MethylScreen: DNA methylation density monitoring using quantitative PCR.

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5' region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.

Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets.

Using a unique microarray platform for cytosine methylation profiling, the DNA methylation landscape of the human genome was monitored at more than 21,000 sites, including 79% of the annotated transcriptional start sites (TSS). Analysis of an oligodendroglioma derived cell line LN-18 revealed more than 4000 methylated TSS. The gene-centric analysis indicated a complex pattern of DNA methylation exists along each autosome, with a trend of increasing density approaching the telomeres. Remarkably, 2% of CpG islands (CGI) were densely methylated, and 17% had significant levels of 5 mC, whether or not they corresponded to a TSS. Substantial independent verification, obtained from 95 loci, suggested that this approach is capable of large scale detection of cytosine methylation with an accuracy approaching 90%. In addition, we detected large genomic domains that are also susceptible to DNA methylation reinforced inactivation, such as the HOX cluster on chromosome 7 (CH7). Extrapolation from the data suggests that more than 2000 genomic loci may be susceptible to methylation and associated inactivation, and most have yet to be identified. Finally, we report six new targets of epigenetic inactivation (IRX3, WNT10A, WNT6, RARalpha, BMP7 and ZGPAT). These targets displayed cell line and tumor specific differential methylation when compared with normal brain samples, suggesting they may have utility as biomarkers. Uniquely, hypermethylation of the CGI within an IRX3 exon was correlated with over-expression of IRX3 in tumor tissues and cell lines relative to normal brain samples.

DNA methylation in lysogens of pathogenic Burkholderia spp. requires prophage induction and is restricted to excised phage DNA.

Burkholderia mallei-specific phage PhiE125 encodes DNA methyltransferases in both the lysogenic and replication modules within its genome. Characterization of DNA methylation in recombinant systems, specifically in PhiE125 lysogenic strains of B. mallei and Burkholderia thailandensis, revealed that, upon induction, cytosine methylation was targeted specifically to the phage episome but not the phage provirus or the host chromosome.

Biodefense-driven murine model of pneumonic melioidosis.

A whole-body mouse model of pneumonic melioidosis was established for future evaluation of biodefense vaccine candidates. The aerosol 50% lethal doses of Burkholderia pseudomallei strain 1026b for BALB/c and C57BL/6 mice and the times to death, dissemination in organs, and tissue loads after exposure of the mice to low- and high-dose aerosols are reported. In addition, rpsL mutant backgrounds were attenuated in this acute model of disease.

Burkholderia pseudomallei kills the nematode Caenorhabditis elegans using an endotoxin-mediated paralysis.

We investigated a non-mammalian host model system for fitness in genetic screening for virulence-attenuating mutations in the potential biowarfare agents Burkholderia pseudomallei and Burkholderia mallei. We determined that B. pseudomallei is able to cause 'disease-like' symptoms and kill the nematode Caenorhabditis elegans. Analysis of killing in the surrogate disease model with B. pseudomallei mutants indicated that killing did not require lipopolysaccharide (LPS) O-antigen, aminoglycoside/macrolide efflux pumping, type II pathway-secreted exoenzymes or motility. Burkholderia thailandensis and some strains of Burkholderia cepacia also killed nematodes. Manipulation of the nematode host genotype suggests that the neuromuscular intoxication caused by both B. pseudomallei and B. thailandensis acts in part through a disruption of normal Ca2+ signal transduction. Both species produce a UV-sensitive, gamma-irradiation-resistant, limited diffusion, paralytic agent as part of their nematode pathogenic mechanism. The results of this investigation suggest that killing by B. pseudomallei is an active process in C. elegans, and that the C. elegans model might be useful for the identification of vertebrate animal virulence factors in B. pseudomallei.

Maintenance of genomic methylation requires a SWI2/SNF2-like protein.

Altering cytosine methylation by genetic means leads to a variety of developmental defects in mice, plants and fungi. Deregulation of cytosine methylation also has a role in human carcinogenesis. In some cases, these defects have been tied to the inheritance of epigenetic alterations (such as chromatin imprints and DNA methylation patterns) that do not involve changes in DNA sequence. Using a forward genetic screen, we identified a gene (DDM1, decrease in DNA methylation) from the flowering plant Arabidopsis thaliana required to maintain normal cytosine methylation patterns. Additional ddm1 alleles (som4, 5, 6, 7, 8) were isolated in a selection for mutations that relieved transgene silencing (E.J.R., unpublished data). Loss of DDM1 function causes a 70% reduction of genomic cytosine methylation, with most of the immediate hypomethylation occurring in repeated sequences. In contrast, many low-copy sequences initially retain their methylation in ddm1 homozygotes, but lose methylation over time as the mutants are propagated through multiple generations by self-pollination. The progressive effect of ddm1 mutations on low-copy sequence methylation suggests that ddm1 mutations compromise the efficiency of methylation of newly incorporated cytosines after DNA replication. In parallel with the slow decay of methylation during inbreeding, ddm1 mutants accumulate heritable alterations (mutations or stable epialleles) at dispersed sites in the genome that lead to morphological abnormalities. Here we report that DDM1 encodes a SWI2/SNF2-like protein, implicating chromatin remodelling as an important process for maintenance of DNA methylation and genome integrity.

The DNA methylation locus DDM1 is required for maintenance of gene silencing in Arabidopsis.

To investigate the relationship between cytosine methylation and gene silencing in Arabidopsis, we constructed strains containing the ddm1 hypomethylation mutation and a methylated and silenced PAI2 tryptophan biosynthetic gene (MePAI2) that results in a blue fluorescent plant phenotype. The ddm1 mutation had both an immediate and a progressive effect on PAI gene silencing. In the first generation, homozygous ddm1 MePAI2 plants displayed a weakly fluorescent phenotype, in contrast to the strongly fluorescent phenotype of the DDM1 MePAI2 parent. After two generations of inbreeding by self-pollination, the ddm1/ddm1 lines became nonfluorescent. The progressive loss of fluorescence correlated with a progressive loss of methylation from the PAI2 gene. These results indicate that methylation is necessary for maintenance of PAI gene silencing and that intermediate levels of DNA methylation are associated with intermediate gene silencing. The results also support our earlier hypothesis that ddm1 homozygotes act as "epigenetic mutators" by accumulating heritable changes in DNA methylation that can lead to changes in gene expression.

Developmental abnormalities and epimutations associated with DNA hypomethylation mutations.

A number of aberrant morphological phenotypes were noted during propagation of the Arabidopsis thaliana DNA hypomethylation mutant, ddm1, by repeated self-pollination. Onset of a spectrum of morphological abnormalities, including defects in leaf structure, flowering time, and flower structure, was strictly associated with the ddm1 mutations. The morphological phenotypes arose at a high frequency in selfed ddm1 mutant lines and some phenotypes became progressively more severe in advancing generations. The transmission of two common morphological trait syndromes in genetic crosses demonstrated that the phenotypes are caused by heritable lesions that develop in ddm1 mutant backgrounds. Loss of cytosine methylation in specific genomic sequences during the selfing regime was noted in the ddm1 mutants. Potential mechanisms for formation of the lesions underlying the morphological abnormalities are discussed.

mCCG methylation in angiosperms.

Two different methods were used to investigate the abundance of cytosine methylation at the outer (5') position in 5'-CCG-3' trinucleotides in angiosperm genomes. Mspl is unable to cut its target site if the outer cytosine is methylated (5'-mCCGG-3'). Using Mspl restriction analysis, it was shown that 5'-mCCG-3' is present in all angiosperm genomes examined, and that the amount of cytosine methylation at this site varies between species. Subsequently, direct measurements were made of the amount of methylation at both cytosines in a subset of 5'-CCG-3' trinucleotides in the Arabidopsis thaliana genome. Based upon these analyses, it was estimated that approximately 20-30% of 5'-CCG-3' trinucleotides in A. thaliana are methylated at the outer cytosine. Approximately 20% of the 5'-CCG-3' trinucleotides contain 5-methyl-cytosine at the inner cytosine position, which corresponds to a previous determination of 5'-mCG-3' methylation in A. thaliana. The implications of 5'-mCCG-3' methylation are discussed.

Characterization of an Arabidopsis thaliana DNA hypomethylation mutant.

We have recently isolated two Arabidopsis thaliana DNA hypomethylation mutations, identifying the DDM1 locus, that cause a 70% reduction in genomic 5-methylcytosine levels [1]. Here we describe further phenotypic and biochemical characterization of the ddm1 mutants. ddm1/ddm1 homozygotes exhibited altered leaf shape, increased cauline leaf number, and a delay in the onset of flowering when compared to non-mutant siblings in a segregating population. Our biochemical characterization investigated two possible mechanisms for DNA hypomethylation. In order to see if ddm1 mutations affect DNA methyltransferase function, we compared DNA methyltransferase activities in extracts from wild-type and ddm1 mutant tissues. The ddm1 mutant extracts had as much DNA methyltransferase activity as that of the wild-type for both the CpI and CpNpG substrates suggesting that the DDM1 locus does not encode a DNA methyltransferase. Moreover, the ddm1 mutations did not affect the intracellular level of S-adenosylmethionine, the methyl group donor for DNA methylation. The possibility that the DDM1 gene product functions as a modifier of DNA methylation is discussed.