DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability1, 2. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis3, 4, 5, 6. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands7, 8. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites9, 10, 11. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain12, 13, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA–DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.

Nature 455, 818-821 (9 October 2008) | doi:10.1038/nature07249

Epigenetic inheritance in mammals is characterized by high-fidelity replication of CpG methylation patterns during development1, 2. UHRF1 (also known as ICBP90 in humans and Np95 in mouse)3 is an E3 ligase important for the maintenance of global and local DNA methylation in vivo 4, 5. The preferential affinity of UHRF1 for hemi-methylated DNA over symmetrically methylated DNA by means of its SET and RING-associated (SRA) domain6 and its association with the maintenance DNA methyltransferase 1 (DNMT1) suggests a role in replication of the epigenetic code4, 5, 7. Here we report the 1.7 Å crystal structure of the apo SRA domain of human UHRF1 and a 2.2 Å structure of its complex with hemi-methylated DNA, revealing a previously unknown reading mechanism for methylated CpG sites (mCpG). The SRA–DNA complex has several notable structural features including a binding pocket that accommodates the 5-methylcytosine that is flipped out of the duplex DNA. Two specialized loops reach through the resulting gap in the DNA from both the major and the minor grooves to read the other three bases of the CpG duplex. The major groove loop confers both specificity for the CpG dinucleotide and discrimination against methylation of deoxycytidine of the complementary strand. The structure, along with mutagenesis data, suggests how UHRF1 acts as a key factor for DNMT1 maintenance methylation through recognition of a fundamental unit of epigenetic inheritance, mCpG.

Nature 455, 822-825 (9 October 2008) | doi:10.1038/nature07273

piRNAs and Piwi proteins have been implicated in transposon control and are linked to transposon methylation in mammals. Here we examined the construction of the piRNA system in the restricted developmental window in which methylation patterns are set during mammalian embryogenesis. We find robust expression of two Piwi family proteins, MIWI2 and MILI. Their associated piRNA profiles reveal differences from Drosophila wherein large piRNA clusters act as master regulators of silencing. Instead, in mammals, dispersed transposon copies initiate the pathway, producing primary piRNAs, which predominantly join MILI in the cytoplasm. MIWI2, whose nuclear localization and association with piRNAs depend upon MILI, is enriched for secondary piRNAs antisense to the elements that it controls. The Piwi pathway lies upstream of known mediators of DNA methylation, since piRNAs are still produced in dnmt3L mutants, which fail to methylate transposons. This implicates piRNAs as specificity determinants of DNA methylation in germ cells.

Molecular Cell, Vol 31, 785-799, 26 September 2008

The factors that guide DNA hypermethylation in cancer are poorly understood. We identified the candidate tumor-suppressor gene, RIL, as a frequent methylation target in cancer. Here, we report on a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Methylation analysis showed that, in aging colon, colon cancer, and leukemias, the short allele had 2.1–3.1-fold higher methylation than the long allele (P<0.001). Short and long alleles had similar expression levels in EBV-transformed cell lines. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors. Transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on, but methylation-seeded constructs showed gradually decreasing transcription from the short allele with eventual spreading of de novo methylation. By contrast, the long allele showed stable expression over time as measured by luciferase, and ~2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.

PLoS Genet 4(8): e1000162. doi:10.1371/journal.pgen.1000162

DNA methylation is essential for normal development1, 2, 3 and has been implicated in many pathologies including cancer4, 5. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing6 and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.

Nature 454, 766-770 (7 August 2008) | doi:10.1038/nature07107; Received 24 March 2008; Accepted 21 May 2008; Published online 6 July 2008

The hypothesis of fetal origins of adult disease posits that early developmental exposures involve epigenetic modifications, such as DNA methylation, that influence adult disease susceptibility. In utero or neonatal exposure to bisphenol A (BPA), a high-production-volume chemical used in the manufacture of polycarbonate plastic, is associated with higher body weight, increased breast and prostate cancer, and altered reproductive function. This study shows that maternal exposure to this endocrine-active compound shifted the coat color distribution of viable yellow agouti (A vy) mouse offspring toward yellow by decreasing CpG (cytosine-guanine dinucleotide) methylation in an intracisternal A particle retrotransposon upstream of the Agouti gene. CpG methylation also was decreased at another metastable locus, the CDK5 activator-binding protein (Cabp IAP). DNA methylation at the A vy locus was similar in tissues from the three germ layers, providing evidence that epigenetic patterning during early stem cell development is sensitive to BPA exposure. Moreover, maternal dietary supplementation, with either methyl donors like folic acid or the phytoestrogen genistein, negated the DNA hypomethylating effect of BPA. Thus, we present compelling evidence that early developmental exposure to BPA can change offspring phenotype by stably altering the epigenome, an effect that can be counteracted by maternal dietary supplements.

PNAS August 7, 2007 vol. 104 no. 32 13056-13061