Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform

DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management.     

A role for poly(ADP-ribosyl)ation in DNA methylation.

The aberrant DNA methylation of promoter regions of housekeeping genes leads to gene silencing. Additional epigenetic events, such as histone methylation and acetylation, also play a very important role in the definitive repression of gene expression by DNA methylation. If the aberrant DNA methylation of promoter regions is the starting or the secondary event leading to the gene silencing is still debated. Mechanisms controlling DNA methylation patterns do exist although they have not been ultimately proven. Our data suggest that poly(ADP-ribosyl)ation might be part of this control mechanism. Thus an additional epigenetic modification seems to be involved in maintaining tissue and cell-type methylation patterns that when formed during embryo development, have to be rigorously conserved in adult organisms.     

Genomic patterns of DNA methylation: targets and function of an epigenetic mark

Methylation of cytosines can mediate epigenetic gene silencing and is the only known DNA modification in eukaryotes. Recent efforts to map DNA methylation across mammalian genomes revealed limited DNA methylation at regulatory regions but widespread methylation in intergenic regions and repeats. This is consistent with the idea that hypermethylation is the default epigenetic state and serves in maintaining genome integrity. DNA methylation patterns at regulatory regions are generally stable, but a minor subset of regulatory regions show variable DNA methylation between cell types, suggesting an additional dynamic component. Such promoter de novo methylation might be involved in the maintenance rather than the initiation of silencing of defined genes during development. How frequently such dynamic methylation occurs, its biological relevance and the pathways involved deserve investigation.     

Connections between epigenetic gene silencing and human disease

Alterations in epigenetic gene regulation are associated with human disease. Here, we discuss connections between DNA methylation and histone methylation, providing examples in which defects in these processes are linked with disease. Mutations in genes encoding DNA methyltransferases and proteins that bind methylated cytosine residues cause changes in gene expression and alterations in the patterns of DNA methylation. These changes are associated with cancer and congenital diseases due to defects in imprinting. Gene expression is also controlled through histone methylation. Altered levels of methyltransferases that modify lysine 27 of histone H3 (K27H3) and lysine 9 of histone H3 (K9H3) correlate with changes in Rb signaling and disruption of the cell cycle in cancer cells. The K27H3 mark recruits a Polycomb complex involved in regulating stem cell pluripotency, silencing of developmentally regulated genes, and controlling cancer progression. The K9H3 methyl mark recruits HP1, a structural protein that plays a role in heterochromatin formation, gene silencing, and viral latency. Cells exhibiting altered levels of HP1 are predicted to show a loss of silencing at genes regulating cancer progression. Gene silencing through K27H3 and K9H3 can involve histone deacetylation and DNA methylation, suggesting cross talk between epigenetic silencing systems through direct interactions among the various players. The reversible nature of these epigenetic modifications offers therapeutic possibilities for a wide spectrum of disease.     

Testing the FMR1 promoter for mosaicism in DNA methylation among CpG sites, strands, and cells in FMR1-expressing males with fragile X syndrome

Variability among individuals in the severity of fragile X syndrome (FXS) is influenced by epigenetic methylation mosaicism, which may also be common in other complex disorders. The epigenetic signal of dense promoter DNA methylation is usually associated with gene silencing, as was initially reported for FMR1 alleles in individuals with FXS. A paradox arose when significant levels of FMR1 mRNA were reported for some males with FXS who had been reported to have predominately methylated alleles. We have used hairpin-bisufite PCR, validated with molecular batch-stamps and barcodes, to collect and assess double-stranded DNA methylation patterns from these previously studied males. These patterns enable us to distinguish among three possible forms of methylation mosaicism, any one of which could explain FMR1 expression in these males. Our data indicate that cryptic inter-cell mosaicism in DNA methylation can account for the presence of FMR1 mRNA in some individuals with FXS.     

Intergenerational genomic DNA methylation patterns in mouse hybrid strains

Background

DNA methylation is a contributing factor to both rare and common human diseases, and plays a major role in development and gene silencing. While the variation of DNA methylation among individuals has been partially characterized, the degree to which methylation patterns are preserved across generations is still poorly understood. To determine the extent of methylation differences between two generations of mice we examined DNA methylation patterns in the livers of eight parental and F1 mice from C57BL/6J and DBA/2J mouse strains using bisulfite sequencing.

Results

We find a large proportion of reproducible methylation differences between C57BL/6J and DBA/2J chromosomes in CpGs, which are highly heritable between parent and F1 mice. We also find sex differences in methylation levels in 396 genes, and 11% of these are differentially expressed between females and males. Using a recently developed approach to identify allelically methylated regions independently of genotypic differences, we identify 112 novel putative imprinted genes and microRNAs, and validate imprinting at the RNA level in 10 of these genes.

Conclusions

The majority of DNA methylation differences among individuals are associated with genetic differences, and a much smaller proportion of these epigenetic differences are due to sex, imprinting or stochastic intergenerational effects. Epigenetic differences can be a determining factor in heritable traits and should be considered in association studies for molecular and clinical traits, as we observed that methylation differences in the mouse model are highly heritable and can have functional consequences on molecular traits such as gene expression.