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In mammals, the correct spatio-temporal patterns of gene expression are coordinated by transcription factor networks in combination with epigenetic signalling pathways. CpG methylation is an epigenetic modification of DNA involved in the heritable transmission of gene silencing patterns. Increasing evidence suggest a primary role for CpG methylation in the direct regulation of gene expression, at least for a subset of promoters. An example of this direct regulation is represented by the ectopic expression of genes involved in genome defence pathways upon global loss of methylation. However, the mechanistic relationship between CpG methylation and transcriptional regulation is not well understood. To explore this, we have applied Cap Analysis of Gene Expression (CAGE), to cells deficient in CpG methylation (Mouse Embryonic Fibroblasts with hypomorphic mutation of Dnmt1 ) and matched controls. This provides a quantitative, single nucleotide resolution, genome wide map of methylation responsive transcription initiation. Integrating this with RNA-seq, genome wide measures of CpG methylation and ChIP-seq for histone modifications in the same system, provides a detailed view of how reduced CpG methylation alters the chromatin and transcriptional landscape of the genome. Our results show dramatic shifts in the cellular RNA pool, with the pronounced up-regulation or de-repression of promoters in a specific sub-family of transposable elements. Tens of other genic and non-coding RNA promoters similarly show dramatic inductions. Contrary to a prior hypothesis, we found no evidence for increased rates of transcriptional initiation from anonymous genomic sites not previously implicated in promoter activity. Transcription initiation at CpG island promoters is generally unaffected by hypomethylation, however a set of TSSs located in CpG island shores and a class to transposable element overlapping TSSs do appear to be sensitive to methylation and are significantly up-regulated upon hypomethylation.

DNA methylation (5-methylcytosine or 5mC) and the 3-dimensional organization of the genome within the cell nucleus are two critical epigenetic features that regulate gene expression and cellular behavior, and aberrant patterns of these epigenetic features are associated with cancer and disease. In cancer, widespread regions of DNA methylation loss, termed hypomethylation, have previously been found positioned at the periphery of the nucleus, the nuclear lamina (NL), suggesting DNA methylation and genome organization could be linked epigenetic states. However, the direct genome-wide relationship between 5mC and genome-NL interactions remains obscured in bulk measurements. To overcome this limitation, we developed a new single-cell sequencing technology (sc5mC+DamID-seq) to simultaneously measure DNA methylation and genome-NL interactions from the same cell. sc5mC+DamID-seq uses mark-specific barcoded adapters and cytosine deamination to identify both epigenetic features and individual cells, enabling us to profile thousands of single cells per day. By applying sc5mC+DamID-seq to chronic myelogenous leukemia cells (KBM7), we observed hypomethylation at regions contacting the NL, known as lamina associated domains (LADs). Interestingly, we also discovered that genomic regions that contact the NL more frequently in single cells display the greatest loss and variability in 5mC. Further, while LADs appear as continuous stretches of contact with the NL in bulk sequencing, LADs at the single-cell level frequently display segments of noncontact with the NL, which we found influences the mean levels of 5mC in LADs. Finally, to test the connection between these two epigenetic features, we globally demethylated the epigenome, which relocated more variable genome-NL contact regions towards the nuclear interior. Thus, simultaneous single-cell measurements in sc5mC+DamID-seq has enabled us to systematically uncover the relationship between DNA methylation and genome organization in cancer cells. As an addition to the protocol, the transcriptome, the ensemble of mRNA produced by a cell, was measured with 5mC and genome-NL contacts, to reveal how epigenetic features directly correlate with gene expression. The mRNA modification m6A was also compared to the epigenetic features to find the relationship between the epigenome and epitranscriptome.

We report a systematic analysis of the DNA methylation variability in 1,595 samples of normal cell subpopulations and 14 tumor subtypes spanning the entire human B-cell lineage. Differential methylation among tumor entities relates to differences in cellular origin and to de novo epigenetic alterations, which allowed us to build an accurate machine learning-based diagnostic algorithm. We identify extensive individual-specific methylation variability in silenced chromatin associated with the proliferative history of normal and neoplastic B cells. Mitotic activity generally leaves both hyper- and hypomethylation imprints, but some B-cell neoplasms preferentially gain or lose DNA methylation. We construct a DNA-methylation-based mitotic clock, called epiCMIT, whose lapse magnitude represents a strong independent prognostic variable in B-cell tumors and is associated with particular driver genetic alterations. Our findings reveal DNA methylation as a holistic tracer of B-cell tumor developmental history, with implications in differential diagnosis and the prediction of clinical outcome. Martin-Subero and colleagues analyze DNA methylation patterns in B-cell tumors and their normal cells of origin, and develop epiCMIT, a methylation-based mitotic clock with prognostic relevance.

Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies.

Aberrant genome-wide hypomethylation is thought to be related to tumorigenesis by promoting genomic instability. Since DNA methylation is considered an important mechanism for the silencingof retroelements, hypomethylation in human tumors may lead to their reactivation. However, the role of DNA hypomethylation in chronic myeloid leukemia (CML) remains to be elucidated. In this study, the methylation status of the LINE-1 (L1) retrotransposon promoter was analysed in CML samples from the chronicphase (CP, n¼140) and the blast crisis (BC, n¼47). L1 hypomethylation was significantly more frequent in BC (74.5%) than in CP (38%) (Po0.0001). Furthermore, L1 hypomethylation led to activation of both ORF1 sense transcription (Po0.0001) and c-MET gene antisense transcription (Po0.0001), and was significantly associated with high levels of BCR–ABL (P¼0.02) and DNMT3b4 (P¼0.001) transcripts. Interestingly, in CP-CML, extensive L1 hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (P¼0.004) or imatinib (P¼0.034) and progression-free survival (P¼0.005). The above results strongly suggest that activation of both sense and antisense transcriptions by aberrant promoter hypomethylation of the L1 elements plays a role in the progression and clinical behavior of the CML.

Uncoupling protein 2 (UCP2) expression in liver is restricted to non-parenchymal cells. By means of differential display screening between normal rat liver and H4IIE hepatoma cells we have isolated a cDNA clone encompassing part of UCP2 cDNA. Northern blot analysis revealed that UCP2 is expressed in some hepatocarcinoma cell lines, while it is absent in adult hepatocytes. UCP2 mRNA in H4IIE cells was downregulated when cells were cultured for 36 h in 0.1% serum and its expression was restored upon addition of 10% serum or phorbol esters. Hypomethylation of UCP2 was observed in transformed UCP2 expressing cells. Our results indicate that UCP2 is expressed in some hepatocarcinoma cell lines and that serum components may participate in maintaining elevated UCP2 levels.