Journal of Molecular Biology
ReviewDNA Modification Readers and Writers and Their Interplay
Graphical abstract
Introduction
Epigenetic modifications of mammalian DNA are crucial regulators of organismic development but also in pathogenesis. The most abundant and most studied mammalian DNA modification was described by Rollin Hotchkiss in 1948, when he identified, in addition to the four canonical DNA bases, a fifth peak on a paper chromatogram from thymus deoxyribonucleic acids. He termed this novel mammalian DNA base “epicytosine” [1], unknowingly anticipating the modern definition of epigenetics and the additional layer of information that epigenetic DNA/chromatin modifications add to the genetic information of every cell. In the same study, Hotchkiss suggested that “epicytosine” corresponded to methylated cytosine. In 1972, a sixth DNA base, named 5-hydroxymethylcytosine (5hmC), was identified in different vertebrate tissues, although this was likely the result of an experimental artifact as in the same study 5-methylcytosine (5mC) was not detected [2]. Hence, 5mC remained the only noncanonical mammalian DNA base that was studied to a larger extent for a long time and helped explaining how different cellular phenotypes can arise from the same nucleotide sequence. The postreplicative covalent addition of a methyl group to the fifth carbon atom of the cytosine pyrimidine ring by the DNA methyltransferase (DNMT) enzymes and oxidation thereof by the Ten-eleven-translocation (TET) enzymes diversify the genome and shape the chromatin landscape (Fig. 1A). DNA cytosine methylation is predominantly found in the context of symmetric CpG dinucleotides and, in the human genome, 70%–80% of all CpGs harbor methylated cytosines [3,4]. This modification is crucial in numerous cellular processes such as X-chromosome inactivation and imprinting, gene repression, control of cellular development and differentiation, silencing of repetitive elements, and genome stability maintenance [[4], [5], [6], [7]]. Meanwhile, two more cytosine variants, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), were identified as oxidation products of 5hmC mediated by TET enzymes [8] (Fig. 1A), shedding light into a possible active DNA demethylation pathway [9]. However, 5hmC and 5fC were also identified as stable epigenetic marks [10,11] and all cytosine modifications are recognized by specific reader proteins [12]. The (hydroxy)methylome is specifically read, amongst others, by members of the methyl-CpG binding domain (MBD) protein family (reviewed in Ref. [13]).
Here, we review the effects of DNA base modifications on DNA double helix structure and the interplay between cytosine methylation readers and modifiers. Although methylation of the nitrogen-6 of deoxyadenosine (m6dA) has been recently described in DNA from mammals [14,15] and linked to nucleosomal positioning in Tetrahymena [16], it will be omitted from this review as its existence is still under debate [17].
Section snippets
Effect of cytosine modifications on DNA double helix structure and recognition by proteins
The flexibility of the dynamic DNA double helix system is a fundamental condition to allow tight protein-DNA contacts, is sequence dependent, and mainly determined by the stacking energy of base steps, hydrogen bondings between base pairs, and exocyclic functional groups pointing within the major or minor groove [18]. Since DNA replication, transcription, and repair all rely on unwinding of the DNA double helix, the influence of base modifications on helix parameters might directly affect DNA
DNMT writer enzymes and the 5mC mark
Methylation of cytosine in eukaryotic DNA relies on the action of the members of the S-adenosyl-l-methionine dependent DNA methyltransferase family (DNMT), which are capable of introducing new DNA methylation sites, as well as maintaining the methylation state upon DNA replication during the cell cycle. DNMT1, the first member of this family, can be considered an epigenetic janitor, since it methylates hemimethylated CpG sites that occur in the course of DNA replication and repair [[59], [60],
DNA methylation and binding of MBD readers
In line with in vitro binding assays [85,88], genome-wide mapping of MBD protein binding in mouse ES and differentiated cells showed a general linear dependence of binding and methylation density for all MBDs except MBD3, as well as the requirement of a functional MBD domain and methylated CGs, CAs, and CACs [56,155]. For some MBDs, additional sequence preference has been reported, with high affinity binding of MeCP2 relying upon an A/T run adjacent to the mCpG [156] and the MBD1 MBD shown to
Interplay of DNA methylation readers and modifiers
Cytosine methylation writers, readers, and modifiers need a tight spatio-temporal regulation to avoid aberrant DNA methylation, a hallmark of numerous human disorders. Deregulation of members of the DNMT, MBD, or TET protein families and their interplay can impair the genomic methylome, as well as chromatin structure and organization, as observed in patients suffering from the neurodegenerative HSANIE disorder (hereditary sensory and autonomic neuropathy, mutations in the TS domain of DNMT1),
Concluding remarks/Perspectives
Altogether these studies unveil new mechanisms of a time and dose-dependent cross-regulation of cytosine modifications and their readers and modifiers, relying on inhibiting each other's binding. Until now, DNA modifications are generally seen as shapers of the reader's binding profiles, and, hence, their genomic localization [160,212]. Conversely, cytosine modification readers do not only play important roles in translating the modifications into distinct chromatin states, thereby regulating
Acknowledgments
We thank all the past and present members of our laboratory for their many contributions along the years. Last but not least, we thank our collaborators over the years, who have made our work so much more enjoyable.
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