Promoter methylation and age-related downregulation of Klotho in rhesus monkey

Abstract

While overall DNA methylation decreases with age, CpG-rich areas of the genome can become hypermethylated. Hypermethylation near transcription start sites typically decreases gene expression. Klotho (KL) is important in numerous age-associated pathways including insulin/IGF1 and Wnt signaling and naturally decreases with age in brain, heart, and liver across species. Brain tissues from young and old rhesus monkeys were used to determine whether epigenetic modification of the KL promoter underlies age-related decreases in mRNA and protein levels of KL. The KL promoter in genomic DNA from brain white matter did not show evidence of oxidation in vivo but did exhibit an increase in methylation with age. Further analysis identified individual CpG motifs across the region of interest with increased methylation in old animals. In vitro methyl modification of these individual cytosine residues confirmed that methylation of the promoter can decrease gene transcription. These results provide evidence that changes in KL gene expression with age may, at least in part, be the result of epigenetic changes to the 5′ regulatory region.

The relevance of α-KLOTHO to the central nervous system: Some key questions

“…Since uremic toxins are believed to act via inflammatory pathways and we see evidence of neuroinflammation in our system, it is quite possible that elevated promoter methylation underlies the decreased brain α-Klotho seen in nephrectomized rats. Supporting this idea further, kl promoter methylation can be modulated in the brain, since an age-dependent increase that correlates with decreased protein expression has been reported in the white matter of rhesus monkeys (King et al, 2012). Altered promoter methylation in the brains of our animals is clearly a hypothesis that can be tested relatively easily.…”

“…Some studies of α-Klotho gene expression have been reported, indicating that the kl promoter can be activated by testosterone and the androgen receptor (Hsu et al, 2014), and by the transcription factor Egr-1 (Choi et al, 2010). Perhaps most intriguingly however, α-Klotho transcription is strongly repressed by promoter methylation (Azuma et al, 2012;King et al, 2012;Sun et al, 2012). This phenomenon has been suggested to underlie the tightly controlled tissue-specific pattern of α-Klotho expression (Azuma et al, 2012) and is reported to increase with age (King et al, 2012).…”

“…Perhaps most intriguingly however, α-Klotho transcription is strongly repressed by promoter methylation (Azuma et al, 2012;King et al, 2012;Sun et al, 2012). This phenomenon has been suggested to underlie the tightly controlled tissue-specific pattern of α-Klotho expression (Azuma et al, 2012) and is reported to increase with age (King et al, 2012). Interestingly, increased KL promoter methylation has also been described in a number of cancers, including cervical carcinoma, colorectal, breast and gastric cancers Pan et al, 2011;Rubinek et al, 2012;Wang et al, 2011).…”

Analysis of DNA modifications in aging research

“…Additionally, pyrosequencing requires multiple sequencing primers and Sanger sequencing traditionally required cloning of PCR products, a laborious task (Zhang et al 2009). Nevertheless, these techniques have been used to great effect to generate base-specific analysis of selected regions and changes in methylation, but not hydroxymethylation, with aging (Noer et al 2007;Maegawa et al 2010;King et al 2012). …”

Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation

“…Additionally the validation approach used can be a model for qualification of other capture probe sets developed in the future. An exciting future direction of BOCS methodology in general will be the application of this technology to other important animal models in biomedical research, such as non-human primates (King et al 2012) and mice, which have been established as important model systems for disease research in the areas of neuroscience, substance abuse, and aging.…”