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Insulin gene expression is regulated by DNA methylation.

Kuroda A, Rauch TA, Todorov I, Ku HT, Al-Abdullah IH, Kandeel F, Mullen Y, Pfeifer GP, Ferreri K - PLoS ONE (2009)

Bottom Line: Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%.Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2).Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes, Endocrinology, & Metabolism, Research Institute of City of Hope, Duarte, California, United States of America.

ABSTRACT

Background: Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression.

Methodology/principal findings: Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro.

Conclusions/significance: Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

Show MeSH
Demethylation of the Ins2 promoter during differentiation of mouse embryonic stem cells to insulin-expressing colonies.Mouse embryonic stem cells from Ngn3-EGFP mice were induced to differentiate through several stages into insulin-expressing colonies as previously described [34]. Genomic DNA from each stage was isolated, bisulfite-treated, and the methylation status was determined by pyrosequencing. The methylation status of the three CpG sites is plotted individually: −171 (white), −182 (black), −414 (hatched). The bars represent the averages and standard deviations of three independent stem cell experiments. Genomic DNA samples from mouse liver and purified beta cells were processed in parallel with each experiment as negative and positive controls, respectively. * p<0.05 compared with previous stage.
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pone-0006953-g004: Demethylation of the Ins2 promoter during differentiation of mouse embryonic stem cells to insulin-expressing colonies.Mouse embryonic stem cells from Ngn3-EGFP mice were induced to differentiate through several stages into insulin-expressing colonies as previously described [34]. Genomic DNA from each stage was isolated, bisulfite-treated, and the methylation status was determined by pyrosequencing. The methylation status of the three CpG sites is plotted individually: −171 (white), −182 (black), −414 (hatched). The bars represent the averages and standard deviations of three independent stem cell experiments. Genomic DNA samples from mouse liver and purified beta cells were processed in parallel with each experiment as negative and positive controls, respectively. * p<0.05 compared with previous stage.

Mentions: As shown in Figure 4, the insulin gene promoter in mESC is methylated on all three CpG sites, similar to the liver DNA control. The methylation status is maintained through embryoid body formation (Fig. 4; EB) and endodermal differentiation in the tertiary culture (Fig. 4; FACS presort). Surprisingly, the insulin promoter in the Ngn3-EGFP+ proendocrine cell population (Fig. 4.; FACS (+) sort), which exhibits detectable Ins2 gene expression [34], was still fully methylated. It was not until the final stage of in vitro differentiation (Fig. 4; colonies) that there was significant demethylation of two (−182 and −414) of the three CpG sites. This result shows that complete demethylation of the insulin promoter is a late step in the differentiation of the beta cell phenotype, and is preceded by low level insulin gene expression. It should be noted that the level of insulin gene expression, even in the final stage of in vitro differentiation, is substantially lower than that of fully differentiated mouse beta cells.


Insulin gene expression is regulated by DNA methylation.

Kuroda A, Rauch TA, Todorov I, Ku HT, Al-Abdullah IH, Kandeel F, Mullen Y, Pfeifer GP, Ferreri K - PLoS ONE (2009)

Demethylation of the Ins2 promoter during differentiation of mouse embryonic stem cells to insulin-expressing colonies.Mouse embryonic stem cells from Ngn3-EGFP mice were induced to differentiate through several stages into insulin-expressing colonies as previously described [34]. Genomic DNA from each stage was isolated, bisulfite-treated, and the methylation status was determined by pyrosequencing. The methylation status of the three CpG sites is plotted individually: −171 (white), −182 (black), −414 (hatched). The bars represent the averages and standard deviations of three independent stem cell experiments. Genomic DNA samples from mouse liver and purified beta cells were processed in parallel with each experiment as negative and positive controls, respectively. * p<0.05 compared with previous stage.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2735004&req=5

pone-0006953-g004: Demethylation of the Ins2 promoter during differentiation of mouse embryonic stem cells to insulin-expressing colonies.Mouse embryonic stem cells from Ngn3-EGFP mice were induced to differentiate through several stages into insulin-expressing colonies as previously described [34]. Genomic DNA from each stage was isolated, bisulfite-treated, and the methylation status was determined by pyrosequencing. The methylation status of the three CpG sites is plotted individually: −171 (white), −182 (black), −414 (hatched). The bars represent the averages and standard deviations of three independent stem cell experiments. Genomic DNA samples from mouse liver and purified beta cells were processed in parallel with each experiment as negative and positive controls, respectively. * p<0.05 compared with previous stage.
Mentions: As shown in Figure 4, the insulin gene promoter in mESC is methylated on all three CpG sites, similar to the liver DNA control. The methylation status is maintained through embryoid body formation (Fig. 4; EB) and endodermal differentiation in the tertiary culture (Fig. 4; FACS presort). Surprisingly, the insulin promoter in the Ngn3-EGFP+ proendocrine cell population (Fig. 4.; FACS (+) sort), which exhibits detectable Ins2 gene expression [34], was still fully methylated. It was not until the final stage of in vitro differentiation (Fig. 4; colonies) that there was significant demethylation of two (−182 and −414) of the three CpG sites. This result shows that complete demethylation of the insulin promoter is a late step in the differentiation of the beta cell phenotype, and is preceded by low level insulin gene expression. It should be noted that the level of insulin gene expression, even in the final stage of in vitro differentiation, is substantially lower than that of fully differentiated mouse beta cells.

Bottom Line: Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%.Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2).Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes, Endocrinology, & Metabolism, Research Institute of City of Hope, Duarte, California, United States of America.

ABSTRACT

Background: Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression.

Methodology/principal findings: Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro.

Conclusions/significance: Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression.

Show MeSH