The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Integrative analysis of 111 reference human epigenomes

An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression.

/pdf/topology-of-the-human-and-mouse-m6a-rna-methylomes-revealed-324vt60ny4.pdf

Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq

Dynamic RNA Modifications in Gene Expression Regulation

The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis.

/pdf/post-transcriptional-gene-regulation-by-mrna-modifications-5984s6cty9.pdf

Post-transcriptional gene regulation by mRNA modifications

Developmental Plasticity and Evolution. In: Mary Jane West-Eberhard, Editor, Oxford University Press (2003) Pp. xi+794. Price $50.00 paper.

The covalent modification of nucleic acids plays an important role in regulating the functions of DNA and RNA. DNA modifications have been analyzed in considerable detail, and the characterization of (cytosine-5) DNA methylation has been crucial for understanding the molecular basis of epigenetic gene regulation (Klose and Bird 2006). (Cytosine-5) methylation has also been documented in various RNA species, including tRNA, but the function of RNA methylation has not been firmly established yet (Motorin et al. 2010).

Dnmt2 proteins were originally assigned to the DNA methyltransferase family, because of their strong sequence conservation of catalytic DNA methyltransferase motifs (Okano et al. 1998; Yoder and Bestor 1998). A recent study has suggested that Dnmt2-mediated DNA methylation is important for transposon silencing in Drosophila (Phalke et al. 2009). However, only a weak and distributive DNA methylation activity has been reported in various systems (Jeltsch et al. 2006).

The ambiguities associated with the DNA methyltransferase activity of Dnmt2 have also prompted the search for alternative enzyme substrates, and resulted in the discovery of a tRNA methyltransferase activity of Dnmt2 (Goll et al. 2006). Purified recombinant human Dnmt2 methylated RNA preparations from Dnmt2 mutant mice, flies, and plants. Further experiments identified C38 in the anti-codon loop of tRNAAsp as the methylation target site of Dnmt2 (Goll et al. 2006). However, the functional relevance of the tRNA methyltransferase activity of Dnmt2 remains to be established. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile (Goll et al. 2006) under standard laboratory conditions. A distinct Dnmt2 mutant phenotype, caused by morpholino knockdown experiments, has so far been reported only in zebrafish, leading to lethal differentiation defects in the retina, liver, and brain (Rai et al. 2007). In addition, two studies have indicated increased stress tolerance in Dnmt2-overexpressing flies and amoebas (Lin et al. 2005; Fisher et al. 2006). However, the underlying molecular mechanisms have not been investigated yet.

/pdf/rna-methylation-by-dnmt2-protects-transfer-rnas-against-3n4nkl0on2.pdf

RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage

Covalent modifications of nucleic acids play an important role in regulating their functions. Among these modifications, (cytosine-5) DNA methylation is best known for its role in the epigenetic regulation of gene expression. Post-transcriptional RNA modification is a characteristic feature of noncoding RNAs, and has been described for rRNAs, tRNAs and miRNAs. (Cytosine-5) RNA methylation has been detected in stable and long-lived RNA molecules, but its function is still unclear, mainly due to technical limitations. In order to facilitate the analysis of RNA methylation patterns we have established a protocol for the chemical deamination of cytosines in RNA, followed by PCR-based amplification of cDNA and DNA sequencing. Using tRNAs and rRNAs as examples we show that cytosine methylation can be reproducibly and quantitatively detected by bisulfite sequencing. The combination of this method with deep sequencing allowed the analysis of a large number of RNA molecules. These results establish a versatile method for the identification and characterization of RNA methylation patterns, which will be useful for defining the biological function of RNA methylation.

/pdf/rna-cytosine-methylation-analysis-by-bisulfite-sequencing-4yzfcg29sr.pdf

RNA cytosine methylation analysis by bisulfite sequencing

The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarus fallax, and also established a triploid AA’B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.

/pdf/clonal-genome-evolution-and-rapid-invasive-spread-of-the-hp5oipv2ce.pdf

Clonal genome evolution and rapid invasive spread of the marbled crayfish

The cytosine analogues azacytidine and decitabine are currently being developed as drugs for epigenetic cancer therapy. Although various studies have shown that both drugs are effective in inhibiting DNA methylation, it has also become clear that their mode of action is not limited to DNA demethylation. Because azacytidine is a ribonucleoside, the primary target of this drug may be cellular RNA rather than DNA. We have now analyzed the possibility that azacytidine inhibits the RNA methyltransferase DNMT2. We found that DNMT2 is variably expressed in human cancer cell lines. RNA bisulfite sequencing showed that azacytidine, but not decitabine, inhibits cytosine 38 methylation of tRNA(Asp), a major substrate of DNMT2. Azacytidine caused a substantially stronger effect than decitabine on the metabolic rate of all the cancer cell lines tested, consistent with an effect of this drug on RNA metabolism. Of note, drug-induced loss of RNA methylation seemed specific for DNMT2 target sites because we did not observe any significant demethylation at sites known to be methylated by other RNA methyltransferases. Our results uncover a novel and quantifiable drug activity of azacytidine and raise the possibility that tRNA hypomethylation might contribute to patient responses.

https://cancerres.aacrjournals.org/content/canres/69/20/8127.full.pdf

Azacytidine inhibits RNA methylation at DNMT2 target sites in human cancer cell lines.

https://www.cell.com/cell-reports/pdf/S2211-1247(13)00408-7.pdf

Katharina Hanna

Papers

RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage

RNA cytosine methylation analysis by bisulfite sequencing

Clonal genome evolution and rapid invasive spread of the marbled crayfish

Azacytidine inhibits RNA methylation at DNMT2 target sites in human cancer cell lines.

The RNA Methyltransferase Dnmt2 Is Required for Efficient Dicer-2-Dependent siRNA Pathway Activity in Drosophila