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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 altered CTCF activity on Pax6 expression in ES cells.(A) Effect of knocking down CTCF mRNA on Pax6 expression. Statistical analysis of suppressed CTCF expression (B) and increased Pax6 expression (C). Data was shown as mean ±S.E. Symbol “*” indicates significant differences (p<0.05, n = 4).
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pone-0020954-g002: Effect of altered CTCF activity on Pax6 expression in ES cells.(A) Effect of knocking down CTCF mRNA on Pax6 expression. Statistical analysis of suppressed CTCF expression (B) and increased Pax6 expression (C). Data was shown as mean ±S.E. Symbol “*” indicates significant differences (p<0.05, n = 4).

Mentions: Our previous studies have demonstrated that CTCF is a negative regulator of Pax6 gene in the cornea and retina. To study whether CTCF also involves regulation of Pax6 in mouse ES cells, CTCF-specific siRNA was transfected to ES cells to knock down CTCF mRNA. We found that expression of CTCF at the protein level was decreased about 75% in ES cells transfected with CTCF-specific siRNA at 72 h (Fig. 2A&2B). At the same time (72 h), expression of Pax6 protein was significantly increased following knockdown of CTCF (Fig. 2A&2C). For control experiments, ES cells were transfected with non-silencing control siRNA following the same procedure and time course as the experimental group. The results demonstrate that altered levels of CTCF by knockdown of CTCF mRNA in ES cells result in up-regulation of 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 altered CTCF activity on Pax6 expression in ES cells.(A) Effect of knocking down CTCF mRNA on Pax6 expression. Statistical analysis of suppressed CTCF expression (B) and increased Pax6 expression (C). Data was shown as mean ±S.E. Symbol “*” indicates significant differences (p<0.05, n = 4).
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Related In: Results  -  Collection

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pone-0020954-g002: Effect of altered CTCF activity on Pax6 expression in ES cells.(A) Effect of knocking down CTCF mRNA on Pax6 expression. Statistical analysis of suppressed CTCF expression (B) and increased Pax6 expression (C). Data was shown as mean ±S.E. Symbol “*” indicates significant differences (p<0.05, n = 4).
Mentions: Our previous studies have demonstrated that CTCF is a negative regulator of Pax6 gene in the cornea and retina. To study whether CTCF also involves regulation of Pax6 in mouse ES cells, CTCF-specific siRNA was transfected to ES cells to knock down CTCF mRNA. We found that expression of CTCF at the protein level was decreased about 75% in ES cells transfected with CTCF-specific siRNA at 72 h (Fig. 2A&2B). At the same time (72 h), expression of Pax6 protein was significantly increased following knockdown of CTCF (Fig. 2A&2C). For control experiments, ES cells were transfected with non-silencing control siRNA following the same procedure and time course as the experimental group. The results demonstrate that altered levels of CTCF by knockdown of CTCF mRNA in ES cells result in up-regulation of 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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