DNA demethylation plays a critical role in transcriptional regulation in differentiated somatic cells. However, there is no experimental evidence that CpG methylation in a small region of a genome restricts gene expression. Here, we show that the anti-CD3repsilon/CD28 antibody stimulation of human CD4+ T cells induces IL2 expression following epigenetic changes, including active demethylation of a specific CpG site, recruitment of Oct-1, and changes in histone modifications. When the stimulatory signal is withdrawn, Oct-1 remains on the enhancer region as a stable marker of the stimulation, causing the second induction to be faster and stronger. Our observations indicate that Oct-1-binding followed by CpG demethylation are key events in the epigenetic regulation of IL2 expression and may act as a memory of the regulatory event.

Recent evidence indicates that the transactivation of estrogen receptor a (ERa) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERa is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERa and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERa and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERa. In CHIPÀ/À cells, the degradation of unliganded ERa was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP þ / þ cells. Our findings suggest that ERa is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERa. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) ␣ and ␤, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptormediated responses. By using the truncated types of ER mutants, we revealed that activation function-1 (AF-1) activity was necessary for the BBP-dependent transactivation function of ER␣. AF-1 is also known to be responsible for the partial agonistic activity of tamoxifen. Whereas tamoxifen exhibits an anti-estrogenic effect on proliferation of the MCF-7 breast cancer cell line, BBP showed an estrogenic effect on MCF-7 to stimulate proliferation. In vivo and in vitro binding assays revealed that whereas 4-hydroxytamoxifen (OHT) induced binding of ER␣ to both an AF-1 coactivator complex (p68/p72 and p300) and corepressor complexes (N-CoR/SMRT), BBP selectively enhanced the binding to the AF-1 coactivators. We also showed that the transcriptional activity of OHT-bound ER␣ was modulated by the ratio between the AF-1 coactivator and corepressor complexes. Expression of a dominant-negative type of N-CoR inhibited the interaction between OHT-bound ER␣ and NCoR/SMRT and enhanced the transcriptional activity of OHT-bound ER␣. Furthermore, the cell growth of MCF-7 stably expressing the dominant-negative type of N-CoR was enhanced by the addition of OHT. These results indicated that fully activated AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen.

Arsenic, a carcinogen, is assumed to induce global DNA hypomethylation by consuming the universal methyl donor S-adenosylmethionine (SAM) in the body. A previous study reported that a methyl-deficient diet (MDD) with arsenic intake greatly reduced global DNA methylation (the content of 5-methylcytosine) in the liver of male C57BL/6 mice. In the present study, we investigated the DNA methylation level, SAM content, and expression of DNA methyltransferases (DNMTs) in the liver of male and female C57BL/6 mice fed a methyl-sufficient diet (MSD), an MDD, or an MDD + arsenic. The DNA methylation level was accurately determined by measuring the content of genomic 5-methyldeoxycytidine (5medC) by high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) using stable-isotope-labeled 5medC and deoxycytidine (dC) as internal standards. The results of this study revealed that while the MDD and arsenic tended to reduce the genomic 5meC content in the male mice livers, the MDD + arsenic significantly increased the 5meC content in the female mice livers. Another unexpected finding was the small differences in 5meC content among the groups. The MDD and MDD + arsenic suppressed DNMT1 expression only in the male mice livers. In contrast, SAM content was reduced by the MDD and MDD + arsenic only in the livers of female mice, showing that the changes in 5meC content were not attributable to SAM content. The sex-dependent changes in 5meC content induced by methyl deficiency and arsenic may be involved in differences in male and female susceptibility to diseases via epigenetic modification of physiological functions.