Global DNA methylation in the mouse liver is affected by methyl deficiency and arsenic in a sex-dependent manner.

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.

Turning off estrogen receptor beta-mediated transcription requires estrogen-dependent receptor proteolysis.

Recent studies have shed light on the ligand-dependent transactivation mechanisms of nuclear receptors (NRs). When the ligand dose is reduced, the transcriptional activity of NRs should be downregulated. Here we show that a ubiquitin-proteasome pathway plays a key role in turning off transcription mediated by estrogen receptor beta (ERbeta). ERbeta shows estrogen-dependent proteolysis, and its degradation is regulated by two regions in the receptor. The N-terminal 37-amino acid-region is necessary for the recruitment of the ubiquitin ligase, i.e., the carboxyl terminus of HSC70-interacting protein (CHIP), to degrade ERbeta. In contrast, the C-terminal F domain protects ligand-unbound ERbeta from proteolysis to abrogate proteasome association. Suppression of CHIP by interfering RNA inhibited this switching off of receptor-mediated transcription when the ligand dose was reduced. Our results suggest that after ligand withdrawal, the active form of the NR is selectively eliminated via ligand-dependent proteolysis to downregulate receptor-mediated transcription.

Arsenite-induced thymus atrophy is mediated by cell cycle arrest: a characteristic downregulation of E2F-related genes revealed by a microarray approach.

Thymus atrophy is induced by a variety of chemicals, including environmental contaminants and is used as a sensitive index to detect their adverse effects on lymphocytes. In the present study we adopted a toxicogenomics approach to identify the pathways that mediate the atrophy induced by arsenite. We also analyzed gene expression changes observed in the course of thymus atrophy by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dexamethasone (DEX), and estradiol (E2), to determine whether arsenite induces atrophy by activating an arsenite-specific pathway or the same pathways as other chemicals. These compounds were intraperitoneally administered to C57BL/6 mice at doses that reduce thymus weight by approximately 30% within 3 days, and gene expression changes in the thymus 24 h after the administration were analyzed by using microarrays and real-time PCR. The microarray analysis showed that arsenite specifically downregulates a variety of E2F target genes that are involved in cell cycle progression. The same genes were also downregulated when mouse B-cell lymphoma A20 cells were exposed to arsenite. Arsenite exposure of the A20 cells was confirmed to induce cell cycle arrest, mainly in the G(1) phase, and reduce cell number. Cell cycle arrest in the G(1) phase was also confirmed to occur in the thymocytes of the arsenite-exposed mice. These results indicate that arsenite induces thymus atrophy through E2F-dependent cell cycle arrest. The results of this study also show that analysis of gene expression in thymuses is a useful method of obtaining clues to the pathways that mediate the effects of atrophy-inducing chemicals.

Late-onset increases in oxidative stress and other tumorigenic activities and tumors with a Ha-ras mutation in the liver of adult male C3H mice gestationally exposed to arsenic.

Tumorigenesis is a complex process involving genetic, epigenetic, and metabolic alterations. Gestational arsenic exposure has been shown to increase hepatic tumors in adult male offspring of C3H mice, which spontaneously develop hepatic tumors often harboring activating Ha-ras mutation. We explored tumor-promoting changes by gestational arsenic exposure with a focus on Ha-ras mutation and gene expression changes. The results of this study demonstrated that gestational arsenic exposure particularly increased hepatic tumors with a C61A Ha-ras mutation. Real-time PCR analyses on the adult normal livers showed that two genes (Creld2, Slc25a30), whose expression are induced by endoplasmic reticulum stress and cellular oxidative stress, respectively, were significantly upregulated and two genes (Fabp4, Ell3), whose products are involved in lipid efflux and apoptosis, respectively, were significantly downregulated more than twofold by gestational arsenic exposure compared with control mice. The expression changes in the four genes were shown to be late-onset events and to some extent to be associated with corresponding histone modifications, and not with DNA methylation changes. The gene expression changes suggested alterations in lipid metabolism and associated oxidative stress augmentation. Consistently, expression of an oxidative-stress-inducible gene heme oxygenase-1 (HO-1) was upregulated in the livers of the arsenic group. We also found increased expression of retrotransposon L1 mRNA in the tumor-bearing livers of the arsenic group in comparison with control mice. These results suggested that gestational arsenic exposure induces tumor-augmenting changes, including oxidative stress and L1 activation, in a late-onset manner, which would particularly promote tumorigenic expansion of cells with a C61A Ha-ras mutation.

A specific CpG site demethylation in the human interleukin 2 gene promoter is an epigenetic memory.

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.

Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor.

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

Full activation of estrogen receptor alpha activation function-1 induces proliferation of breast cancer cells.

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) alpha and beta, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptor-mediated 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 ERalpha. 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 ERalpha 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 ERalpha 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 ERalpha and N-CoR/SMRT and enhanced the transcriptional activity of OHT-bound ERalpha. 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.