These studies demonstrate an age-dependent demethylation and overexpression of genes normally suppressed by DNA methylation when T cells are cultured in media with low Met or folate concentrations, and that Hcy potentiates the effect. While multiple age-dependent changes in transmethylation biochemistry may contribute, age-dependent decreases in Dnmt1 are likely important, since Dnmt1 siRNA knockdowns in T cells from young people reproduced the effect. T cells also express DNA methyltransferase 3a, and Dnmt3a levels decrease with aging (Liu et al., 2009b
). However, selective Dnmt1 and Dnmt3a knockdowns demonstrate that Dnmt3a has only a minor role in maintenance T cell DNA methylation (Liu et al., 2009b
) and so is unlikely to have a significant role in the age-dependent effects reported here. T cells express very little Dnmt3b (Zhang et al., 2002
) and its role in T cell function is uncertain.
There was little effect of varying folate or Met concentrations on T cells from subjects up to ~50 years of age, after which methylation sensitive gene expression increased steadily with age, particularly when dietary transmethylation factors were limiting. Since Dnmt1 levels decrease throughout life (Zhang et al., 2002
) the change may reflect a threshold effect of Dnmt1, such that Dnmt1 levels must drop below a critical level before alterations in the micronutrient concentrations have an effect.
Hcy also contributed to gene overexpression in T cells from older people, but only when folate levels were low. Hcy is converted intracellularly to SAH, and SAH binds the catalytic domain of most SAM-dependent methyltransferases with high affinity, making it a potent inhibitor of transmethylation reactions (James et al., 2002
). Folate is required for the conversion of Hcy to Met (James et al., 2002
), and the lower folate levels used in these studies may thus decrease the efficiency of this clearance mechanism.
In these studies Met/folate restriction had a smaller effect on KIR expression in CD4+ than CD8+ T cells from older people. Similarly, greater numbers of CD8+ T cells overexpressed CD70 relative to CD4+ cells. We and others have reported that greater numbers of CD8+ cells overexpress methylation sensitive genes in lupus, aging and following treatment with DNA methylation inhibitors (Li et al., 2008
; Liu et al., 2009a
; Lu et al., 2003
). The reason is unknown, but may reflect a differential stringency in the maintenance of methylation patterns in these subsets. However, the partial effects on KIR and CD70 in both populations suggest the existence of subsets with different Dnmt1 levels. This possibility prompted studies comparing micronutrient restriction in CD28+ and CD28− T cells, and demonstrated a greater effect on CD28− T cells. We recently reported that this subset expresses lower Dnmt1 levels than CD28+ cells from the same individuals (Liu et al., 2009b
), supporting the contention that decreased Dnmt1 levels make CD28− T cells more sensitive to nutrient restriction. However, the observation that some CD28+ cells can overexpress KIR suggests that Dnmt1 levels may vary within this subset as well. Since CD4+CD28+ cells do not aberrantly express methylation-sensitive markers like KIR though, the subset affected is not immediately obvious.
The aberrant overexpression of methylation sensitive genes can have pathologic consequences. The “senescent” CD4+CD28− T cell subset, which infiltrates atherosclerotic plaques and is implicated in plaque rupture and myocardial infarctions (Nakajima et al., 2003
), also overexpresses genes normally suppressed by DNA methylation in CD4+CD28+ T cells. The genes include the KIR gene family, CD70, IFN-γ and perforin (Liu et al., 2009b
; Nakajima et al., 2003
). This subset increases with aging as well as with proliferative stress in vitro and in chronic inflammatory diseases like rheumatoid arthritis (Vallejo, 2005
). Interestingly, serum Hcy levels increase with aging (Refsum et al., 2006
) and in patients with lupus (Roman et al., 2007
), and Hcy is implicated in atherosclerotic vascular disease (Selhub et al., 1995
) which accelerates in aging and lupus (Refsum et al., 2006
; Roman et al., 2007
). The present studies raise the possibility that increased Hcy levels may contribute to atherosclerosis and myocardial infarctions by inhibiting DNA methylation in T lymphocytes or other cell types, and raise the possibility that diets poor in Met or folate, together with effects of replicative stress on T cell Dnmt1 levels (Liu et al., 2009b
), may contribute to these conditions.
Overexpression of methylation sensitive genes may also contribute to some forms of autoimmunity. Murine T cells treated with 5-azacytidine demethylate and overexpress genes like LFA-1, making them autoreactive, and these cells cause a lupus-like disease in vivo
. T cells overexpressing LFA-1 by transfection also become autoreactive and cause a lupus-like disease in vivo (Quddus et al., 1993
; Yung et al., 1996
), suggesting that demethylation of LFA-1 and likely other genes contribute to the development of lupus-like autoimmunity . The lupus inducing drugs procainamide and hydralazine are also DNA methylation inhibitors (Cornacchia et al., 1988
), and murine T cells treated with these drugs and other DNA methylation inhibitors cause a lupus-like disease in adoptive transfer models (Quddus et al., 1993
; Yung et al., 1997
). Finally, CD4+ T cells from patients with active lupus demethylate and overexpress the same genes and demonstrate the same functional changes as those caused by DNA methylation inhibitors in vitro, indicating a role for T cell DNA demethylation in lupus-like human autoimmunity as well (Richardson, 2007
). Interestingly, a recent study of more than 1600 men and women with lupus demonstrated an age dependent increase in disease onset, with a linear increase in incidence through age 74 in men, and a steady increase in women up to menopause (Somers et al., 2007
), suggesting an age contribution to lupus.
In conclusion, our results suggest that aging, a poor diet, and increased serum Hcy levels can have functionally significant effects on the expression of genes normally suppressed by DNA methylation in T lymphocytes and perhaps other cells. Further, the methylation errors may accumulate over time. This mechanism could potentially contribute to a number of poorly understood diseases of aging, and clinical trials of folate/Met supplementation may be indicated.