Methylation-mediated silencing of TMS1/ASC is accompanied by histone hypoacetylation and CpG island-localized changes in chromatin architecture.

Aberrant methylation of CpG-dense islands in the promoter regions of genes is an acquired epigenetic alteration associated with the silencing of tumor suppressor genes in human cancers. In a screen for endogenous targets of methylation-mediated gene silencing, we identified a novel CpG island-associated gene, TMS1, which is aberrantly methylated and silenced in response to the ectopic expression of DNA methyltransferase-1. TMS1 functions in the regulation of apoptosis and is frequently methylated and silenced in human breast cancers. In this study, we characterized the methylation pattern and chromatin architecture of the TMS1 locus in normal fibroblasts and determined the changes associated with its progressive methylation. In normal fibroblasts expressing TMS1, the CpG island is defined by an unmethylated domain that is separated from densely methylated flanking DNA by distinct 5' and 3' boundaries. Analysis of the nucleoprotein architecture of the locus in intact nuclei revealed three DNase I-hypersensitive sites that map within the CpG island. Strikingly, two of these sites coincided with the 5'- and 3'-methylation boundaries. Methylation of the TMS1 CpG island was accompanied by loss of hypersensitive site formation, hypoacetylation of histones H3 and H4, and gene silencing. This altered chromatin structure was confined to the CpG island and occurred without significant changes in methylation, histone acetylation, or hypersensitive site formation at a fourth DNase I-hypersensitive site 2 kb downstream of the TMS1 CpG island. The data indicate that there are sites of protein binding and/or structural transitions that define the boundaries of the unmethylated CpG island in normal cells and that aberrant methylation overcomes these boundaries to direct a local change in chromatin structure, resulting in gene silencing.     

The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa

The circadian clock of Neurospora broadly regulates gene expression and is synchronized with the environment through molecular responses to changes in ambient light and temperature. It is generally understood that light entrainment of the clock depends on a functional circadian oscillator comprising the products of the wc-1 and wc-2 genes as well as those of the frq gene (the FRQ/WCC oscillator). However, various models have been advanced to explain temperature regulation. In nature, light and temperature cues reinforce one another such that transitions from dark to light and/or cold to warm set the clock to subjective morning. In some models, the FRQ/WCC circadian oscillator is seen as essential for temperature-entrained clock-controlled output; alternatively, this oscillator is seen exclusively as part of the light pathway mediating entrainment of a cryptic "driving oscillator" that mediates all temperature-entrained rhythmicity, in addition to providing the impetus for circadian oscillations in general. To identify novel clock-controlled genes and to examine these models, we have analyzed gene expression on a broad scale using cDNA microarrays. Between 2.7 and 5.9% of genes were rhythmically expressed with peak expression in the subjective morning. A total of 1.4-1.8% of genes responded consistently to temperature entrainment; all are clock controlled and all required the frq gene for this clock-regulated expression even under temperature-entrainment conditions. These data are consistent with a role for frq in the control of temperature-regulated gene expression in N. crassa and suggest that the circadian feedback loop may also serve as a sensor for small changes in ambient temperature.     

Temperature-sensitive and circadian oscillators of Neurospora crassa share components

In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ΔFWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO.     

DNA and histone methylation in plants

Heritable patterns of gene activity and gene silencing arise by the formation and the propagation of specific chromatin states that restrict or permit gene expression. In mammals and in plants, restrictive heterochromatin is associated with the hypermethylation of DNA at CG sites and with the specific modification of histones, such as the methylation of histone H3 at lysine 9 (H3K9(Me)). In addition to CG methylation, plant nuclear DNA packaged in restrictive chromatin is also usually methylated in cytosines outside a CG sequence context. The functional relationship between an unexpectedly complex plant DNA-methylation system and histone modifications that lead to chromatin compaction and gene silencing is under intense scrutiny. The results of recent studies indicate intriguing links between chromatin remodeling, histone methylation, DNA methylation and RNA interference.     

Analysis of target sequences of DDM1s in Brassica rapa by MSAP

DNA methylation is an important epigenetic modification regulating gene expression and transposon silencing. Although epigenetic regulation is involved in some agricultural traits, there has been relatively little research on epigenetic modifications of genes in Brassica rapa, which includes many important vegetables. In B. rapa, orthologs of DDM1, a chromatin remodeling factor required for maintenance of DNA methylation, have been characterized and DNA hypomethylated knock-down plants by RNAi (ddm1-RNAi plants) have been generated. In this study, we investigated differences of DNA methylation status at the genome-wide level between a wild-type (WT) plant and a ddm1-RNAi plant by methylation-sensitive amplification polymorphism (MSAP) analysis. MSAP analysis detected changes of DNA methylation of many repetitive sequences in the ddm1-RNAi plant. Search for body methylated regions in the WT plant revealed no difference in gene body methylation levels between the WT plant and the ddm1-RNAi plant. These results indicate that repetitive sequences are preferentially methylated by DDM1 genes in B. rapa.     

The Neurospora circadian clock: simple or complex?

The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity. We conclude that there may be many gene products involved in the clock mechanism, there may be multiple interacting oscillators comprising the clock mechanism, there may be feedback from output pathways onto the oscillator(s) and from the oscillator(s) onto input pathways, and there may be several independent clocks coexisting in one organism. Thus even a relatively simple lower eukaryote can be used to address questions about a complex, networked circadian system.     

A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.     

A diverse epigenetic landscape at human exons with implication for expression

DNA methylation is an important epigenetic marker associated with gene expression regulation in eukaryotes. While promoter methylation is relatively well characterized, the role of intragenic DNA methylation remains unclear. Here, we investigated the relationship of DNA methylation at exons and flanking introns with gene expression and histone modifications generated from a human fibroblast cell-line and primary B cells. Consistent with previous work we found that intragenic methylation is positively correlated with gene expression and that exons are more highly methylated than their neighboring intronic environment. Intriguingly, in this study we identified a unique subset of hypomethylated exons that demonstrate significantly lower methylation levels than their surrounding introns. Furthermore, we observed a negative correlation between exon methylation and the density of the majority of histone modifications. Specifically, we demonstrate that hypo-methylated exons at highly expressed genes are associated with open chromatin and have a characteristic histone code comprised of significantly high levels of histone markings. Overall, our comprehensive analysis of the human exome supports the presence of regulatory hypomethylated exons in protein coding genes. In particular our results reveal a previously unrecognized diverse and complex role of the epigenetic landscape within the gene body.