, CTCF, Dmnt3a and β-actin were determined by Western analysis. (C) Data was shown as mean ±S.E. obtained from three independent experiments. Symbol “*” indicates significant differences between treated and un-treated groups.

Mentions: Effects of altered DNA methylation on Pax6 expression in 5-azadCyd-treated and Dmnt3a-transfected cells were monitored by Western analysis. Treatment of 5-azadCyd induced DNA de-methylation down-regulated Pax6 expression (Fig. 7A). In contrast, over-expression of Dnmt3a by transfecting cells with full-length cDNA encoding Dnmt3 up-regulated Pax6 expression following a time course (Fig. 7B). Statistical analysis showed that Pax6 expression was significantly increased after over-expression of Dnmt3. The result is consistent with the observation that there was an increased DNA methylation in the CTCF binding motifs in the Pax6 promoter region, and that there was an increased Pax6 expression on the 8th day of EB formation during ES cell differentiation to radial glial cells. Taken together, our data support the notion that there are increased DNA methylation in the region of CTCF binding motifs upstream from the Pax6 promoter can exclude CTCF binding in the region during the period of ES cell differentiation to radial glial cells, which abolishes the repressor effect of CTCF on Pax6 promoter activity, resulting in an enhanced Pax6 expression.


Regulation of Pax6 by CTCF during induction of mouse ES cell differentiation.

Gao J, Wang J, Wang Y, Dai W, Lu L - PLoS ONE (2011)

Effect of DNA methylation or de-methylation on Pax6 gene expression.(A) Effect of decreasing DNA methylation on Pax6 gene expression in ES cells. ES cells were treated with 1 µM 5-azadCyd for 24 and 48 h and subjected to Western analysis to compare the Pax6 expression w/wo 5-azadCyd-mediated de-methylation treatment. (B) Effect of increasing DNA methylation on Pax6 gene expression in ES cells. ES cells were transfected with full-length cDNA encoding Dmnt3a for 24–72 h and protein levels of Pax6, CTCF, Dmnt3a and β-actin were determined by Western analysis. (C) Data was shown as mean ±S.E. obtained from three independent experiments. Symbol “*” indicates significant differences between treated and un-treated groups.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113856&req=5

pone-0020954-g007: Effect of DNA methylation or de-methylation on Pax6 gene expression.(A) Effect of decreasing DNA methylation on Pax6 gene expression in ES cells. ES cells were treated with 1 µM 5-azadCyd for 24 and 48 h and subjected to Western analysis to compare the Pax6 expression w/wo 5-azadCyd-mediated de-methylation treatment. (B) Effect of increasing DNA methylation on Pax6 gene expression in ES cells. ES cells were transfected with full-length cDNA encoding Dmnt3a for 24–72 h and protein levels of Pax6, CTCF, Dmnt3a and β-actin were determined by Western analysis. (C) Data was shown as mean ±S.E. obtained from three independent experiments. Symbol “*” indicates significant differences between treated and un-treated groups.
Mentions: Effects of altered DNA methylation on Pax6 expression in 5-azadCyd-treated and Dmnt3a-transfected cells were monitored by Western analysis. Treatment of 5-azadCyd induced DNA de-methylation down-regulated Pax6 expression (Fig. 7A). In contrast, over-expression of Dnmt3a by transfecting cells with full-length cDNA encoding Dnmt3 up-regulated Pax6 expression following a time course (Fig. 7B). Statistical analysis showed that Pax6 expression was significantly increased after over-expression of Dnmt3. The result is consistent with the observation that there was an increased DNA methylation in the CTCF binding motifs in the Pax6 promoter region, and that there was an increased Pax6 expression on the 8th day of EB formation during ES cell differentiation to radial glial cells. Taken together, our data support the notion that there are increased DNA methylation in the region of CTCF binding motifs upstream from the Pax6 promoter can exclude CTCF binding in the region during the period of ES cell differentiation to radial glial cells, which abolishes the repressor effect of CTCF on Pax6 promoter activity, resulting in an enhanced Pax6 expression.

Bottom Line: Instead, the interaction of CTCF with Pax6 gene was regulated by decreased CTCF occupancy in its binding motifs upstream from Pax6 P0 promoter following the course of ES cell differentiation.Furthermore, changes in DNA methylation levels in vitro and in vivo effectively altered methylation status of these identified CpG sites, which affected ability of CTCF to interact with the P0 promoter, resulting in increases in Pax6 expression.We conclude that there is an epigenetic mechanism involving regulations of Pax6 gene during ES cell differentiation to neural stem cells, which is through increases or decreases in methylation levels of Pax6 gene to effectively alter the ability of CTCF in control of Pax6 expression, respectively.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Torrance, California, United States of America.

ABSTRACT
Pax6 plays an important role in embryonic cell (ES) differentiation during embryonic development. Expression of Pax6 undergoes from a low level to high levels following ES cell differentiation to neural stem cells, and then fades away in most of the differentiated cell types. There is a limited knowledge concerning how Pax6 is regulated in ES cell differentiation. We report that Pax6 expression in mouse ES cells was controlled by CCCTC binding factor (CTCF) through a promoter repression mechanism. Pax6 expression was significantly enhanced while CTCF activity was kept in the constant during ES cell differentiation to radial glial cells. Instead, the interaction of CTCF with Pax6 gene was regulated by decreased CTCF occupancy in its binding motifs upstream from Pax6 P0 promoter following the course of ES cell differentiation. Reduced occupancy of CTCF in the binding motif region upstream from the P0 promoter was due to increased DNA methylations in the CpG sites identified in the region. Furthermore, changes in DNA methylation levels in vitro and in vivo effectively altered methylation status of these identified CpG sites, which affected ability of CTCF to interact with the P0 promoter, resulting in increases in Pax6 expression. We conclude that there is an epigenetic mechanism involving regulations of Pax6 gene during ES cell differentiation to neural stem cells, which is through increases or decreases in methylation levels of Pax6 gene to effectively alter the ability of CTCF in control of Pax6 expression, respectively.

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