Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an ‘epigenetic memory’ of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

Andy Feinberg and colleagues show in a colon cancer model that most DNA methylation alterations occur in sequence regions distinct from promoters or canonical CpG islands, termed 'CpG island shores', and that this methylation is strongly related to gene expression and can discriminate tissue types regardless of species of origin. For the past 25 years, it has been known that alterations in DNA methylation (DNAm) occur in cancer, including hypomethylation of oncogenes and hypermethylation of tumor suppressor genes. However, most studies of cancer methylation have assumed that functionally important DNAm will occur in promoters, and that most DNAm changes in cancer occur in CpG islands. Here we show that most methylation alterations in colon cancer occur not in promoters, and also not in CpG islands, but in sequences up to 2 kb distant, which we term 'CpG island shores'. CpG island shore methylation was strongly related to gene expression, and it was highly conserved in mouse, discriminating tissue types regardless of species of origin. There was a notable overlap (45–65%) of the locations of colon cancer–related methylation changes with those that distinguished normal tissues, with hypermethylation enriched closer to the associated CpG islands, and hypomethylation enriched further from the associated CpG island and resembling that of noncolon normal tissues. Thus, methylation changes in cancer are at sites that vary normally in tissue differentiation, consistent with the epigenetic progenitor model of cancer, which proposes that epigenetic alterations affecting tissue-specific differentiation are the predominant mechanism by which epigenetic changes cause cancer.

Andrew Feinberg and colleagues report whole-genome bisulfite sequencing of three colon cancers and matched normal tissue and two adenomatous polyps. The authors identify large blocks of relative hypomethylation over half of the genome. Tumor heterogeneity is a major barrier to effective cancer diagnosis and treatment. We recently identified cancer-specific differentially DNA-methylated regions (cDMRs) in colon cancer, which also distinguish normal tissue types from each other, suggesting that these cDMRs might be generalized across cancer types. Here we show stochastic methylation variation of the same cDMRs, distinguishing cancer from normal tissue, in colon, lung, breast, thyroid and Wilms' tumors, with intermediate variation in adenomas. Whole-genome bisulfite sequencing shows these variable cDMRs are related to loss of sharply delimited methylation boundaries at CpG islands. Furthermore, we find hypomethylation of discrete blocks encompassing half the genome, with extreme gene expression variability. Genes associated with the cDMRs and large blocks are involved in mitosis and matrix remodeling, respectively. We suggest a model for cancer involving loss of epigenetic stability of well-defined genomic domains that underlies increased methylation variability in cancer that may contribute to tumor heterogeneity.

Andrew Feinberg and colleagues identify large chromatin domains marked by histone H3 lysine 9 dimethylation in human and mouse cells. These blocks show tissue specificity, and their appearance is correlated with differentiation status. Higher eukaryotes must adapt a totipotent genome to specialized cell types with stable but limited functions. One potential mechanism for lineage restriction is changes in chromatin, and differentiation-related chromatin changes have been observed for individual genes1,2. We have taken a genome-wide view of histone H3 lysine 9 dimethylation (H3K9Me2) and find that differentiated tissues show surprisingly large K9-modified regions (up to 4.9 Mb). These regions are highly conserved between human and mouse and are differentiation specific, covering only ∼4% of the genome in undifferentiated mouse embryonic stem (ES) cells, compared to 31% in differentiated ES cells, ∼46% in liver and ∼10% in brain. These modifications require histone methyltransferase G9a and are inversely related to expression of genes within the regions. We term these regions large organized chromatin K9 modifications (LOCKs). LOCKs are substantially lost in cancer cell lines, and they may provide a cell type–heritable mechanism for phenotypic plasticity in development and disease.

This study was originally conceived to test in a rigorous way the specificity of three major approaches to high-throughput array-based DNA methylation analysis: (1) MeDIP, or methylated DNA immunoprecipitation, an example of antibody-mediated methyl-specific fractionation; (2) HELP, or HpaII tiny fragment enrichment by ligation-mediated PCR, an example of differential amplification of methylated DNA; and (3) fractionation by McrBC, an enzyme that cuts most methylated DNA. These results were validated using 1466 Illumina methylation probes on the GoldenGate methylation assay and further resolved discrepancies among the methods through quantitative methylation pyrosequencing analysis. While all three methods provide useful information, there were significant limitations to each, specifically bias toward CpG islands in MeDIP, relatively incomplete coverage in HELP, and location imprecision in McrBC. However, we found that with an original array design strategy using tiling arrays and statistical procedures that average information from neighboring genomic locations, much improved specificity and sensitivity could be achieved, e.g., ∼100% sensitivity at 90% specificity with McrBC. We term this approach “comprehensive high-throughput arrays for relative methylation” (CHARM). While this approach was applied to McrBC analysis, the array design and computational algorithms are fractionation method-independent and make this a simple, general, relatively inexpensive tool suitable for genome-wide analysis, and in which individual samples can be assayed reliably at very high density, allowing locus-level genome-wide epigenetic discrimination of individuals, not just groups of samples. Furthermore, unlike the other approaches, CHARM is highly quantitative, a substantial advantage in application to the study of human disease.