While the methylation of DNA in 5′ promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear1–5. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5′ CpG island (CGI) promoters, while a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences5–10. Tissue-specific intragenic methylation might reduce,3 or, paradoxically, enhance transcription elongation efficiency1,2,4,5. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes11–15. To investigate the role of intragenic methylation, we generated a map of DNA methylation from human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were revealed to be in intragenic and intergenic regions, while less than 3% of CpG islands in 5′ promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters16. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus17,18 and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.

Cytosine methylation, a common form of DNA modification that antagonizes transcription, is found at transposons and repeats in vertebrates, plants and fungi. Here we have mapped DNA methylation in the entire Arabidopsis thaliana genome at high resolution. DNA methylation covers transposons and is present within a large fraction of A. thaliana genes. Methylation within genes is conspicuously biased away from gene ends, suggesting a dependence on RNA polymerase transit. Genic methylation is strongly influenced by transcription: moderately transcribed genes are most likely to be methylated, whereas genes at either extreme are least likely. In turn, transcription is influenced by methylation: short methylated genes are poorly expressed, and loss of methylation in the body of a gene leads to enhanced transcription. Our results indicate that genic transcription and DNA methylation are closely interwoven processes.

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

Eukaryotic chromatin is separated into functional domains differentiated by posttranslational histone modifications, histone variants, and DNA methylation [1][2][3][4][5][6] . Methylation is associated with repression of transcriptional initiation in plants and animals, and is frequently found in transposable elements. Proper methylation patterns are critical for eukaryotic development 4,5 , and aberrant methylationinduced silencing of tumor suppressor genes is a common feature of human cancer 7 . In contrast to methylation, the histone variant H2A.Z is preferentially deposited by the Swr1 ATPase complex near 5′ ends of genes where it promotes transcriptional competence [8][9][10][11][12][13][14][15][16][17][18][19][20] . How DNA methylation and H2A.Z influence transcription remains largely unknown. Here we show that in the plant Arabidopsis thaliana, regions of DNA methylation are quantitatively deficient in H2A.Z. Exclusion of H2A.Z is seen at sites of DNA methylation in the bodies of actively transcribed genes and in methylated transposons. Mutation of the MET1 DNA methyltransferase, which causes both losses and gains of DNA methylation 4,5 , engenders opposite changes in H2A.Z deposition, while mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z 17 leads to genome-wide hypermethylation. Our findings indicate that DNA methylation can influence chromatin structure and effect gene silencing by excluding H2A.Z, and that H2A.Z protects genes from DNA methylation.To investigate H2A.Z deposition in plant chromatin, we generated a high resolution genomewide map of H2A.Z in Arabidopsis by adapting the in vivo biotinylation system we used to affinity-purify Drosophila chromatin 21 . We tagged Arabidopsis H2A.Z with a peptide specifically recognized by the E. coli biotin ligase BirA (biotin ligase recognition peptide, BLRP), and created transgenic plants co-expressing BLRP-H2A.Z with BirA. Cytological localization revealed that BLRP-H2A.Z has a diffuse nuclear distribution, but is excluded from heterochromatic chromocenters ( Supplementary Fig. 1), the same pattern as that of endogenous H2A.Z 17 . Following digestion with micrococcal nuclease to mostly mononucleosomes (Supplementary Fig. 1), we purified biotinylated chromatin from root tissue and co-hybridized Author information Microarray data are deposited in GEO with accession number GSE12212. Methods summaryWe adapted the biotin-mediated affinity purification system we developed in Drosophila tissue culture cells 21 to allow protein purification from Arabidopsis plants. Biotinylated H2A.Z was purified largely as described 21 . Endogenous H2A.Z was immunopurified as described 17 , except the IP was performed in TNE. Our methylated DNA IP protocol (MeDIP), microarray design and labeling protocol are described in 22 . All labeled samples were sent to NimbleGen Systems (Madison, WI) for hybridization, except the pie1 samples, which were hybridized at the FHCRC DNA array facility. For bisulfite sequencing, 2 μg of genomic DNA for each sample wer...