Expanded methyl-sensitive cut counting reveals hypomethylation as an epigenetic state that highlights functional sequences of the genome

Laboratory of Neurobiology, Library and Information Services, and Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2011; 108(23):9715-20. DOI: 10.1073/pnas.1105713108
Source: PubMed


Methyl-sensitive cut counting (MSCC) with the HpaII methylation-sensitive restriction enzyme is a cost-effective method to pinpoint unmethylated CpGs at single base-pair resolution. However, it has the drawback of addressing only CpGs in the context of the CCGG site, leaving out the remainder of the possible 16 XCGX tetranucleotides in which CpGs are found. We expanded MSCC to include three additional enzymes to address a total of 5 of the 16 XCGX combinations. This allowed us to survey methylation at about one-third of all a mammalian genome's CpGs. Applied to mouse liver DNA, we correctly confirmed data reported with other methods showing hypomethylation to be concentrated at promoters and in CpG islands (CGIs), with gene bodies and intergenic regions being mostly methylated. Grouping unmethylated CpGs, characterized by high MSCC scores (7% false discovery rate), we found a large number of unmethylated regions not qualifying as CGIs located in intergenic and intronic regions, which are highly enriched in functional DNA sequences (open regulatory annotation database) as well as in noncoding yet highly conserved mammalian sequences thought to be important but with as yet unknown function. About 50% of MSCC-defined unmethylated regions do not overlap algorithm-defined CGIs and offer a novel search space in which new functionalities of DNA may be found in health and disease.

Download full-text


Available from: Alejandro Cesar Colaneri
  • Source
    • "Genome-wide cysteine methylation profiling was carried out using methyl sensitive cut counting (MSCC) assay as previously described [16,17]. Briefly, the sequencing libraries were generated using 0.65μg of genomic DNA for each library. "
    [Show abstract] [Hide abstract]
    ABSTRACT: While the effects of hypoxia on gene expression have been investigated in the CNS to some extent, we currently do not know what role epigenetics plays in the transcription of many genes during such hypoxic stress. To start understanding the role of epigenetic changes during hypoxia, we investigated the long-term effect of hypoxia on gene expression and DNA methylation in hippocampal neuronal cells. Primary murine hippocampal neuronal cells were cultured for 7 days. Hypoxic stress of 1% O2, 5% CO2 for 24 hours was applied on Day 3, conditions we found to maximize cellular hypoxic stress response without inducing cell death. Cells were returned to normoxia for 4 days following the period of hypoxic stress. On Day 7, Methyl-Sensitive Cut Counting (MSCC) was used to identify a genome-wide methylation profile of the hippocampal cell lines to assess methylation changes resulting from hypoxia. RNA-Seq was also done on Day 7 to analyze changes in gene transcription. Phenotypic analysis showed that neuronal processes were significantly shorter after 1 day of hypoxia, but there was a catch-up growth of these processes after return to normoxia. Transcriptome profiling using RNA-Seq revealed 369 differentially expressed genes with 225 being upregulated, many of which form networks shown to affect CNS development and function. Importantly, the expression level of 59 genes could be correlated to the changes in DNA methylation in their promoter regions. CpG islands, in particular, had a strong tendency to remain hypomethylated long after hypoxic stress was removed. From this study, we conclude that short-term, sub-lethal hypoxia results in long-lasting changes to genome wide DNA methylation status and that some of these changes can be highly correlated with transcriptional modulation in a number of genes involved in functional pathways that have been previously implicated in neural growth and development.
    Full-text · Article · Oct 2013 · PLoS ONE
  • Source
    • "However, the use of MSCC to quantify absolute levels of methylation is limited by the existence of biases that are site specific, i.e. sites in the genome, which tend to be harder to digest in a manner that is independent of its state of methylation [21], [22]. It has been shown that these site-specific biases are systematic and reproducible among replicates [22]. Since we contrasted the digestion frequencies in a site-by-site basis, these systematic biases are expected to cancel out. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background In eukaryotes, the combinatorial usage of cis-regulatory elements enables the assembly of composite genetic switches to integrate multifarious, convergent signals within a single promoter. Plants as sessile organisms, incapable of seeking for optimal conditions, rely on the use of this resource to adapt to changing environments. Emerging evidence suggests that the transcriptional responses of plants to stress are associated with epigenetic processes that govern chromatin accessibility. However, the extent at which specific chromatin modifications contribute to gene regulation has not been assessed. Methodology/Principal Findings In the present work, we combined methyl-sensitive-cut counting and RNA-seq to follow the transcriptional and epigenetic response of plants to simulated drought. Comprehensive genome wide evidence supports the notion that the methylome is widely reactive to water potential. The predominant changes in methylomes were loci in the promoters of genes encoding for proteins suited to cope with the environmental challenge. Conclusion/Significance These selective changes in the methylome with corresponding changes in gene transcription suggest drought sets in motion an instructive mechanism guiding epigenetic machinery toward specific effectors genes.
    Full-text · Article · Apr 2013 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.
    Full-text · Article · Dec 2011 · Nature
Show more