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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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Correlation of CTCF and Pax6 in ES cell differentiation to radial glial cells.(A) Expressions of CTCF and Pax6 mRNAs during ES cell differentiation to radial glial cells. Total RNA samples were harvested from undifferentiated ES cells (0 d) and embryonic body (EB) cells after 4 day or 8 day induction. RT-PCR assays were performed to measure the expression of CTCF, Pax6 and GAPDH. (B) Expressions of CTCF and Pax6 proteins during ES cell differentiation to radial glial cells. Western assays were performed to detect expression levels of CTCF, Pax6 and β-actin. (C) Illustration of ChIP-based PCR designs. (D) Detection of specific protein-nucleotide interaction between CTCF and Pax6 gene during ES cell differentiation and EB formation. Chromatin samples were immuno-pulled down by CTCF-specific antibody. ChIP-based PCR was performed to amplify CTCF binding DNA fragment in Pax6 P0 promoter region. All ChIP and pCR experiments were repeated for at least three times.
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pone-0020954-g003: Correlation of CTCF and Pax6 in ES cell differentiation to radial glial cells.(A) Expressions of CTCF and Pax6 mRNAs during ES cell differentiation to radial glial cells. Total RNA samples were harvested from undifferentiated ES cells (0 d) and embryonic body (EB) cells after 4 day or 8 day induction. RT-PCR assays were performed to measure the expression of CTCF, Pax6 and GAPDH. (B) Expressions of CTCF and Pax6 proteins during ES cell differentiation to radial glial cells. Western assays were performed to detect expression levels of CTCF, Pax6 and β-actin. (C) Illustration of ChIP-based PCR designs. (D) Detection of specific protein-nucleotide interaction between CTCF and Pax6 gene during ES cell differentiation and EB formation. Chromatin samples were immuno-pulled down by CTCF-specific antibody. ChIP-based PCR was performed to amplify CTCF binding DNA fragment in Pax6 P0 promoter region. All ChIP and pCR experiments were repeated for at least three times.

Mentions: Based on the observation that Pax6 expression was regulated by CTCF in ES cells, we investigated whether CTCF activity was also changed to correlate to the altered Pax6 expression during the time course of ES cell differentiation to radial glial cells. The effect of RA-induced ES differentiation on CTCF activity was investigated by focusing on two respects that include the CTCF expression level and its DNA binding capacity. First, both mRNA and protein expressions of CTCF were monitored following the time course of ES cell differentiation (Fig. 3A&3B). We found that Pax6 expression markedly increased on the 8th day of EB formation, and that there were no changes in CTCF expression at either the protein or mRNA level. Second, association of CTCF with the binding motifs in the promoter of Pax6 gene was examined. It has been shown that CTCF binds as a repressor to the region of −1.2 kb upstream from Pax6 P0 promoter to suppress Pax6 transcription [16]. A set of primers was designed accordingly for chromatin immunoprecipitation (ChIP)-based PCR experiments to generate a 260 bp DNA fragment based on consensus DNA sequences upstream from the Pax6 P0 promoter within the region of CTCF binding motifs (Fig. 3C). Results of ChIP-based PCR using specific antibodies against CTCF revealed that there were remarkable decreases in CTCF binding on DNA motifs in Pax6 promoter region on the 8th day of EB formation. For controls, non-immunoprecipitated chromatins were used as DNA templates in PCR experiments (labeled as input) (Fig. 3D). Taken together, CTCF expression was not affected by ES cell differentiation, instead its binding ability to DNA motifs in Pax6 promoter was diminished, suggesting that increases in Pax6 expression during differentiation to radial glial cells are very likely resulted from disengaging CTCF from its binding motifs and releasing Pax6 promoter from the repressor effect of CTCF.


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)

Correlation of CTCF and Pax6 in ES cell differentiation to radial glial cells.(A) Expressions of CTCF and Pax6 mRNAs during ES cell differentiation to radial glial cells. Total RNA samples were harvested from undifferentiated ES cells (0 d) and embryonic body (EB) cells after 4 day or 8 day induction. RT-PCR assays were performed to measure the expression of CTCF, Pax6 and GAPDH. (B) Expressions of CTCF and Pax6 proteins during ES cell differentiation to radial glial cells. Western assays were performed to detect expression levels of CTCF, Pax6 and β-actin. (C) Illustration of ChIP-based PCR designs. (D) Detection of specific protein-nucleotide interaction between CTCF and Pax6 gene during ES cell differentiation and EB formation. Chromatin samples were immuno-pulled down by CTCF-specific antibody. ChIP-based PCR was performed to amplify CTCF binding DNA fragment in Pax6 P0 promoter region. All ChIP and pCR experiments were repeated for at least three times.
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Related In: Results  -  Collection

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pone-0020954-g003: Correlation of CTCF and Pax6 in ES cell differentiation to radial glial cells.(A) Expressions of CTCF and Pax6 mRNAs during ES cell differentiation to radial glial cells. Total RNA samples were harvested from undifferentiated ES cells (0 d) and embryonic body (EB) cells after 4 day or 8 day induction. RT-PCR assays were performed to measure the expression of CTCF, Pax6 and GAPDH. (B) Expressions of CTCF and Pax6 proteins during ES cell differentiation to radial glial cells. Western assays were performed to detect expression levels of CTCF, Pax6 and β-actin. (C) Illustration of ChIP-based PCR designs. (D) Detection of specific protein-nucleotide interaction between CTCF and Pax6 gene during ES cell differentiation and EB formation. Chromatin samples were immuno-pulled down by CTCF-specific antibody. ChIP-based PCR was performed to amplify CTCF binding DNA fragment in Pax6 P0 promoter region. All ChIP and pCR experiments were repeated for at least three times.
Mentions: Based on the observation that Pax6 expression was regulated by CTCF in ES cells, we investigated whether CTCF activity was also changed to correlate to the altered Pax6 expression during the time course of ES cell differentiation to radial glial cells. The effect of RA-induced ES differentiation on CTCF activity was investigated by focusing on two respects that include the CTCF expression level and its DNA binding capacity. First, both mRNA and protein expressions of CTCF were monitored following the time course of ES cell differentiation (Fig. 3A&3B). We found that Pax6 expression markedly increased on the 8th day of EB formation, and that there were no changes in CTCF expression at either the protein or mRNA level. Second, association of CTCF with the binding motifs in the promoter of Pax6 gene was examined. It has been shown that CTCF binds as a repressor to the region of −1.2 kb upstream from Pax6 P0 promoter to suppress Pax6 transcription [16]. A set of primers was designed accordingly for chromatin immunoprecipitation (ChIP)-based PCR experiments to generate a 260 bp DNA fragment based on consensus DNA sequences upstream from the Pax6 P0 promoter within the region of CTCF binding motifs (Fig. 3C). Results of ChIP-based PCR using specific antibodies against CTCF revealed that there were remarkable decreases in CTCF binding on DNA motifs in Pax6 promoter region on the 8th day of EB formation. For controls, non-immunoprecipitated chromatins were used as DNA templates in PCR experiments (labeled as input) (Fig. 3D). Taken together, CTCF expression was not affected by ES cell differentiation, instead its binding ability to DNA motifs in Pax6 promoter was diminished, suggesting that increases in Pax6 expression during differentiation to radial glial cells are very likely resulted from disengaging CTCF from its binding motifs and releasing Pax6 promoter from the repressor effect of CTCF.

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.

Show MeSH