Uncovering the DNA methylation landscape in key regulatory regions within the FADS cluster

Abstract

Genetic variants near and within the fatty acid desaturase (FADS) cluster are associated with polyunsaturated fatty acid (PUFA) biosynthesis, levels of several disease biomarkers and risk of human disease. However, determining the functional mechanisms by which these genetic variants impact PUFA levels remains a challenge. Utilizing an Illumina 450K array, we previously reported strong allele-specific methylation (ASM) associations (p = 2.69×10−29) between a single nucleotide polymorphism (SNP) rs174537 and DNA methylation of CpG sites located in the putative enhancer region between FADS1 and FADS2, in human liver tissue. However, this array only featured 20 CpG sites within this 12kb region. To better understand the methylation landscape within this region, we conducted bisulfite sequencing of the region between FADS1 and FADS2. Liver tissues from 50 male subjects (27 European Americans, 23 African Americans) were obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, and used to ascertain the genotype at rs174537 and methylation status across the region of interest. Associations between rs174537 genotype and methylation status of 136 CpG sites were determined. Age-adjusted linear regressions were used to assess ASM associations with rs174537 genotype. The majority of CpG sites (117 out of 136, 86%) exhibited high levels of methylation with the greatest variability observed at three key regulatory regions–the promoter regions for FADS1 and FADS2 and a putative enhancer site between the two genes. Eight CpG sites within the putative enhancer region displayed significant (FDR p <0.05) ASM associations with rs174537. These data support the concept that both genetic and epigenetic factors regulate PUFA biosynthesis, and raise fundamental questions as to how genetic variants such as rs174537 impact DNA methylation in distant regulatory regions, and ultimately the capacity of tissues to synthesize PUFAs.

Allele-specific methylation in the FADS genomic region in DNA from human saliva, CD4+ cells, and total leukocytes

“…Interestingly, DNA methylation at the CpG site located in the putative enhancer region (i.e., chr11:61587979 (cg27386326)) was highest in DNA extracted from total blood leukocyte samples. DNA methylation levels quantified from total leukocytes at this CpG site was comparable to the levels obtained from liver tissue specimens, as previously published [ 10 , 24 ]. Consistently, the degree of DNA methylation was significantly different between DNA sources, as seen in Table 1 .…”

“…Based on our previous studies, DNA methylation was quantified at six key CpG sites between FADS1 and FADS2. These sites were previously identified to have strong ASM with rs174537 from liver tissues [ 10 , 24 ]. While our previous paper identified eight CpG sites, we were limited by sample material to analyze six out of the eight CpG sites in this paper.…”

“…DNA methylation is known to alter gene expression, especially when located in critically regulatory regions, such as a promoter or enhancer [ 22 , 23 ]. We recently performed a genome-wide, allele-specific (GWAS) methylation (ASM) analysis with rs174537 to test the hypothesis that rs174537 is associated with DNA methylation levels in human liver tissues, since the liver is believed to be the primary organ for fatty acid biosynthesis [ 10 , 24 ]. Indeed, we observed significant associations between rs174537 and methylation at eight CpG sites, spanning the FADS1 promoter, a putative enhancer, and FADS2 promoter region, all located in a 12-kb regulatory region between FADS1 and FADS2 genes [ 24 ].…”

“…We recently performed a genome-wide, allele-specific (GWAS) methylation (ASM) analysis with rs174537 to test the hypothesis that rs174537 is associated with DNA methylation levels in human liver tissues, since the liver is believed to be the primary organ for fatty acid biosynthesis [ 10 , 24 ]. Indeed, we observed significant associations between rs174537 and methylation at eight CpG sites, spanning the FADS1 promoter, a putative enhancer, and FADS2 promoter region, all located in a 12-kb regulatory region between FADS1 and FADS2 genes [ 24 ]. However, since DNA methylation is known to be highly tissue-dependent, the objective of this study was to determine if the strong ASM between rs174537 and these key CpG sites could be detected in easily accessible peripheral samples and biological fluids, such as saliva and whole blood.…”

Biological Role of Unsaturated Fatty Acid Desaturases in Health and Disease

“…Several transcription factor binding site motifs were identified in promoters regions of unsaturated fatty acid desaturases, including nuclear factor Y (NF-Y), CCAAT enhancer binding protein (C/EBP), sterol regulatory element (SRE), nuclear factor 1 (NF-1), stimulatory protein 1 (Sp1), activated protein 1 (AP1), hepatocyte nuclear factor 4α (HNF4α), and peroxisome proliferator activated receptor γ (PPARγ) [42][43][44]. Epigenetic alterations (i.e., methylation of DNA) may also contribute to changes in desaturase activity [45,46]. Furthermore, recent findings suggest that D5D and D6D activity can change in response to the cytosolic NAD + /NADH ratio.…”