The Mechanisms of Generation, Recognition, and Erasure of DNA 5-Methylcytosine and Thymine Oxidations

Abstract

One of the most fundamental questions in the control of gene expression in mammals is how the patterns of epigenetic modifications of DNA are generated, recognized, and erased. This includes covalent cytosine methylation of DNA and its associated oxidation states. An array of AdoMet-dependent methyltransferases, Fe(II)-and ␣-ketoglutarate-dependent dioxygenases, base excision glycosylases, and sequence-specific transcription factors is responsible for changing, maintaining, and interpreting the modification status of specific regions of chromatin. This review focuses on recent developments in characterizing the functional and structural links between the modification status of two DNA bases 5-methylcytosine and thymine (5-methyluracil).In the DNA of higher organisms, cytosine exists in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5mC), 2 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) (1-5). These forms are genetically equivalent in terms of base-pairing and protein-coding, but differ in how they interact with macromolecules and influence gene expression. There is much interest in the effects of these modifications in epigenetic regulation, in development and differentiation, in neuron function, and in diseases. In general, the modifications (or "marks") are added to cytosine in situ, following its incorporation into DNA in the unmodified form. DNA methyltransferases (Dnmt) convert certain cytosines to 5mC, usually within the sequence context CpG (6, 7) or CpA (8). A subset of these 5mC residues is then converted to 5hmC, 5fC, and 5caC in consecutive Fe(II)-and ␣-ketoglutarate-dependent oxidation reactions by the teneleven translocation (Tet) dioxygenases (2-4). The Tet dioxygenases are widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi (9), mushroom (Coprinopsis cinerea) (10), and honey bee (Apis mellifera) (11). High-throughput methods for characterizing 5mC oxidation states at single base resolution are becoming available, so our understanding of 5mC oxidation is expected to develop rapidly. In this minireview, we focus on the mechanisms of generating, recognizing, and possibly erasing 5mC and its oxidative forms in DNA. Tet Proteins Are 5-Methylpyrimidine Dioxygenases, but Not Demethylating EnzymesChromatin regulates transcriptional processes through postsynthetic modifications of both of its components: DNA and histones (Fig. 1a). Much remains to be learned about how the combination of these modifications (or lack thereof) facilitates or silences transcription. One broad theme has emerged that a web of interactions tightly coordinates the modification of a segment of DNA and its associated histones, affecting local chromatin structure and determining the functional states. The Tet enzymes belong to a family of Fe(II)-and ␣-ketoglutaratedependent dioxygenases that also includes the Jumonji domain-containing histone lysine demethylases, the N-methyl nucleic acid demethylase includin...

Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications

“…It is possible that WT1 either recruits Tet2 to its target sites and/or binds the sequences modified by Tet2 enzymatic activity. Accumulating evidence supports the view that 5hmC exists as a relatively stable modification, constituting a distinct epigenetic signal (60,61) that marks lowly expressed genes, gene bodies, and intragenic regions (reviewed in (62,63)). 5fC and 5caC are found at much lower levels and at fewer sites, but are enriched at mono-methylated histone H3 lysine 4 (H3K4me1)-marked enhancers (64–69), implying that these higher oxidized forms might play an important role in enhancer activity.…”

DNA Base Flipping: A General Mechanism for Writing, Reading, and Erasing DNA Modifications

“…On the other hand, FTO and ALKBH5 likely generate N6-hydroxymethyladenine intermediate in which the oxidized carbon is attached to a reactive nitrogen atom (adenine-N6). This intermediate spontaneously releases the hydroxymethyl group as formaldehyde and decomposes to adenine—the final “demethylated” product (Hashimoto et al 2015b) (Fig. 1b).…”

“…Along with the previously mentioned MBD family proteins that recognize 5mC within the simple dinucleotide CpG sequence, certain mammalian zinc-finger family proteins such as Kaiso (Buck-Koehntop et al 2012), Zfp57 (Liu et al 2012), Klf4 (Liu et al 2014), and Egr1 (Hashimoto et al 2014a) bind 5mC within specific sequences via a common structural motif (Liu et al 2013; Hashimoto et al 2015b). In addition, another zinc-finger transcription factor WT1 (Hashimoto et al 2014a) and the basic helix-loop-helix (bHLH) family Tcf3-Ascl1 heterodimer (Golla et al 2014) can specifically bind 5caC within their consensus sequences.…”

Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

“…The best-known modified DNA-recognition domains are two that recognize methylated cytosine: methyl-binding domains (MBDs) recognize fully methylated CpG dinucleotides (Dhasarathy & Wade, 2008; Guy, Cheval, Selfridge, & Bird, 2011), and “SET and RING finger-associated” (SRA) domains that bind hemimethylated CpG sites generated transiently by DNA replication (Hashimoto, Horton, Zhang, & Cheng, 2009; Sharif & Koseki, 2011; reviewed in Hashimoto, Zhang, Vertino, & Cheng, 2015; Liu, Zhang, Blumenthal, & Cheng, 2013). Both MBD and SRA domains have been structurally characterized in complexes with 5mC (Arita, Ariyoshi, Tochio, Nakamura, & Shirakawa, 2008; Avvakumov et al, 2008; Hashimoto et al, 2008; Ho et al, 2008; Ohki et al, 2001; Scarsdale, Webb, Ginder, & Williams, 2011).…”