DNA methylation within the I.4 promoter region correlates with CYPl19A1 gene expression in human ex vivo mature omental and subcutaneous adipocytes

Abstract

BackgroundDNA methylation at specific CpG sites within gene promoter regions is known to regulate transcriptional activity in vitro. In human adipose tissue, basal transcription of the aromatase (CYP19A1) gene is driven primarily by the I.4 promoter however the role of DNA methylation in regulating expression in ex vivo mature adipocytes is unknown. This observational study reports the correlation of DNA methylation within the I.4 promoter region of human mature subcutaneous and omental adipocytes with aromatase expression and body composition measures.MethodsOmental and subcutaneous adipose tissue were collected from 25 obese subjects undergoing bariatric surgery and the mature adipocyte fraction purified. DNA methylation status of 5 CpG sites within a 550 base pair region encompassing the transcription start site (TSS) of promoter I.4 was determined using pyrosequencing. Relative aromatase and I.4 promoter specific mRNA expression was determined by qRT-PCR and whole body DXA performed in 25 participants.ResultsSite-specific DNA methylation varied from 21 ± 10% to 81 ± 11%. In omental adipocytes percentage methylation at the I.4.1 and I.4.2 CpG sites, but not other nearby sites, was negatively correlated with relative aromatase mRNA expression (R = - 0.52, P = 0.017 and R = - 0.52, P = 0.015). In contrast subcutaneous adipocytes percentage DNA methylation at the I.4.3 and I.4.5 sites were positively correlated with relative aromatase mRNA expression (R = 0.47, P = 0.022 and R = 0.55, P = 0.004). In a small subset of patients DNA methylation at the I.4.5 site was also positively correlated with whole body lean mass, bone mineral content and density.ConclusionsIn conclusion in mature adipocytes, the primary source of estradiol after menopause, increasing DNA methylation was correlated with aromatase mRNA expression and thus estradiol biosynthesis. These findings support a tissue-specific epigenetic regulation of the basal promoter activity in mature adipocytes; the mechanisms influencing this regulation and its physiological role remain to be elucidated.

Developmental vitamin D deficiency increases foetal exposure to testosterone

“…Consistent with this we show DVDdeficiency produced a significant reduction in brain aromatase which is the principal catabolic enzyme in the elimination of testosterone [50]. We next explored the methylation of the aromatase promoter as one plausible mechanism [51][52][53]. We show one of the two brain aromatase promoters PII is hyper-methylated in male DVDdeficient foetal brains.…”

The role of estrogens in the adipose tissue milieu

“…Using this method, DNA methylation was found to regulate aromatase expression in primary APs, 75 breast adipose fibroblasts, 76 skin fibroblasts, 77 endometrial stromal cells, 78 placental choriocarcinoma, 79 placenta, 80 and neuroglia. 93 Aromatase expression was positively correlated with DNA methylation in subcutaneous depots but inversely correlated in visceral adipose tissue, potentially indicating a fundamental divide between subcutaneous and visceral epigenetic mechanisms. 91 The mechanism by which DNA methylation regulates aromatase expression may be direct, that is, by changes at the aromatase promoter itself, 75,77 or indirect, by epigenetic alterations at unknown genomic loci.…”

“…In one study that used mature adipocytes isolated ex vivo from patients undergoing bariatric surgery, DNA methylation of the adipose-specific aromatase promoter (I.4) was measured in both subcutaneous and visceral depots, along with the expression of aromatase itself. 93 Aromatase expression was positively correlated with DNA methylation in subcutaneous depots but inversely correlated in visceral adipose tissue, potentially indicating a fundamental divide between subcutaneous and visceral epigenetic mechanisms. 93 This study is one of the few to examine actual patient samples, rather than cultured cells, and to show epigenetic differences at specific genomic sites that may be of clinical relevance.…”

“…93 Aromatase expression was positively correlated with DNA methylation in subcutaneous depots but inversely correlated in visceral adipose tissue, potentially indicating a fundamental divide between subcutaneous and visceral epigenetic mechanisms. 93 This study is one of the few to examine actual patient samples, rather than cultured cells, and to show epigenetic differences at specific genomic sites that may be of clinical relevance. An important caveat is that the expression of aromatase within adipose tissues is primarily driven by APs within the depot, 94 not mature adipocytes, suggesting that other important primary estrogen sources within the tissue remain unsampled.…”

“…81 Epigenetic regulation of aromatase by DNA methylation appears widely conserved across evolution, including in chicken, 82 rat, 83 alligator, 84 sea bass, 85 zebrafish, 86 perch, 87 eel, 88 buffalo, 89,90 sheep, and cattle. 93 This study is one of the few to examine actual patient samples, rather than cultured cells, and to show epigenetic differences at specific genomic sites that may be of clinical relevance. 76 There is evidence that one of the ERs, estrogen receptor alpha (ERα), is epigenetically regulated by DNA methylation in rat brain; 92 however, substantially less is known about the DNA methylation patterns of the aromatase promoter in subcutaneous versus visceral adipose tissues in vivo.…”