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Allele-specific, age-dependent and BMI-associated DNA methylation of human MCHR1.

Stepanow S, Reichwald K, Huse K, Gausmann U, Nebel A, Rosenstiel P, Wabitsch M, Fischer-Posovszky P, Platzer M - PLoS ONE (2011)

Bottom Line: The AC haplotype shows a significantly higher methylation level than the GT haplotype.Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype.We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription.

View Article: PubMed Central - PubMed

Affiliation: Genome Analysis, Leibniz Institute for Age Research, Jena, Germany. stepanow@fli-leibniz.de

ABSTRACT

Background: Melanin-concentrating hormone receptor 1 (MCHR1) plays a significant role in regulation of energy balance, food intake, physical activity and body weight in humans and rodents. Several association studies for human obesity showed contrary results concerning the SNPs rs133072 (G/A) and rs133073 (T/C), which localize to the first exon of MCHR1. The variations constitute two main haplotypes (GT, AC). Both SNPs affect CpG dinucleotides, whereby each haplotype contains a potential methylation site at one of the two SNP positions. In addition, 15 CpGs in close vicinity of these SNPs constitute a weak CpG island. Here, we studied whether DNA methylation in this sequence context may contribute to population- and age-specific effects of MCHR1 alleles in obesity.

Principal findings: We analyzed DNA methylation of a 315 bp region of MCHR1 encompassing rs133072 and rs133073 and the CpG island in blood samples of 49 individuals by bisulfite sequencing. The AC haplotype shows a significantly higher methylation level than the GT haplotype. This allele-specific methylation is age-dependent. In young individuals (20-30 years) the difference in DNA methylation between haplotypes is significant; whereas in individuals older than 60 years it is not detectable. Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype. Heterozygous lymphoblastoid cell lines show the same pattern of allele-specific DNA methylation. The cell line, which exhibits the highest difference in methylation levels between both haplotypes, also shows allele-specific transcription of MCHR1, which can be abolished by treatment with the DNA methylase inhibitor 5-aza-2'-deoxycytidine.

Conclusions: We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription. Conceivably, this epigenetic regulation contributes to the age- and/or population specific effects reported for MCHR1 in several human obesity studies.

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Allele-specific mRNA expression of LCLs.Mean frequency of the GT allele in cDNA is shown for three heterozygous LCLs GM12760, GM12864 and C0913 as measured by pyrosequencing at five time points, which were also analyzed for methylation level. GM12760 and GM12864 showed an equal transcription of both alleles, whereby GM12760 exhibited on average 47.4±5.6% of GT alleles and GM12864 50±4.2%. The LCL C0913, which exhibits the highest AC allele methylation level showed an average frequency of 75.7±10.4% of GT alleles. This preferential GT allele expression is significantly different from the equal transcription observed in GM12760 and GM12860 (P<0.05, ANOVA). Allele frequencies were confirmed by cloning and sequencing of a single passage of the LCL.
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pone-0017711-g007: Allele-specific mRNA expression of LCLs.Mean frequency of the GT allele in cDNA is shown for three heterozygous LCLs GM12760, GM12864 and C0913 as measured by pyrosequencing at five time points, which were also analyzed for methylation level. GM12760 and GM12864 showed an equal transcription of both alleles, whereby GM12760 exhibited on average 47.4±5.6% of GT alleles and GM12864 50±4.2%. The LCL C0913, which exhibits the highest AC allele methylation level showed an average frequency of 75.7±10.4% of GT alleles. This preferential GT allele expression is significantly different from the equal transcription observed in GM12760 and GM12860 (P<0.05, ANOVA). Allele frequencies were confirmed by cloning and sequencing of a single passage of the LCL.

Mentions: By pyrosequencing, we analyzed MCHR1 ASE in these cell lines at five time points. Both GM12760 and GM12864 did not show allele-specific transcription (Figure 7). This was confirmed by cloning and subsequent sequencing of cDNA from a single passage, which allowed calling of the respective alleles (number of GT/AC clones: GM12760 = 74/82, GM12864 = 33/28). In contrast, in LCL C0913, which showed the highest difference in methylation intensity between GT and AC alleles, we observed allelic imbalance of MCHR1 transcription. Mean frequency of transcripts representing the GT allele was 75.7±10.4% (Figure 7). This was confirmed by cloning and sequencing using cDNA from a single passage (GT/AC = 51/16, GT-fraction: 76%). The preferential GT allele expression of C0913 is significantly different from the equal expression of both alleles observed in GM12760 and GM12864 (P<0.05, ANOVA). To check if the observed allele-specific transcription is not due to a different number of allele copies in these LCLs, we measured allelic status in genomic DNA by pyrosequencing. All three LCLs have equal copies of both alleles (Figure S2).


Allele-specific, age-dependent and BMI-associated DNA methylation of human MCHR1.

Stepanow S, Reichwald K, Huse K, Gausmann U, Nebel A, Rosenstiel P, Wabitsch M, Fischer-Posovszky P, Platzer M - PLoS ONE (2011)

Allele-specific mRNA expression of LCLs.Mean frequency of the GT allele in cDNA is shown for three heterozygous LCLs GM12760, GM12864 and C0913 as measured by pyrosequencing at five time points, which were also analyzed for methylation level. GM12760 and GM12864 showed an equal transcription of both alleles, whereby GM12760 exhibited on average 47.4±5.6% of GT alleles and GM12864 50±4.2%. The LCL C0913, which exhibits the highest AC allele methylation level showed an average frequency of 75.7±10.4% of GT alleles. This preferential GT allele expression is significantly different from the equal transcription observed in GM12760 and GM12860 (P<0.05, ANOVA). Allele frequencies were confirmed by cloning and sequencing of a single passage of the LCL.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102661&req=5

pone-0017711-g007: Allele-specific mRNA expression of LCLs.Mean frequency of the GT allele in cDNA is shown for three heterozygous LCLs GM12760, GM12864 and C0913 as measured by pyrosequencing at five time points, which were also analyzed for methylation level. GM12760 and GM12864 showed an equal transcription of both alleles, whereby GM12760 exhibited on average 47.4±5.6% of GT alleles and GM12864 50±4.2%. The LCL C0913, which exhibits the highest AC allele methylation level showed an average frequency of 75.7±10.4% of GT alleles. This preferential GT allele expression is significantly different from the equal transcription observed in GM12760 and GM12860 (P<0.05, ANOVA). Allele frequencies were confirmed by cloning and sequencing of a single passage of the LCL.
Mentions: By pyrosequencing, we analyzed MCHR1 ASE in these cell lines at five time points. Both GM12760 and GM12864 did not show allele-specific transcription (Figure 7). This was confirmed by cloning and subsequent sequencing of cDNA from a single passage, which allowed calling of the respective alleles (number of GT/AC clones: GM12760 = 74/82, GM12864 = 33/28). In contrast, in LCL C0913, which showed the highest difference in methylation intensity between GT and AC alleles, we observed allelic imbalance of MCHR1 transcription. Mean frequency of transcripts representing the GT allele was 75.7±10.4% (Figure 7). This was confirmed by cloning and sequencing using cDNA from a single passage (GT/AC = 51/16, GT-fraction: 76%). The preferential GT allele expression of C0913 is significantly different from the equal expression of both alleles observed in GM12760 and GM12864 (P<0.05, ANOVA). To check if the observed allele-specific transcription is not due to a different number of allele copies in these LCLs, we measured allelic status in genomic DNA by pyrosequencing. All three LCLs have equal copies of both alleles (Figure S2).

Bottom Line: The AC haplotype shows a significantly higher methylation level than the GT haplotype.Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype.We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription.

View Article: PubMed Central - PubMed

Affiliation: Genome Analysis, Leibniz Institute for Age Research, Jena, Germany. stepanow@fli-leibniz.de

ABSTRACT

Background: Melanin-concentrating hormone receptor 1 (MCHR1) plays a significant role in regulation of energy balance, food intake, physical activity and body weight in humans and rodents. Several association studies for human obesity showed contrary results concerning the SNPs rs133072 (G/A) and rs133073 (T/C), which localize to the first exon of MCHR1. The variations constitute two main haplotypes (GT, AC). Both SNPs affect CpG dinucleotides, whereby each haplotype contains a potential methylation site at one of the two SNP positions. In addition, 15 CpGs in close vicinity of these SNPs constitute a weak CpG island. Here, we studied whether DNA methylation in this sequence context may contribute to population- and age-specific effects of MCHR1 alleles in obesity.

Principal findings: We analyzed DNA methylation of a 315 bp region of MCHR1 encompassing rs133072 and rs133073 and the CpG island in blood samples of 49 individuals by bisulfite sequencing. The AC haplotype shows a significantly higher methylation level than the GT haplotype. This allele-specific methylation is age-dependent. In young individuals (20-30 years) the difference in DNA methylation between haplotypes is significant; whereas in individuals older than 60 years it is not detectable. Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype. Heterozygous lymphoblastoid cell lines show the same pattern of allele-specific DNA methylation. The cell line, which exhibits the highest difference in methylation levels between both haplotypes, also shows allele-specific transcription of MCHR1, which can be abolished by treatment with the DNA methylase inhibitor 5-aza-2'-deoxycytidine.

Conclusions: We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription. Conceivably, this epigenetic regulation contributes to the age- and/or population specific effects reported for MCHR1 in several human obesity studies.

Show MeSH
Related in: MedlinePlus