The Tet/JBP family

This family is defined by all 2OGFeDOs that are closer to the kinetoplastid JBP proteins and the metazoan Tets than any other family of dioxygenases. They are characterized by a shared derived character (synapomorphy) in the form of an extended α-helix just N-terminal to the first core strand (Fig. 1). This long α-helix appears to be kinked in most members of the Tet/JBP family by a conserved proline in the middle of the helix. This family can be divided into 5 distinct subfamilies (Table 1). At least a subset of members of each of the families shows either fusions to DNA-binding or chromatin-associated protein domains, or gene-neighborhood/genome-context associations with known DNA-binding domains (Table 1, Fig. 2). This strongly supports a role for most members of the family in modifying DNA. In the first experimentally studied subfamily of this group, the JBP subfamily, JBP2 is fused to a Swi2/Snf2 ATPase module, which is consistent with the role for ATP-dependent chromatin reorganization in synthesis of the J base in kinetoplastid DNA.³⁵ The kinetoplastid JBP1 is fused to a previously uncharacterized C-terminal domain, which is also present as a solo version in other uncharacterized kinetoplastid proteins. Its predicted secondary structure indicates an α + β topology and it contains several strongly conserved polar residues including an absolutely conserved GGTRY motif (Suppl. material). This implies that it could possess an uncharacterized enzymatic activity or could constitute a specific base J-binding domain.

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Figure 2

Representative domain architectures of the newly identified versions of nucleic-acid-modifying 2OGFeDO proteins. Architectures are arranged by their phylogenetic and family affinities, and are labeled by their gene and species names. Domain architectures within a family are boxed. Domains are typically denoted by their standard names. Non-standard domain nomenclatures are clarified in the inset key at the bottom of the figure. Operons are shown as arrows where the arrow head points from the 5′ to the 3′ direction of the coding frame of the gene. Gene neighborhoods are labeled by the gene coding for the 2OGFeDO protein.

The DNA-binding CXXC domain found in the Tet subfamily proteins also occurs in several chromatin proteins, including the animal DNA methylase DNMT1 and the methylated DNA-binding protein MBD1^11,39,40 (Fig. 2). Given the domain architectural parallel to the DNMT1 methyltransferase and the precedence of the pyrimidine modification catalyzed by the related JBP subfamily we proposed that this subfamily might catalyze oxidative modification of 5-methylcytosines in animal DNA. Further experimental characterization of the human Tet1 protein showed that it indeed catalyzes this reaction to generate 5-hydroxymethylcytosine both in vitro and in cells. Studies based on overexpression of Tet1 in cultured human cells support its role in potential demethylation of 5-methylcytosines directly or indirectly via this oxidative intermediate.³⁸ Based on the crystal structure of the AlkB protein (PDB: 2fd8) we observed that the unique cysteine-rich extension found at the N-terminus of the Tet subfamily 2OGFeDOs (Table 1) is likely, in part, to occupy a position similar to the N-terminal DNA-binding extensions of the AlkB protein.³⁶ Hence, we speculate that this domain might similarly be involved in forming a DNA-recognition surface. The low complexity insert in the core double stranded β-helix of the Tet subfamily is exactly in the same position as an unstructured insert seen in the prolyl hydroxylases (PDB: 2JIJ, Fig. 1) and is inferred to be located on the exterior surface on one side of the 2OGFeDO catalytic domain. Its persistence across the entire Tet subfamily, despite lack of sequence conservation, suggests that it might form a generalized protein-protein interaction surface. In most members we also identified one or more high confidence sumoylation sites in this insert suggesting that the Tet family might be regulated through this protein modification.

In the gnathostome lineage, after the divergence of agnathan vertebrates, the Tet subfamily underwent a triplication to spawn the Tet1, Tet2 and Tet3 genes, which are conserved in all gnathostomes. Of these Tet1 and Tet3 retained the ancestral CXXC domain, whereas Tet2 appears to have lost the CXXC domain. However, analysis of the chromosome neighborhood shows that a standalone CXXC domain protein (CXXC4) is encoded in the same chromosome as a neighboring gene usually in the opposite orientation. This suggests that in Tet2 a local chromosomal inversion detached the ancestral CXXC domain-encoding exon to form a separate gene. In light of this we speculate that CXXC4 could possibly function as an independent protein interacting with Tet2 in a complex. Given the regulation of CXXC4 by the Wnt pathway,⁴¹ it would be interesting to investigate if this might constitute a specific regulatory mechanism feeding into Tet2 enzymatic action. The exon-intron structure of the Tet family is also largely retained across animals. Except Tet2, in all cases the first conserved coding exon encodes the CXXC domain, which probably represents the ancestral gene bearing the CXXC domain that was fused to the 2OGFeDO domain-coding segment. Thus, the Tet progenitor appears to have been acquired prior to the divergence of extant metazoans and underwent a gene-fusion event with the progenitor of the N-terminal exon encoding the CXXC domain. The sequence between the CXXC and the 2OGFeDO domains is a large low complexity region, which is extremely fast evolving and shows poor conservation of exon-intron boundaries across metazoa. This region is also subject to insertions of microsatellite DNA repeats as seen in Tet1 of the platypus, where it appears to have been incorporated into the coding sequence (Suppl. material). In zebrafish Tet3 this region shows a large insertion of an integrated retrovirus into the intron following the CXXC exon. These observations suggest that this intervening low-complexity region between the CXXC domain and the catalytic module appears to be under little evolutionary constraint. The catalytic module comprising of the cysteine-rich extension and the 2OGFeDO domain are encoded by 7 highly conserved exons. However, within the low complexity insert in the 2OGFeDO domain there is considerable variability in exon-intron boundaries and exon numbers.

The expansions of the Tet/JBP homologs in mushrooms and algae define a distinct subfamily where, with a few exceptions, most representatives are standalone proteins (Table 1, Figs. 2 and ​and3).3). However, their genomic context suggests that they are genes within a novel DNA transposon, which appears to have proliferated in some of these organisms (See below). The bacteriophage gp2 subfamily (Table 1) is found close to the viral origin of replication associated with a gene encoding the ParB protein, which belongs to a superfamily of DNA-binding proteins implicated in bacterial and phage chromosome segregation.⁴² Typically, the bacteriophage chromosome origins are enriched in genes related to chromosome-segregation, partitioning and packaging, suggesting that gp2 might interact with the ParB protein in these functions. In other phages the ParB protein shows fusions with DNA methylases, and these enzymes have been implicated in regulation of replication or chromosome partitioning in enter-obacteriophages such as P1.⁴³ Hence, the actinophage gp2 might modify methylated bases or reverse their methylation to regulate chromosome partitioning in these viruses. Given the presence of 5-hmC in DNA of other phages, it is possible that, like Tet1, these phage proteins oxidize 5-methylcytosine to 5-hmC directly on DNA. On the whole the Tet/JBP family shows a sporadic distribution: in prokaryotes it is restricted to certain bacteriophages of the caudoviral group or their prophage derivatives integrated in various genomes. The bacteriophage gp2s are the smallest versions of these proteins and represent more-or-less the minimal 2OGFeDO catalytic domain. Hence, they could potentially be the ancestral versions, which spawned the different eukaryotic versions through lateral transfer. The four remaining eukaryotic subfamilies of the Tet/JPB family are not particularly closely related to each other in terms of sequence similarity or domain architectures and have very patchy phyletic patterns (see Table 1). Thus, the eukaryotic versions could have emerged either via multiple transfers from the bacteriophage/bacterial source and might have also disseminated via cross-species transposition within eukaryotes (see below).

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Figure 3

Genomic organization and domain architectures of predicted transposons encoding DNA-modifying enzymes. Genes are depicted as arrows with the arrow head pointing from the 5′ to the 3′ direction of the coding sequence. Gene neighborhoods of the predicted transposase are typically labeled with the gene name of the 2OGFeDO containing protein, the species name and the gi number. In potential fragmentary elements where the 2OGFeDO is absent, the gene neighborhood is labeled with the gene name of the predicted transposase-containing gene. The key at the bottom of the figure explains non-standard domain and gene names, while other gene and domains names are as commonly used in literature.

The algal RNA-modification associated family

This family is defined by the presence of a synapomorphic tryptophan just N-terminal to the strand prior to the first helix of the core catalytic domain (Suppl. material). In sequence searches, these proteins also tend to recover the Tet/JBP family prior to any other version of the 2OGFeDO superfamily, suggesting that there might be distant relationship between the two families. This family is currently found only in two phylogenetically distinct groups of algae, namely chlorophytes and stramenopiles such as diatoms, pelagophytes and haptophytes. This phyletic pattern suggests that they probably emerged in the primary endosymbiotic photosynthetic chlorophytes and were transferred to stramenopiles during the one or more endosymbiotic associations with the primary photosynthetic lineages. Almost all of these proteins show fusions of the 2OGFeDO domain to domains related to RNA-binding or enzymatic domains involved in RNA metabolism (Table 1 and Fig. 2). The two independent fusions of these 2OGFeDO domains to RNA methylases (Fig. 2) suggests that, like the JBP/Tet family, they might also catalyze further modification of methylated bases, possibly generating a hydroxymethyl derivative like hmC or hmU. Further, fusions to the cysteinyl tRNA synthetase C-terminal domain, which recognizes the anticodon of tRNACys⁴⁴ and the pseudouridine synthase which modifies tRNA⁴⁵ suggests that these enzymes catalyze the formation of hydroxylated bases unique to the tRNAs of algae. 2OGFeDO domains of this family are also fused to a TIM-barrel domain, which belongs to a superfamily that includes decarboxylases and amidohydrolases.⁴⁶ This enzyme could hence potentially act in conjunction with the 2OGFeDO domain in catalyzing removal of a methyl group on a base through an oxidation-decarboxylation mechanism that was proposed earlier for the thymine-7 hydroxylase.³² A member of this family from the pelagophyte Aureococcus contains an interesting C-terminal fusion to a second 2OGFeDO domain, which is however of the AlkB family (see below), suggesting that it might catalyze two distinct oxidative modifications. Members of this family in haptophytes, pelagophytes and diatoms are also fused to a distinctive leucine-rich repeat domain (Fig. 2). The domain architectural diversity of this family (Fig. 2) suggests that it has radiated to catalyze a range of unique RNA modifications, which might have a distinctive adaptive role unique to these algae. In contrast to the majority of these fusions, a representative of this family from Aureococcus (Fig. 2, Suppl. material) is fused to the methylated-DNA binding TAM(MBD) domain. This fusion implies that like Tet1, this protein might catalyze the synthesis of hmC from methylated cytosine in DNA.

The AlkB family

The AlkB family has been previously described and has been extensively characterized both in biochemical and structural terms.^34,47 In addition to the versions acting on DNA, we had also described versions from RNA viruses and eukaryotes that are likely to act on RNA. Representatives of the latter group from animals are fused to RNA methylase domains.² This family is defined by the synapomorphy in the form of a conserved arginine in place of the usual aromatic residue at the end of the C-terminal strand of the core domain³⁴ (Fig. 1). Members of this family also have a unique β-hairpin insert, just N-terminal to the first helix of the core catalytic domain, which interacts with nucleic acids³⁶ (Fig. 1). In this study we discovered a novel subfamily of AlkB proteins in fungi. While the classical AlkB proteins, which act on DNA are not combined with any additional specific DNA-binding domains, this fungal subfamily is typified by a remarkable fusion to multiple N-terminal domains (Table 1 and Fig. 2), including a SAD(SRA) domain.⁴⁸ Some exemplars of the SAD(SRA) domains have been shown to specifically recognize DNA with methylated cytosines;⁴⁹ however, representatives of this AlkB subfamily are found in fungi, such as Cryptococcus neoformans, which apparently lack any known DNA cytosine methylation system (Suppl. material). Further, all characterized representatives of the AlkB family appear to function on N-methylated bases rather than C-5 methylated pyrimidines.⁴⁷ Hence, this version of the SAD(SRA) domain might not recognize 5-methylcytosine, but perhaps some other methylated base. The presence of additional N-terminal DNA-binding domains in this subfamily suggests that, unlike classical AlkBs, it might bind either specific DNA sequences or distinctive DNA structures. Hence, unlike the regular AlkB proteins which repair methylated-DNA in a non-specific manner, members of this subfamily might be involved in a localized DNA repair via recognition of specific sequences or structures of DNA. In evolutionary terms, this fungal AlkB subfamily appears to have been derived through duplication and divergence of the ancestral fungal AlkB, prior to the divergence of ascomycetes and basidiomycetes.

The R3H domain-associated family

This novel family identified in the current study is defined by a RxxW signature at the N-terminus of the first helix of the core catalytic domain and an additional conserved domain just C-terminal to the 2OGFeDO domain (Fig. 2, Suppl. material). This conserved C-terminal domain contains a strongly-conserved signature in the form of HxY and GxD motifs at the N-terminus and a GNxG motif followed by a conserved tyrosine at the C-terminus. This strongly conserved pattern suggests that the domain is likely to be enzymatic. These two N-terminal domains are usually further linked in the same polypeptide to a C-terminal cysteine cluster, predicted to chelate Zn, and a R3H domain (Table 1). R3H domains have been shown to bind both single stranded DNA and RNA,⁵⁰ indicating that this family is likely to act on bases in single stranded nucleic acids, probably in conjunction with the unknown activity catalyzed by the second conserved domain. Interestingly, this family shows a very patchy phyletic pattern, being found in several phylogenetically distant eukaryotes and bacteria (Table 1). For example, it is only found in Daphnia amongst animals or only in Phytophthora among stramenopiles, but none of their close sister groups among completely sequenced genomes. However, it is fairly widespread in the fungi suggesting that it was at least present in the common ancestor of ascomycetes and basidiomycetes. On the whole the phyletic pattern suggests that the family has either undergone extensive lateral transfer between certain lineages and/or multiple instances of gene loss. This family is also notable for lineage-specific expansions in certain lineages, such as the crustacean Daphnia, the mushroom Coprinopsis, the heterolobosean amoeboflagellate Naegleria and the moss Selaginella (Table 1). Multiple copies have often emerged via local gene-duplications and show no evidence for any association with a conserved transposase or transposon encoded proteins. Such expansions are typical of families that provide an adaptive value by being present in multiple diversified copies, usually in the context of counter-pathogen strategies or detoxification of diverse environmental compounds.⁵¹ Hence, a possible role for these proteins could be in the defense against viral nucleic acids or genomic parasites via a novel oxidative modification of nucleic acids.

The DNA glycosylase associated family of 2OGFeDO domains

This family is prototyped by proteins from species of the chlorophyte alga Ostreococcus (e.g., OSTLU_17228), which combine a novel version of the 2OGFeDO domain with a C-terminal DNA glycosylase module. The DNA glycosylase module is orthologous to the animal MBD4,⁵² and like it combines a EndoIII-superfamily DNA glycosylase domain with a divergent TAM(MBD) domain (Fig. 2). However, the fusion with the 2OGFeDO domain appears to be a lineage-specific one that is not represented in multicellular plants. The domain architecture suggests that the 2OGFeDO domain might function in conjunction with the DNA glycosylase domain, with the former domain oxidatively modifying a base and the latter probably carrying out excision of the modified base. Given that members of this family are also found in photosynthetic stramenopile algae and cyanobacteria (Table 1, Fig. 3), it is possible that they were first acquired by the primary endosymbiotic plant lineages from the cyanobacteria and subsequently transmitted to stramenopiles. However, these versions are usually small proteins that do not show the fusions to the DNA glycosylase domain, making it unclear if they actually modify bases in nucleic acids.

L.A. and L.M.I. acknowledge the Intramural research program of the National Library of Medicine, National Institutes of Health, USA for funding their research. M.T. and A.R. are supported by a pilot grant from the Harvard Stem Cell Institute.