Single nucleotide extension technology for quantitative site-specific evaluation of (met)C/C in GC-rich regions

The development and use of high throughput technologies for detailed mapping of methylated cytosines ((met)C) is becoming of increasing importance for the expanding field of epigenetics. The single nucleotide primer extension reaction used for genotyping of single nucleotide polymorphisms has been recently adapted to interrogate the bisulfite modification induced ‘quantitative’ C/T polymorphism that corresponds to (met)C/C in the native DNA. In this study, we explored the opportunity to investigate C/T (and G/A) ratios using the Applied Biosystems (ABI) SNaPshot technology. The main effort of this study was dedicated to addressing the complexities in the analysis of DNA methylation in GC-rich regions where interrogation of the target cytosine can be confounded by variable degrees of methylation in other cytosines (resulting in variable C/T or G/A ratios after treatment with bisulfite) in the annealing site of the interrogating primer. In our studies, the mismatches of the SNaPshot primer with the target DNA sequence resulted in a biasing effect of up to 70% while these effects decreased as the location of the polymorphic site moved upstream of the target cytosine. We demonstrated that the biasing effect can be corrected with the SNaPshot primers containing degenerative C/T and G/A nucleotides. A series of experiments using various permutations of quantitative C/T and G/A polymorphisms at various positions of the target DNA sequence demonstrated that SNaPshot is able to accurately report cytosine methylation levels with <5% average SD from the true values. Given the relative simplicity of the method and the possibility to multiplex C/T and G/A interrogations, the SNaPshot approach may become a useful tool for large-scale mapping of (met)C

Epigenetic dysregulation of enhancers in neurons is associated with Alzheimer’s disease pathology and cognitive symptoms

Epigenetic control of enhancers alters neuronal functions and may be involved in Alzheimer’s disease (AD). Here, we identify enhancers in neurons contributing to AD by comprehensive fine-mapping of DNA methylation at enhancers, genome-wide. We examine 1.2 million CpG and CpH sites in enhancers in prefrontal cortex neurons of individuals with no/mild, moderate, and severe AD pathology (n = 101). We identify 1224 differentially methylated enhancer regions; most of which are hypomethylated at CpH sites in AD neurons. CpH methylation losses occur in normal aging neurons, but are accelerated in AD. Integration of epigenetic and transcriptomic data demonstrates a pro-apoptotic reactivation of the cell cycle in post-mitotic AD neurons. Furthermore, AD neurons have a large cluster of significantly hypomethylated enhancers in the DSCAML1 gene that targets BACE1. Hypomethylation of these enhancers in AD is associated with an upregulation of BACE1 transcripts and an increase in amyloid plaques, neurofibrillary tangles, and cognitive decline

Microarray-based DNA methylation profiling: technology and applications

This work is dedicated to the development of a technology for unbiased, high-throughput DNA methylation profiling of large genomic regions. In this method, unmethylated and methylated DNA fractions are enriched using a series of treatments with methylation sensitive restriction enzymes, and interrogated on microarrays. We have investigated various aspects of the technology including its replicability, informativeness, sensitivity and optimal PCR conditions using microarrays containing oligonucleotides representing 100 kb of genomic DNA derived from the chromosome 22 COMT region in addition to 12 192 element CpG island microarrays. Several new aspects of methylation profiling are provided, including the parallel identification of confounding effects of DNA sequence variation, the description of the principles of microarray design for epigenomic studies and the optimal choice of methylation sensitive restriction enzymes. We also demonstrate the advantages of using the unmethylated DNA fraction versus the methylated one, which substantially improve the chances of detecting DNA methylation differences. We applied this methodology for fine-mapping of methylation patterns of chromosomes 21 and 22 in eight individuals using tiling microarrays consisting of over 340 000 oligonucleotide probe pairs. The principles developed in this work will help to make epigenetic profiling of the entire human genome a routine procedure

Allele-skewed DNA modification in the brain:relevance to a schizophrenia GWAS

Numerous recent studies have suggested that phenotypic effects of DNA sequence variants can be mediated or modulated by their epigenetic marks, such as allele-skewed DNA modification (ASM). Using Affymetrix SNP microarrays, we performed a comprehensive search of ASM effects in human post-mortem brain and sperm samples (total n = 256) from individuals with major psychosis and control individuals. Depending on the phenotypic category of the brain samples, 1.4%-7.5% of interrogated SNPs exhibited ASM effects. Next, we investigated ASM in the context of genetic studies of schizophrenia and detected that brain ASM SNPs were significantly overrepresented among sub-threshold SNPs from a schizophrenia genome-wide association study (GWAS). Brain ASM SNPs showed a much stronger enrichment in a schizophrenia GWAS than in 17 large GWASs of non-psychiatric diseases and traits, arguing that ASM effects are at least partially tissue specific. Studies of germline and control brain ASM SNPs supported a causal association between ASM and schizophrenia. Finally, significantly higher proportions of ASM SNPs than of non-ASM SNPs were detected at loci exhibiting epigenetic signatures of enhancers and promoters, and they were overrepresented within transcription factor binding regions and DNase I hypersensitive sites. All of these findings collectively indicate that ASM SNPs should be prioritized in follow-up GWASs

Gene-body hypermethylation of ATM in peripheral blood DNA of bilateral breast cancer patients

Bilaterality of breast cancer is an indicator of constitutional cancer susceptibility; however, the molecular causes underlying this predisposition in the majority of cases is not known. We hypothesize that epigenetic misregulation of cancer-related genes could partially account for this predisposition. We have performed methylation microarray analysis of peripheral blood DNA from 14 women with bilateral breast cancer compared with 14 unaffected matched controls throughout 17 candidate breast cancer susceptibility genes including BRCA1, BRCA2, CHEK2, ATM, ESR1, SFN, CDKN2A, TP53, GSTP1, CDH1, CDH13, HIC1, PGR, SFRP1, MLH1, RARB and HSD17B4. We show that the majority of methylation variability is associated with intragenic repetitive elements. Detailed validation of the tiled region around ATM was performed by bisulphite modification and pyrosequencing of the same samples and in a second set of peripheral blood DNA from 190 bilateral breast cancer patients compared with 190 controls. We show significant hypermethylation of one intragenic repetitive element in breast cancer cases compared with controls (P = 0.0017), with the highest quartile of methylation associated with a 3-fold increased risk of breast cancer (OR 3.20, 95% CI 1.78–5.86, P = 0.000083). Increased methylation of this locus is associated with lower steady-state ATM mRNA level and correlates with age of cancer patients but not controls, suggesting a combined age–phenotype-related association. This research demonstrates the potential for gene-body epigenetic misregulation of ATM and other cancer-related genes in peripheral blood DNA that may be useful as a novel marker to estimate breast cancer risk

A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation