Age-dependent decreases in DNA methyltransferase levels and low transmethylation micronutrient levels synergize to promote overexpression of genes implicated in autoimmunity and acute coronary syndromes

Abstract

T cell DNA methylation levels decline with age, activating genes such as KIR and TNFSF7 (CD70), implicated in lupus-like autoimmunity and acute coronary syndromes. The mechanisms causing age-dependent DNA demethylation are unclear. Maintenance of DNA methylation depends on DNA methyltransferase 1 (Dnmt1) and intracellular S-adenosylmethionine (SAM) levels, and is inhibited by S-adenosylhomocysteine (SAH). SAM levels depend on dietary micronutrients including folate and methionine. SAH levels depend on serum homocysteine concentrations. T cell Dnmt1 levels also decline with age. We hypothesized that age-dependent Dnmt1 decreases synergize with low folate, low methionine or high homocysteine levels to demethylate and activate methylation-sensitive genes. T cells from healthy adults ages 22–81, stimulated and cultured with low folate, low methionine, or high homocysteine concentrations showed demethylation and overexpression of KIR and CD70 beginning at age ∼50 and increased further with age. The effects were reproduced by Dnmt1 knockdowns in T cells from young subjects. These results indicate that maintenance of T cell DNA methylation patterns is more sensitive to low folate and methionine levels in older than younger individuals, due to low Dnmt1 levels, and that homocysteine further increases aberrant gene expression. Thus, attention to proper nutrition may be particularly important in the elderly.

Introduction

DNA methylation is a post-synthetic modification that silences gene expression. DNA methylation patterns are established during differentiation, and serve to suppress genes unnecessary or detrimental to the function of any given cell, but for which the cell expresses activating transcription factors. DNA methylation is particularly important in T lymphocytes, which differentiate throughout life into multiple subsets with distinct functions but express a partially overlapping repertoire of transcription factors. Consequently, some subset-specific genes are silenced primarily by DNA methylation. For example, inhibiting T lymphocyte DNA methylation activates the Th1 cytokine IFN-γ in Th2 cells, the Th2 cytokine IL-4 in Th1 cells, the cytotoxic molecule perforin in CD4 cells and the KIR gene family, normally confined to natural killer (NK) lymphocytes, on CD4 and CD8 cells (Basu et al., 2009, Liu et al., 2009a, Lu et al., 2003, Richardson, 2007). These studies support the importance of DNA methylation in maintaining subset-specific T lymphocyte gene expression.

DNA methylation patterns must be replicated each time a cell divides. However, the maintenance of DNA methylation patterns deteriorates with age in most cells, causing aberrant methylation of some CpG islands that can lead to malignant transformation, as well as demethylation of other regions that promotes gene overexpression (Yung and Julius, 2008). In T cells a small number of CpG islands methylate with age, suppressing gene expression (Tra et al., 2002). However, the predominant effect is a decline in total deoxymethylcytosine (d^mC) content, causing aberrant overexpression of genes normally silenced by methylation (Richardson, 2003). The mechanisms causing T cell DNA demethylation are important to understand, because the demethylation may contribute to the development of lupus-like autoimmunity as well as acute coronary syndromes, by causing aberrant overexpression of genes including the KIR gene family, perforin, CD70, IFN-γ, LFA-1 and others (Jones and Chen, 2006, Kaplan et al., 2004, Liu et al., 2009b, Liu et al., 2009a, Lu et al., 2002, Lu et al., 2005, Nakajima et al., 2003).

DNA methylation patterns are replicated during mitosis by the maintenance DNA methyltransferase Dnmt1. Dnmt1 binds proliferating cell nuclear antigen (PCNA) in the replication fork and recognizes CpG pairs (Iida et al., 2002). If the parent DNA strand is methylated, Dnmt1 catalyzes the transfer of the methyl group from S-adenosylmethionine (SAM) to the corresponding dC in the daughter strand, producing 5-methylcytosine (^mC) and S-adenosylhomocysteine (SAH) (Ross and Poirier, 2002):

$$\text{SAM}+-\text{CpG}-\stackrel{\text{Dnmt}1}{\to }\text{SAH}+-{}^{\text{m}}\text{CpG}-\text{.}$$

The forward velocity of this reaction is directly proportional to SAM concentrations and Dnmt1 activity, and inversely proportional to SAH:

$$V=\frac{\text{k}[\text{Dnmt}1][\text{SAM}]}{[\text{SAH}]}$$

T cell Dnmt1 levels decrease with age, which contributes to the decline in overall d^mC content (Zhang et al., 2002). However, diet is also important in replicating DNA methylation patterns, and is a mechanism by which the environment can modify the epigenome. SAM levels depend on micronutrients such as folate and methionine (Met) (Ross and Poirier, 2002), and restricting dietary methyl donors can demethylate DNA in tissues like the liver (Pogribny et al., 1995) and peripheral blood leukocytes (Rampersaud et al., 2000). SAH levels are also affected by diet. SAH concentrations are dependent on homocysteine (Hcy) levels, and folate is required for the metabolism of Hcy to Met (Ross and Poirier, 2002). Elevated serum Hcy levels contribute to DNA demethylation in conditions such as chronic renal failure, and folate supplementation can reverse this effect (Ingrosso et al., 2003). How dietary transmethylation micronutrients, Hcy and decreased Dnmt1 levels interact to cause decreases in DNA methylation and aberrant gene overexpression in aging is unknown.

We hypothesized that decreases in folate or Met, increases in Hcy, and age-dependent decreases in Dnmt1 levels may be synergistic in inhibiting T cell DNA methylation. We therefore cultured T cells from healthy young and older individuals, as well as T cell Dnmt1 “knockdowns”, in media containing variable amounts of folate, Met, and/or Hcy, and examined the effects on the expression and methylation of genes normally suppressed by DNA methylation. The results suggest a potentially important mechanism increasing sensitivity of older people, and perhaps people with some forms of autoimmunity, to adverse effects from a diet poor in methyl donors.