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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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Identification of methylation sites within CTCF binding motifs.(A) Putative methylation sites in the region of CTCF binding motifs upstream from Pax6 promoter. (B) Quantitative determination of methylation status at cytosines in CTCF binding sties after inductions of demethylation and methylation. Genomic DNA is treated with sodium bisulfite followed by PCR of the target sequence to generate the template for Ms-SNuPE assays. Methylation statuses were evaluated by the radio of radial incorporation of [32]P-dCTP (representing methylated cytosine) and [32]P-dTTP (representing unmethylated cytosines). DNA methylations were detected by using bisulfite sodium modifications and PCR. Demethylation and methylation were induced by 5-azadCyd (1 µM) and by over-expression of Dnmt3a, respectively. (C) Statistical analysis of methylation percentages in site1–3. Data was shown as mean ±S.E. and represented results from three independent Ms-SNuPE assays. Symbol “*” indicates significant differences comparing with cells without treatment. (p<0.05, n = 3).
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pone-0020954-g004: Identification of methylation sites within CTCF binding motifs.(A) Putative methylation sites in the region of CTCF binding motifs upstream from Pax6 promoter. (B) Quantitative determination of methylation status at cytosines in CTCF binding sties after inductions of demethylation and methylation. Genomic DNA is treated with sodium bisulfite followed by PCR of the target sequence to generate the template for Ms-SNuPE assays. Methylation statuses were evaluated by the radio of radial incorporation of [32]P-dCTP (representing methylated cytosine) and [32]P-dTTP (representing unmethylated cytosines). DNA methylations were detected by using bisulfite sodium modifications and PCR. Demethylation and methylation were induced by 5-azadCyd (1 µM) and by over-expression of Dnmt3a, respectively. (C) Statistical analysis of methylation percentages in site1–3. Data was shown as mean ±S.E. and represented results from three independent Ms-SNuPE assays. Symbol “*” indicates significant differences comparing with cells without treatment. (p<0.05, n = 3).

Mentions: It has shown that the interaction of CTCF with DNA sequence can be inhibited if the binding motif is methylated [18]. Next question is whether DNA methylation affects associations of CTCF with Pax6 DNA since CTCF binding capacity to Pax6 DNA decreased during radial glial cell differentiation. First, several potential 5-methylcytosines were located in the 80 bp region of CTCF binding motifs through DNA sequence analyzing (Fig. 4A). Genomic DNAs from ES cells were isolated and treated with sodium bisulfite that converts unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. DNA fragments containing CTCF binding sites were obtained by PCR with primers specific to bisulfite-converted DNA and sequenced for conformation. Normal genomic DNAs without sodium bisulfite treatment were also isolated for control experiments. In the PCR products, only 5-methylcytosines remained as cytosines (C) while un-methylcytosines were converted to thymines (T). Sequencing results revealed that within the tested DNA region, there were mixed C/T pairs occurring in 3 positions (sites 1–3 shown in Fig. 4A), indicating an uneven methylation of cytosine residues. All of other cytosine residues in CG-rich islands were completely converted to T (data not shown). Sites 1–3 were subjects to further quantitative analysis by performing Ms-SNuPE assays. These sites were differentially mehylated showing that methylations occurred about 15% in site 1, 55% in sties 2 and 3 (Fig. 4B&4C). To further verify methylation data, methylation responses of these sites were examined by altering cellular DNA methylation levels. ES cells were treated with 1 µM of 5-azadCyd to suppress methyltransferase resulting in decrease in DNA methylation. In contrast, ES cells were transfected with full-length cDNA encoding methyltranferase Dnmt3a to increase DNA methylation. Results showed: 1) site 1 was about 15% methylated in control cells compared to 12% and 18% methylated in 5-azadCyd treated and Dnmt3a-transfected cells, respectively; and 2) both sites 2 and 3 were approximate 55% methylated in control cells compared to 32% and 80% methylated in 5-azadCyd-treated and Dnmt3a-transfected cells, respectively (Fig. 4C). These results suggest that there are at least three cytosine residues that respond to alterations of cellular DNA methylation levels located in the region of CTCF binding motifs.


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)

Identification of methylation sites within CTCF binding motifs.(A) Putative methylation sites in the region of CTCF binding motifs upstream from Pax6 promoter. (B) Quantitative determination of methylation status at cytosines in CTCF binding sties after inductions of demethylation and methylation. Genomic DNA is treated with sodium bisulfite followed by PCR of the target sequence to generate the template for Ms-SNuPE assays. Methylation statuses were evaluated by the radio of radial incorporation of [32]P-dCTP (representing methylated cytosine) and [32]P-dTTP (representing unmethylated cytosines). DNA methylations were detected by using bisulfite sodium modifications and PCR. Demethylation and methylation were induced by 5-azadCyd (1 µM) and by over-expression of Dnmt3a, respectively. (C) Statistical analysis of methylation percentages in site1–3. Data was shown as mean ±S.E. and represented results from three independent Ms-SNuPE assays. Symbol “*” indicates significant differences comparing with cells without treatment. (p<0.05, n = 3).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113856&req=5

pone-0020954-g004: Identification of methylation sites within CTCF binding motifs.(A) Putative methylation sites in the region of CTCF binding motifs upstream from Pax6 promoter. (B) Quantitative determination of methylation status at cytosines in CTCF binding sties after inductions of demethylation and methylation. Genomic DNA is treated with sodium bisulfite followed by PCR of the target sequence to generate the template for Ms-SNuPE assays. Methylation statuses were evaluated by the radio of radial incorporation of [32]P-dCTP (representing methylated cytosine) and [32]P-dTTP (representing unmethylated cytosines). DNA methylations were detected by using bisulfite sodium modifications and PCR. Demethylation and methylation were induced by 5-azadCyd (1 µM) and by over-expression of Dnmt3a, respectively. (C) Statistical analysis of methylation percentages in site1–3. Data was shown as mean ±S.E. and represented results from three independent Ms-SNuPE assays. Symbol “*” indicates significant differences comparing with cells without treatment. (p<0.05, n = 3).
Mentions: It has shown that the interaction of CTCF with DNA sequence can be inhibited if the binding motif is methylated [18]. Next question is whether DNA methylation affects associations of CTCF with Pax6 DNA since CTCF binding capacity to Pax6 DNA decreased during radial glial cell differentiation. First, several potential 5-methylcytosines were located in the 80 bp region of CTCF binding motifs through DNA sequence analyzing (Fig. 4A). Genomic DNAs from ES cells were isolated and treated with sodium bisulfite that converts unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. DNA fragments containing CTCF binding sites were obtained by PCR with primers specific to bisulfite-converted DNA and sequenced for conformation. Normal genomic DNAs without sodium bisulfite treatment were also isolated for control experiments. In the PCR products, only 5-methylcytosines remained as cytosines (C) while un-methylcytosines were converted to thymines (T). Sequencing results revealed that within the tested DNA region, there were mixed C/T pairs occurring in 3 positions (sites 1–3 shown in Fig. 4A), indicating an uneven methylation of cytosine residues. All of other cytosine residues in CG-rich islands were completely converted to T (data not shown). Sites 1–3 were subjects to further quantitative analysis by performing Ms-SNuPE assays. These sites were differentially mehylated showing that methylations occurred about 15% in site 1, 55% in sties 2 and 3 (Fig. 4B&4C). To further verify methylation data, methylation responses of these sites were examined by altering cellular DNA methylation levels. ES cells were treated with 1 µM of 5-azadCyd to suppress methyltransferase resulting in decrease in DNA methylation. In contrast, ES cells were transfected with full-length cDNA encoding methyltranferase Dnmt3a to increase DNA methylation. Results showed: 1) site 1 was about 15% methylated in control cells compared to 12% and 18% methylated in 5-azadCyd treated and Dnmt3a-transfected cells, respectively; and 2) both sites 2 and 3 were approximate 55% methylated in control cells compared to 32% and 80% methylated in 5-azadCyd-treated and Dnmt3a-transfected cells, respectively (Fig. 4C). These results suggest that there are at least three cytosine residues that respond to alterations of cellular DNA methylation levels located in the region of CTCF binding motifs.

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
Related in: MedlinePlus