Protein methylation pathways comprise methionine adenosyltransferase (MAT), which produces S-adenosylmethionine (SAM) and SAM-dependent substrate-specific methyltransferases. However, the function of MAT in the nucleus is largely unknown. MafK represses or activates expression of heme oxygenase-1 (HO-1) gene, depending on its heterodimer partners. Proteomics analysis of MafK revealed its interaction with MATIIα, a MAT isozyme. MATIIα was localized in nuclei and found to form a dense network with chromatin-related proteins including Swi/Snf and NuRD complexes. MATIIα was recruited to Maf recognition element (MARE) at HO-1 gene. When MATIIα was knocked down in murine hepatoma cell line, expression of HO-1 was derepressed at both basal and induced levels. The catalytic activity of MATIIα, as well as its interacting factors such as MATIIβ, BAF53a, CHD4, and PARP1, was required for HO-1 repression. MATII serves as a transcriptional corepressor of MafK by interacting with chromatin regulators and supplying SAM for methyltransferases.

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Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stress-induced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.

Biologicalactivities of four chemically synthesized trichostatin-related compounds, (R)-trichostatin A, (S)-trichostatin A, (It )-trichostatic acid, and (S)-trichostatic acid, were investigated. Assays of differentiation-inducing activity in Friend leukemia cells and G2-arresting activity in the cell cycle of normal rat fibroblast cells were used as monitoring systems for comparing the bioactivities of these compounds.The results clearly showedthat both of the enantiomers of trichostatic acid had no activity in both the assay systems. In the case of (S)-trichostatin A, the antipode of naturally occurring trichostatin A, 50% effective concentrations were determined to be 50~70-fold higher than those of (R )-trichostatin A. The relationship between this ratio and the value of enantiomeric excess strongly suggests that (5)-trichostatin A is also biologically inactive. These results indicate that the absolute configuration and the presence of the hydroxamate group of trichostatin A are essential for its biological activity. TrichostatinsA and C (Fig. 1), which had been originally discovered as fungistatic antibiotics by Tsuji et ah, Tsuji and Kobayashi1 2), were also found by our group to be very potent inducers of erythroid differentiation in mouse Friend leukemia cells3>4). More interestingly, we found that a low concentration of trichostatin A reversibly blocked the cell cycle of normal fibroblast cells at both the G! and G2 phases and induced the formation of proliferative tetraploid cells after release from the G2-arrest by this drug5). These observations suggest that trichostatin A will be useful for studying the mechanisms of cell differentiation and the eukaryotic cell cycle.MORIOKA et ah reported independently that trichostatic acid (Fig. 1) showed a similar effect on Friend leukemia cells, although its effective concentration was about 1,000-fold higher than that of trichostatin A6). Recently, they have also reported that the racemic form of trichostatic acid had no activity as a differentiation inducer7). To verify these observations and the relationship of stereochemistry to biological activity, MORI and Koseki synthesized both enantiomers of trichostatin A and trichostatic acid8). In this paper, we compared their biological activities, and concluded that (i^-trichostatin A, a naturally occurring substance, was the only active agent among these four compounds. Materials and Methods Chemicals and Physico-chemical MeasurementsPurified preparations of (R )-trichostatin A ( > 93% enantiomeric excess), (S)-trichostatin A ( >93 % enantiomeric excess), (i? )-trichostatic acid (98 %

Although several molecular markers for human breast cancer exist, their versatility is limited. Here we demonstrate, through a differential proteome analysis utilizing the fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) method between seven cancer cells and one normal cell, that the presence of cooperatively expressed annexin-2 and galectin-1 without tropomyosin-1 in a tissue could be used to diagnose metastatic breast cancer. Interestingly, in a metastatic cancer cell, the expression of the former two together with highly expressed cofilin-1 activates the Rho signal pathway to aggressively form disorganized actin filaments. Despite the excess expression of annexin-2 and galectin-1 in the normal cell, the highly expressed tropomyosin-1 counteracted the activity of cofilin-1 and stabilized the filaments, resulting in the restoration of the disorganization. This phenomenon suggests that enhancement of tropomyosin-1 should be used as therapy for metastatic breast cancer.