Background: Structural changes in chromatin play essential roles in regulating eukaryotic gene expression. Silencing, potent repression of transcription in Saccharomyces cerevisiae, occurs near telomeres and at the silent mating-type loci, as well as at rDNA loci. This type of repression relates to the condensation of chromatin that occurs in the heterochromatin of multicellular organisms. Anti-silencing is a reaction by which silenced loci are de-repressed. Genetic studies revealed that several factors participate in the anti-silencing reaction. However, actions of factors and molecular mechanisms underlying anti-silencing remain unknown.

The significance of catechins, the main constituent of green tea, is being increasingly recognized with regard to cancer prevention. Catechins have been studied for interactions with various proteins, but the mechanisms of the various catechins are not yet elucidated. Based on our previous observation that nucleic acids extracted from catechintreated cells are colored, we studied whether catechins directly interact with nucleic acids using surface plasmon resonance assay (Biacore) and cold spray ionization-mass spectrometry. These two methods clearly showed that (؊)-epigallocatechin gallate (EGCG) binds to both DNA and RNA molecules: the Biacore assay indicated that four catechins bound to DNA oligomers, and cold spray ionization-mass spectrometry analysis showed one to three EGCG molecules bound to single strand 18 mers of DNA and RNA. Moreover, one or two molecules of EGCG bound to double-stranded (AG-CT) oligomers of various nucleotide lengths. These results suggest that multiple binding sites of EGCG are present in DNA and RNA oligomers. Double-stranded DNA (dsDNA) oligomers were detected only as EGCG-bound forms at high temperature, whereas at low temperature both the free and bound forms were detected, suggesting that EGCG protects dsDNA oligomers from dsDNA melting to single-stranded DNA. Because both galloyl and catechol groups of EGCG are essential for DNA binding, both groups seem to hold strands of DNA via their branching structure. These findings reveal for the first time the link between catechins and polynucleotides and will intensify our understanding of the effects of catechins on DNA in terms of cancer prevention.Green tea is an acknowledged cancer preventive in Japan (1-7). Most of the active principles are assumed to be green tea catechins because they show various cancer-preventive activities in vitro in cell culture and in vivo, including anti-oxidant, anti-cancer, and antimutagenic activities (1-7). (Ϫ)-Epigallocatechin gallate (EGCG), 2 (Ϫ)-epigallocatechin (EGC), (Ϫ)-epicatechin gallate (ECG), (Ϫ)-epicatechin (EC), (Ϫ)-gallocatechin gallate (GCG), and (ϩ)-catechin are the major components of green tea polyphenols (8), and EGCG is the main constituent of green tea (9). Since we first reported in 1987 that topical applications of EGCG inhibited tumor promotion with teleocidin of the 12-O-tetradecanoylphorbol-13-acetate types and okadaic acid on mouse skin in two-stage carcinogenesis experiments (10, 11), we have studied the mechanisms of action of green tea catechins in human cancer cell lines (12-18). During our study of EGCG, we often observed that nucleic acids extracted from EGCGtreated human cancer cells were catechin colored, suggesting that EGCG binds to DNA and RNA molecules in cells. Our speculation was strengthened by results showing that 3 H-EGCG was found in nuclei of human lung cancer cell line PC-9 1 h after treatment (13). Although EGCG apparently protects against DNA damage induced by free radicals, ionization, and ultraviolet radiation, as well as DNA methylation (5,19,20),...

We report a novel chromatin-modulating factor, nuclear FK506-binding protein (FKBP). It is a member of the peptidyl prolyl cis-trans isomerase (PPIase) family, whose members were originally identified as enzymes that assist in the proper folding of polypeptides. The endogenous FKBP gene is required for the in vivo silencing of gene expression at the rDNA locus and FKBP has histone chaperone activity in vitro. Both of these properties depend on the N-terminal non-PPIase domain of the protein. The C-terminal PPIase domain is not essential for the histone chaperone activity in vitro, but it regulates rDNA silencing in vivo. Chromatin immunoprecipitation showed that nuclear FKBP associates with chromatin at rDNA loci in vivo. These in vivo and in vitro findings in nuclear FKBPs reveal a hitherto unsuspected link between PPIases and the alteration of chromatin structure.

The H1N1 influenza A virus of swine-origin caused pandemics throughout the world in 2009 and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. The threat of influenza A thus remains a serious global health issue and novel drugs that target these viruses are highly desirable. Influenza A possesses an endonuclease within its RNA polymerase which comprises PA, PB1 and PB2 subunits. To identify potential new anti-influenza compounds in our current study, we screened 33 different types of phytochemicals using a PA endonuclease inhibition assay in vitro and an anti-influenza A virus assay. The marchantins are macrocyclic bisbibenzyls found in liverworts, and plagiochin A and perrottetin F are marchantin-related phytochemicals. We found from our screen that marchantin A, B, E, plagiochin A and perrottetin F inhibit influenza PA endonuclease activity in vitro. These compounds have a 3,4-dihydroxyphenethyl group in common, indicating the importance of this moiety for the inhibition of PA endonuclease. Docking simulations of marchantin E with PA endonuclease suggest a putative “fitting and chelating model” as the mechanism underlying PA endonuclease inhibition. The docking amino acids are well conserved between influenza A and B. In a cultured cell system, marchantin E was further found to inhibit the growth of both H3N2 and H1N1 influenza A viruses, and marchantin A, E and perrotein F showed inhibitory properties towards the growth of influenza B. These marchantins also decreased the viral infectivity titer, with marchantin E showing the strongest activity in this assay. We additionally identified a chemical group that is conserved among different anti-influenza chemicals including marchantins, green tea catechins and dihydroxy phenethylphenylphthalimides. Our present results indicate that marchantins are candidate anti-influenza drugs and demonstrate the utility of the PA endonuclease assay in the screening of phytochemicals for anti-influenza characteristics.

The influenza A RNA polymerase possesses endonuclease activity to digest the host mRNA. Thus this endonuclease domain can be a target of anti-influenza A virus drug. Here we report that green tea catechins inhibit this viral endonuclease activity and that their galloyl group is important for their function. Docking simulations revealed that catechins with galloyl group fit well into the active pocket of the endonuclease domain to enable stable binding. Our results provide useful data that make it possible to refine and optimize catechin-based drug design more readily for stability.