Inhibition of promoter activity by methylation: possible involvement of protein mediators.

To study the relationship between DNA methylation and promoter activity we have methylated in vitro the promoters of the mouse metallothionein I gene and the herpes simplex virus thymidine kinase gene. We have transiently transfected these promoters fused to the human growth hormone in their methylated or unmethylated state into mouse L or F9 cells. Promoters methylated by methylase (M.) Hpa II and M.Hha I caused inhibition of reporter gene expression in L cells but not in F9 cells, while methylation of all CpGs by M.Sss I caused inhibition in both cell lines. Repression of promoter activity by M.Hpa II and M.Hha I methylation, but not by M.Sss I methylation, could be alleviated by cotransfection with an excess of untranscribable DNA methylated with M.Sss I. The methylated sites in nuclei isolated from the transfected L cells, but not F9 cells, were found to be protected from Msp I digestion. Taken together these results suggest that a factor present in L cells and missing in F9 cells mediates the methylation-directed inhibition of promoter activity. The ability of methylated DNA to overcome the inhibition seems to reflect competition for the mediator factor. Interestingly, treatment with Zn2+ ions brought about activation of the methylated promoter of the metallothionein gene. Similarly, butyrate could override the repression of the thymidine kinase methylated promoter. These activations were not accompanied by demethylation of the promoter or displacement of the mediator factor.     

Differentially methylated alleles in a distinct region of the human interleukin-1alpha promoter are associated with allele-specific expression of IL-1alpha in CD4+ T cells

Cytokine secretion profiles of activated T cells are critical for maintaining the immunologic balance between protection and tolerance. In mice, several cytokines have been reported to exhibit monoallelic expression. Previously, we found that the human interleukin-1 alpha (IL1A) gene exhibits a stable allele-specific expression pattern in CD4+ T-cell clones. We investigated whether DNA methylation is involved in the allele-specific expression of IL-1alpha. Here, we show that differential methylation of CpGs in the proximal promoter region is associated with allele-specific expression of IL-1alpha in CD4+ T cells. The differential methylation pattern is already observed in naive T cells. In keratinocytes, which constitutively produce IL-1alpha, the proximal promoter is hypomethylated. CpGs located further upstream and in intron 4 were almost all methylated, irrespective of expression. Treatment of nonexpressing cells and of T-cell clones with 5-aza-2'deoxycytidine induced IL-1alpha expression in the nonexpressing cells and induced expression of the formerly silent allele in T-cell clones. In addition, electrophoretic mobility shift assays showed that methylation of CpGs in the proximal promoter resulted in direct inhibition of binding of nuclear factor(s). Taken together, these results suggest that allele-specific expression of IL-1alpha in CD4+ cells is achieved, at least in part, by differential methylation of the promoter.     

Regulation of the expression of the tissue transglutaminase gene by DNA methylation

We have investigated the role of DNA methylation in the regulation of the expression of the human tissue transglutaminase gene. Studies on the methylation of the transglutaminase promoter in normal and neoplastic human cells demonstrated that the promoter is methylated in vivo and hypomethylation of the promoter is correlated with constitutive gene expression. Demethylation of the promoter in vivo by treatment of the cells with 5-azacytidine increased transglutaminase expression and hypermethylation of the promoter in vitro suppressed its activity. These studies suggest that alternations in DNA methylation may be one of the mechanisms regulating the tissue-specific expression of the tissue transglutaminase gene.     

Epigenetic control of vasopressin expression is maintained by steroid hormones in the adult male rat brain

Although some DNA methylation patterns are altered by steroid hormone exposure in the developing brain, less is known about how changes in steroid hormone levels influence DNA methylation patterns in the adult brain. Steroid hormones act in the adult brain to regulate gene expression. Specifically, the expression of the socially relevant peptide vasopressin (AVP) within the bed nucleus of the stria terminalis (BST) of adult brain is dependent upon testosterone exposure. Castration dramatically reduces and testosterone replacement restores AVP expression within the BST. As decreases in mRNA expression are associated with increases in DNA promoter methylation, we explored the hypothesis that AVP expression in the adult brain is maintained through sustained epigenetic modifications of the AVP gene promoter. We find that castration of adult male rats resulted in decreased AVP mRNA expression and increased methylation of specific CpG sites within the AVP promoter in the BST. Similarly, castration significantly increased estrogen receptor α (ERα) mRNA expression and decreased ERα promoter methylation within the BST. These changes were prevented by testosterone replacement. This suggests that the DNA promoter methylation status of some steroid responsive genes in the adult brain is actively maintained by the presence of circulating steroid hormones. The maintenance of methylated or demethylated states of some genes in the adult brain by the presence of steroid hormones may play a role in the homeostatic regulation of behaviorally relevant systems.     

GATA3 up-regulation associated with surface expression of CD294/CRTH2: a unique feature of human Th cells

GATA-3 and T-box expressed in T cells (T-bet) play central roles in Th-cell development and function. Consistently, studies in mice document their selective expression in Th1 and Th2 cells, respectively. In contrast, it is not clear whether these genes are regulated in human Th cells. Here we show that T-bet expression is polarized to a comparable degree in human and mouse Th-cell cultures, while only mouse GATA3 is subject to substantial regulation. This did not reflect differential skewing efficiency in human versus mouse cultures, as these contained similar frequencies of IFN-gamma- and IL-4-producing cells. However, GATA-3 was expressed at significantly higher levels in human IL-4-producing cells enriched via capture with monoclonal antibodies (mAbs) against the PGD(2) receptor, CRTH2, the best selective Th2-cell surface marker to date. Along with increased IL-4 and GATA-3, CRTH2(+) Th cells isolated from Th2-skewed cultures or the circulating memory pool exhibited markedly decreased IFN-gamma and T-bet expression. Thus, the human GATA-3 gene is not regulated in response to polarizing signals that are sufficient to direct Th2-specific expression in mouse cells. This postulates the involvement of an additional level of complexity in the regulation of human GATA-3 expression and stresses the existence of nontrivial differences in the regulation of human versus mouse T-cell function.