An Alu element-associated hypermethylation variant of the POMC gene is associated with childhood obesity

Abstract

The individual risk for common diseases not only depends on genetic but also on epigenetic polymorphisms. To assess the role of epigenetic variations in the individual risk for obesity, we have determined the methylation status of two CpG islands at the POMC locus in obese and normal-weight children. We found a hypermethylation variant targeting individual CpGs at the intron 2-exon 3 boundary of the POMC gene by bisulphite sequencing that was significantly associated with obesity. POMC exon 3 hypermethylation interferes with binding of the transcription enhancer P300 and reduces expression of the POMC transcript. Since intron 2 contains Alu elements that are known to influence methylation in their genomic vicinity, the exon 3 methylation variant seems to result from an Alu element-triggered default state of methylation boundary definition. Exon 3 hypermethylation in the POMC locus represents the first identified DNA methylation variant that is associated with the individual risk for obesity.

Figure 1. POMC CpG methylation pattern of CpG island 1 and 2.

Analysis example of the DNA methylation pattern of human peripheral blood cells (PBC) from a normal weight individual after sequencing of 10 different clones of the PCR amplification product. Filled cycles represent methylated CpGs, open cycles non-methylated CpG positions, which are numbered according to their relative position to the start of the next exon. Alu element positions and P300 binding site are marked.

Figure 2. POMC DNA methylation pattern of CpG island 1 and 2 in normal-weight adolescents (n = 12), laser-microdissected MSH positive cells of the human arcuate nucleus (n = 5) and human newborn samples (n = 8).

CpG positions are numbered according to their relative position to the next exon start.

Figure 3. The DNA methylation was analysed with two different bisulphite based protocols in two independent cohorts.

A, B, C agarose-embedded DNA was analysed by bisulphite sequencing. In D, E, F DNA was bisulphite treated in a non-agarose-bead protocol. A/D Diagram of the DNA methylation score (%) of POMC CpG island 2 within PBC of normal weight (red) and obese individuals (blue) (p<0,001). CpG positions are numbered according to their relative position to the next exon start. B/E Box plots analysis represents the statistic differences from the mean CpG methylation score (%) of CpG position −4 to +6 in normal weight individuals versus obese patients. C/F Percent of normal weight (red) and obese (blue) individuals, who showed DNA methylation at the annotated single CpG position (−1 to +5) in relation to the analysed number of samples.

Figure 4. Longitudinal analysis of POMC DNA methylation score (%) (CpG position −4 to +6) before and after the development of obesity in altogether 21 participants of the MAS birth cohort.

Each line represents one individual course of DNA methylation score prior to and after weight gain.

Figure 5. POMC intron2 exon3 genomic sequence, P300 ChIP assay, real-time PCR analysis, and real-time POMC PCR.

(A) POMC intron2 exon3 genomic sequence with annotated ChIP primer localization and P300 binding site. (B) P300 ChIP assay was performed at three independent occasions within human peripheral blood cells. POMC fragment was amplified with two different primer pairs (fragment 1 and 2). Confirmation of a published P300 binding site within the insulin gene promoter in β-TC3 cells was used as a positive control (Figure S5). (C) Real time PCR analysis (PCR fragment 2) of the P300 ChIP results of 4 obese patients with a hypermethylation variant and 6 individuals with hypomethylated intron 2-exon 3 intersection at POMC CpG island 2. (D) Real-time POMC PCR (POMC exon 2–3 and POMC exon3) of cDNA extracted from PBC of hypomethylated normal weight individuals (n = 20), hypermethylated obese patients (n = 20), and obese individuals without the hypermethylated variant (n = 20) reveal reduced POMC gene expression in the hypermethylated samples.

Figure 6. Species-specific POMC methylation pattern POMC DNA methylation pattern after bisulphite genomic sequencing of CpG island 2.

In (A) normal-weight human individuals (n = 36), (B) chimpanzee PBC (n = 2), (C) lemur PBC (n = 3), and (D) mice PBC (n = 3). CpG positions are numbered according to their relative position to the exon 3 start.