Epigenetic signature and enhancer activity of the human APOE gene

Abstract

The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ε2/ε3/ε4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ε4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ε2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ε2/ε3/ε4 allele-carrying 3'-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ε2/ε3/ε4 allele-specific manner. These findings implicate a novel functional role for a 3'-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI.

Figure 1.

Gene map with CGIs at the APOE locus. (A) Genomic position of exons and CGIs. (B) Full-genomic sequence of the APOE CGI with 90 CpG dinucleotides (underlined). Numbers to the right represent CpG counts within each line of 100 nucleotides, and a potential core region (lines 3–6) is marked by numbers in bold font. (C) The two SNPs (rs429358 and rs7412) determining APOE ɛ2/ɛ3/ɛ4 alleles change the CpG content and landscape of the CGI; their positions are indicated by arrows #1 and #2 in (B).

Figure 2.

Methylation profiles of the APOE CGI from human PMB tissues and lymphocytes. (A) The mean percentage of methylation at the 75 targeted CpG sites across tissues (n = 14 or 15) and the SNP rs429358 site for ɛ4/ɛ4 homozygous samples only (denoted by filled diamonds, n = 2 or 3). (B) For each tissue type, methylation percentages from all 15 subjects were averaged at each of the 75 CpG sites. The range and mean (denoted by filled black diamonds) methylation percentages across these 75 sites are displayed in a box-and-whisker plot. Mean methylation proportions across all 75 CpG sites were transformed, and statistically significant differences between all five tissues tested were detected (Holm-adjusted P < 10⁻¹⁰ for all 10 pair-wise comparisons).

Figure 3.

Enhancer/silencer effect of un-methylated APOE CGI in pGL4 reporter assays. (A) The diagram of reporter constructs with promoters of either APOE (left panel) or TOMM40 (right panel) and a 3′ APOE CGI fragment (491 bp) in the forward orientation. Haplotype compositions are also shown. (B) Constructs were transiently transfected into three cell lines (HepG2, SH-SY5Y and U118). Luciferase activity of each construct was measured and compared with its promoter-only construct counterpart (set at 1.0). Each reporter activity was generated from five experimental replicates. Expression levels between constructs were compared by one-way ANOVA [APOE promoter: HepG2, F(8, 45) = 2.99, P = 0.008; SH-SY5Y, F(8, 45) = 0.79, P = 0.61; U118, F(8, 63) = 0.39, P = 0.92; TOMM40 promoter: HepG2, F(8, 45) = 4.06, P = 0.001; SH-SY5Y, F(8, 45) = 23.81, P < 0.001; U118, F(8, 63) = 0.54, P = 0.82)]. Pair-wise comparisons were made with a post hoc Bonferroni test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4.

Enhancer effect of the methylated APOE CGI in pCpG-free vector reporter assays. (A) The diagram of reporter constructs with an APOE CGI fragment (1483 bp) in either the forward or reverse orientation, 5′ of the hEF-1 promoter. (B) The APOE CGI was either methylated by DNA methyltransferase treatment (methylation section) or left un-methylated and transiently transfected into three cell lines (HepG2, SH-SY5Y and U118). AP activity of each construct was measured and compared with its promoter-only construct counterpart (set at 1.0). Each reporter activity was generated from five experimental replicates. Expression levels between constructs were compared by one-way ANOVA [Forward: HepG2, F(5, 78) = 0.54, P = 0.74; SH-SY5Y, F(5, 66) = 7.39, P < 0.001; U118, F(5, 48) = 4.04, P = 0.004; Reverse: HepG2, F(5, 84) = 0.98, P = 0.43; SH-SY5Y, F(5, 66) = 2.84, P = 0.02; U118, F(5, 48) = 4.56, P = 0.002]. Pair-wise comparisons were made with a post hoc Bonferroni test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5.

Enhancer effect of the APOE CGI in humanized APOE mice. (A) An outline of the genomic structure of the APOE locus in transgenic mice, including the human APOE gene Ex2-4. (B) Relative quantification of mRNA expression levels in neurons and astrocytes. Expression levels in each condition were normalized with an endogenous control (ACTB), and compared with ɛ3 counterparts (set at 1.0) using the ΔΔC_T calculation. Each expression level was generated from at least four experimental replicates. Expression levels of APOE and TOMM40 between APOE haplotypes were compared by one-way ANOVA [APOE: neurons, F(2, 21) = 8.5, P = 0.002; astrocytes, F(2,12) = 17.07, P < 0.001; TOMM40: neurons, F(2, 21) = 5.07, P = 0.016; astrocytes, F(2, 12) = 1.46, P = 0.27]. Pair-wise comparisons were made with a post hoc Tukey honest significance test (*P < 0.05, **P < 0.01, ***P < 0.001). Error bars represent standard deviation from the mean (neurons, n = 8; astrocytes, n = 5).

Figure 6.

Relative mRNA expression levels and their correlation with APOE CGI DNA methylation levels in PMB tissues. Expression levels of (A) APOE and (B) TOMM40 were normalized with an endogenous control (ACTB) and calibrated to expression levels in the frontal lobe (set to 1.0). Gene expression between tissues was evaluated by one-way ANOVA [APOE, F(3, 54) = 3.48, P = 0.022; TOMM40, F(3, 55) = 13.41, P < 0.001]. Post hoc pair-wise comparsions were made using Tukey's honest significance test (*P < 0.05, ***P ≤ 0.001). Error bars were calculated using standard propagation of error methods. The average percentage of methylation, at the APOE CGI, for each PMB sample was correlated to gene expression of (C) APOE and (D) TOMM40. A positive trend was observed between levels of methylation and APOE expression that did not reach the cutoff value for statistical significance (Pearson's correlation, r = 0.219, n = 58, P < 0.098). A positive correlation between the levels of methylation and TOMM40 expression was observed (Pearson's correlation, r = 0.347, n = 59, P < 0.007).