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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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Induction of radial glial cells from embryonic stem cells.(A) Morphological changes of neural differentiation from ES cells. (B) Altered mRNA expression of ES cell markers (Oct4) and radial gial cells markers (nestin, BLBP and Pax6). (C) Staining for radial gial cells markers. ES cells growing on gelatin-coated plates were dissociated and transferred to nonadherent bacterial dishes to form embryonic body. After 4-day of embryonic body formation, 5 µM retinoid acid was added to the culture media and cells were allowed to grow for another 4 days. Embryonic bodies were dissociated after a total of 8-day induction and plated to poly-D-lysine/laminin-coated dishes in N2 medium. N2 medium is changed after 2 h. Cell samples were collected before and after the 4 days and days induction as shown as ES (0 d), EB (4 d) and EB (8 d) respectively. Photos of RG cells were taken 6 h after passage. Total RNAs were extracted at indicated time points and altered expressions of cell lineage-specific markers were monitored by RT-PCR. Cells were immunostained with antibodies specific to Nestin (green) and Pax6 (red). Cell nuclei were stained by DAPI (Blue). Results were plotted as a percentage of positively stained cells with Nestin and Pax6 antibodies vs numbers of DAPI-positive nuclei.
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pone-0020954-g001: Induction of radial glial cells from embryonic stem cells.(A) Morphological changes of neural differentiation from ES cells. (B) Altered mRNA expression of ES cell markers (Oct4) and radial gial cells markers (nestin, BLBP and Pax6). (C) Staining for radial gial cells markers. ES cells growing on gelatin-coated plates were dissociated and transferred to nonadherent bacterial dishes to form embryonic body. After 4-day of embryonic body formation, 5 µM retinoid acid was added to the culture media and cells were allowed to grow for another 4 days. Embryonic bodies were dissociated after a total of 8-day induction and plated to poly-D-lysine/laminin-coated dishes in N2 medium. N2 medium is changed after 2 h. Cell samples were collected before and after the 4 days and days induction as shown as ES (0 d), EB (4 d) and EB (8 d) respectively. Photos of RG cells were taken 6 h after passage. Total RNAs were extracted at indicated time points and altered expressions of cell lineage-specific markers were monitored by RT-PCR. Cells were immunostained with antibodies specific to Nestin (green) and Pax6 (red). Cell nuclei were stained by DAPI (Blue). Results were plotted as a percentage of positively stained cells with Nestin and Pax6 antibodies vs numbers of DAPI-positive nuclei.

Mentions: In the present study, a previous established protocol was used to induce ES cell differentiation toward radial glial cells [4], [5]. Cultured mouse ES cells were transferred to petri-dishes and treated with RA. Photos were taken by a Nikon invert microscope to document morphological changes of RA-induced ES cells in different stages of differentiation (Fig. 1A). Results of RA-induced ES differentiation followed a pattern that was consistent with previous reports. After formation of embryonic body (EB), RA-induced cells were successfully differentiated to spindle-shaped radial glial cells. Results from RT-PCR revealed that mRNAs of nestin (neural precursor marker) and BLBP (radial glial cell marker) were highly expressed, while expression of Oct4 mRNA (an ES cell marker) was suppressed in 8 days in RA-induced cells (Fig. 1B). More importantly, expression of Pax6 mRNA was dramatically increased in the 8th day in RA-induced cells. Nestin- and Pax6-positive cells were identified by immunostaining experiments in RA-induced cells, demonstrating that there was a significantly high conversion of ES cells to the radial glial cells (Fig. 1C). These results indicate that Pax6 is regulated during ES cell differentiation toward radial glial cells.


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)

Induction of radial glial cells from embryonic stem cells.(A) Morphological changes of neural differentiation from ES cells. (B) Altered mRNA expression of ES cell markers (Oct4) and radial gial cells markers (nestin, BLBP and Pax6). (C) Staining for radial gial cells markers. ES cells growing on gelatin-coated plates were dissociated and transferred to nonadherent bacterial dishes to form embryonic body. After 4-day of embryonic body formation, 5 µM retinoid acid was added to the culture media and cells were allowed to grow for another 4 days. Embryonic bodies were dissociated after a total of 8-day induction and plated to poly-D-lysine/laminin-coated dishes in N2 medium. N2 medium is changed after 2 h. Cell samples were collected before and after the 4 days and days induction as shown as ES (0 d), EB (4 d) and EB (8 d) respectively. Photos of RG cells were taken 6 h after passage. Total RNAs were extracted at indicated time points and altered expressions of cell lineage-specific markers were monitored by RT-PCR. Cells were immunostained with antibodies specific to Nestin (green) and Pax6 (red). Cell nuclei were stained by DAPI (Blue). Results were plotted as a percentage of positively stained cells with Nestin and Pax6 antibodies vs numbers of DAPI-positive nuclei.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113856&req=5

pone-0020954-g001: Induction of radial glial cells from embryonic stem cells.(A) Morphological changes of neural differentiation from ES cells. (B) Altered mRNA expression of ES cell markers (Oct4) and radial gial cells markers (nestin, BLBP and Pax6). (C) Staining for radial gial cells markers. ES cells growing on gelatin-coated plates were dissociated and transferred to nonadherent bacterial dishes to form embryonic body. After 4-day of embryonic body formation, 5 µM retinoid acid was added to the culture media and cells were allowed to grow for another 4 days. Embryonic bodies were dissociated after a total of 8-day induction and plated to poly-D-lysine/laminin-coated dishes in N2 medium. N2 medium is changed after 2 h. Cell samples were collected before and after the 4 days and days induction as shown as ES (0 d), EB (4 d) and EB (8 d) respectively. Photos of RG cells were taken 6 h after passage. Total RNAs were extracted at indicated time points and altered expressions of cell lineage-specific markers were monitored by RT-PCR. Cells were immunostained with antibodies specific to Nestin (green) and Pax6 (red). Cell nuclei were stained by DAPI (Blue). Results were plotted as a percentage of positively stained cells with Nestin and Pax6 antibodies vs numbers of DAPI-positive nuclei.
Mentions: In the present study, a previous established protocol was used to induce ES cell differentiation toward radial glial cells [4], [5]. Cultured mouse ES cells were transferred to petri-dishes and treated with RA. Photos were taken by a Nikon invert microscope to document morphological changes of RA-induced ES cells in different stages of differentiation (Fig. 1A). Results of RA-induced ES differentiation followed a pattern that was consistent with previous reports. After formation of embryonic body (EB), RA-induced cells were successfully differentiated to spindle-shaped radial glial cells. Results from RT-PCR revealed that mRNAs of nestin (neural precursor marker) and BLBP (radial glial cell marker) were highly expressed, while expression of Oct4 mRNA (an ES cell marker) was suppressed in 8 days in RA-induced cells (Fig. 1B). More importantly, expression of Pax6 mRNA was dramatically increased in the 8th day in RA-induced cells. Nestin- and Pax6-positive cells were identified by immunostaining experiments in RA-induced cells, demonstrating that there was a significantly high conversion of ES cells to the radial glial cells (Fig. 1C). These results indicate that Pax6 is regulated during ES cell differentiation toward radial glial cells.

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