5-hydroxymethylcytosine (5-hmC), a derivative of 5-methylcytosine (5-mC), is abundant in the brain for unknown reasons. Our goal was to characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC using glucosylation coupled with restriction enzyme digestion, and interrogation on microarrays. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary, in both human and mouse. This boundary change was mainly due to 5-hmC in the brain, but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent datasets and with single molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC, relative to alternatively-spliced exons. Our study suggests a novel role for 5-hmC in RNA splicing and synaptic function in the brain.

Inability to digest lactose due to lactase non-persistence is a common trait in adult mammals, with the exception of certain human populations that exhibit lactase persistence. It is not clear how the lactase gene can be dramatically downregulated with age in most individuals, but remains active in some. We performed a comprehensive epigenetic study of the human and mouse intestine using chromosome-wide DNA modification profiling and targeted bisulfite sequencing. Epigenetically-controlled regulatory elements were found to account for the differences in lactase mRNA levels between individuals, intestinal cell types and species. The importance of these regulatory elements in modulating lactase mRNA levels was confirmed by CRISPR-Cas9-induced deletions. Genetic factors contribute to epigenetic changes occurring with age at the regulatory elements, as lactase persistence- and non-persistence-DNA haplotypes demonstrated markedly different epigenetic aging. Thus, genetic factors facilitate a gradual accumulation of epigenetic changes with age to affect phenotypic outcome.

Dynamic patterns of cytosine-5 methylation and successive hydroxylation are part of epigenetic regulation in eukaryotes, including humans, which contributes to normal phenotypic variation and disease risk. Here we present an approach for the mapping of unmodified regions of the genome, which we call the unmethylome. Our technique is based on DNA methyltransferase-directed transfer of activated groups and covalent biotin tagging of unmodified CpG sites followed by affinity enrichment and interrogation on tiling microarrays or next generation sequencing. Control experiments and pilot studies of human genomic DNA from cultured cells and tissues demonstrate that, along with providing a unique cross-section through the chemical landscape of the epigenome, the methyltransferase-directed transfer of activated groups-based approach offers high precision and robustness as compared with existing affinity-based techniques.

Over the past decade, epigenetic phenomena claimed a central role in cell regulatory processes and proved important factors for understanding complex human diseases. One of the best understood epigenetic mechanisms is DNA methylation. In the mammalian genome, cytosines (C) were long known to exist in two functional states: unmethylated or methylated at the 5-position of the pyrimidine ring (5mC). Recent studies of genomic DNA from the human and mouse brain, neurons and from mouse embryonic stem cells found that a substantial fraction of 5mC in CpG dinucleotides is converted to 5-hydroxymethyl-cytosine (hmC) by the action of 2-oxoglutarate- and Fe(II)-dependent oxygenases of the TET family. These findings provided important clues in a long elusive mechanism of active DNA demethylation and bolstered a fresh wave of studies in the area of epigenetic regulation in mammals. This 15 review is dedicated to critical assessment of the most popular techniques with respect to their suitability for analysis of hmC in mammalian genomes. It also discusses the most recent data on biochemical and chemical aspects of the formation and further conversion of this nucleobase in DNA and its possible biological roles in cell differentiation, embryogenesis and brain function.

S-Adenosylmethionine-dependent DNA methyltransferases (MTases) perform direct methylation of cytosine to yield 5-methylcytosine (5mC), which serves as part of the epigenetic regulation mechanism in vertebrates. Active demethylation of 5mC by TET oxygenases produces 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which were shown to be enzymatically excised and then replaced with an unmodified nucleotide. Here we find that both bacterial and mammalian C5-MTases can catalyze the direct decarboxylation of caC yielding unmodified cytosine in DNA in vitro but are inert toward fC. The observed atypical enzymatic C-C bond cleavage reaction provides a plausible precedent for a direct reversal of caC to the unmodified state in DNA and offers a unique approach for sequence-specific analysis of genomic caC.