Dear Editor,
Human MettL3 and MettL14 are two members of the MT-A70 family of S-adenosyl-l-methionine
(SAM)-dependent methyltransferases (MTases). They form a heterodimer
1
known to function as an mRNA adenine-N6 MTase (ref.
2
and references therein). In addition, MettL3 and MettL14 associate with chromatin
and localize to the transcription start sites of active genes
3
. Here we show human MettL3–MettL14 complex is active in vitro as a DNA adenine-N6
MTase and methylates GGACT in single-strand DNA and double-strand DNA-containing mismatches.
DNA methylation in bacteria and archaea is common, occurring at carbon C5 of cytosine
ring or exocyclic amino groups of cytosine (at N4) and adenine (at N6). The bacterial
‘orphan’ MTases—which are not coupled with a restriction endonuclease as part of a
self-defense restriction-modification system (Supplementary Ref. S1)—are generally
involved in epigenetic gene regulation, chromosome replication and DNA repair. Examples
of orphan MTases include the DNA adenine MTase (Dam) in Escherichia coli and cell
cycle-regulated DNA MTase (CcrM) in Caulobacter crescentus which are, respectively,
responsible for maintenance of adenine methylation of GATC or GA
nTC (n = any nucleotide) immediately after replication (Supplementary Ref. S2, S3).
In mammals, the epigenetic DNA methylation marks have been generated and maintained
by DNA cytosine-C5 MTases Dnmt1 and Dnmt3 family (Supplementary Ref. S4), whereas
DNA adenine methylation was reported only recently. Low levels of N6-methyladenine
(N6mA) in DNA have been observed in mouse embryonic stem cells
4
and human glioblastoma
5
. However, the observations of existence of DNA N6mA in the genomes of higher organisms
are controversial (Supplementary Ref. S4.1-2) and the identity of the mammalian DNA
adenine MTase(s) has not yet been convincingly established. Whereas mammalian HemK2
had been documented to be a DNA adenine-N6 MTase (Supplementary Ref. S5) (renamed
as N6AMT1), we and others found that human HemK2 is not active on DNA (Supplementary
Ref. S6, S7).
The following considerations prompted us to investigate whether human MettL3–MettL14
heterodimer (termed MettL3-14 thereafter) also possesses methyl transfer activity
on DNA adenine. First, while MettL3 is preferentially enriched at the 3′ end of protein-coding
genes (Supplementary Ref. S8), echoing its involvement in mRNA adenine methylation,
MettL3 and MettL14 are also associated with chromatin and localize to the transcriptional
start sites of active genes
3
. Reanalyzing published ChIP-seq datasets of MettL3 and MettL14 from human leukemia
MOLM13 cells showed that 37% of MettL3 and 85% of MettL14 binding sites contain DNA
sequences equivalent to the RNA-recognition motif of MettL3-14, represented by RRACH
(R = G/A and H = A/C/U in RNA and A/C/T in DNA) (Supplementary Fig. S1). Upon ultraviolet
irradiation, MettL3 and MettL14 are recruited rapidly (within 2 min) to the damaged
sites, and MettL3 activity is required for the DNA repair
6
. MettL14 has been reported to recognize trimenthylation of histone lysine 36 (H3K36me3)
7
and loss of MettL3 results in loss of trimethylation of histone H3 lysine 4 (H3K4me3)
8
. In addition, the Drosophila MettL3 homolog Ime4 (Inducer of meiosis 4) is localized
to the sites of transcription (Supplementary Ref. S9).
Second, while mammalian MettL3-14 is active on single-strand mRNA
2
, many nucleic acids-modifying enzymes are able to modify both DNA and RNA (ref.
9
and references therein), including members of AlkB family involved in the direct reversal
of alkylation damage to DNA and RNA (Supplementary Ref. S10), and members of Apobec
family of cytidine deaminases (Supplementary Ref. S11). Tet2, one of the ten-eleven
translocation proteins initially discovered as DNA 5-methylcytosine (5mC) dioxygenases
(Supplementary Ref. S12), mediates oxidation of 5mC in mRNA (Supplementary Ref. S13,
S14).
Third, MettL3 and MettL14 belong to a functionally diverse MT-A70 family of SAM-dependent
MTases (Supplementary Ref. S15). Another family member (murine MettL4) was reported
to be responsible for N6-methyladenine deposition in genic elements corresponds with
transcriptional silencing
10
(though the in vitro enzymatic activity of MettL4 was not reported). In addition,
a DNA adenine MTase complex in ciliates (single-celled eukaryotes), consisting of
two MT-A70 proteins (MTA1 and MTA9), methylates double-stranded DNA
11
. We note that four out of five nucleotides within the consensus motif of MettL3-14
overlap with the recognition sequence of CcrM (GG
A
CT vs. G
A
n
TC), an enzyme active on both double-stranded (ds) and single-stranded (ss) DNA (Supplementary
Ref. S16). We thus included CcrM as a positive control. Both CcrM and MettL3-14 are
β-class MTases
12
.
We designed three short dsDNA oligos (the oligo numbers refer to our laboratory code
and letters T and B designate the top and the bottom strand respectively): #4 contains
the CcrM recognition sequence GA(g/c)TC, #5 contains the Dam recognition sequence
GATC and used as a negative control, and #6 contains GGACT, the DNA equivalent of
the RNA-recognition sequence of MettL3-14 and an overlapping CcrM recognition sequence
(GGA
c
TC) (Fig. 1a). Using purified recombinant enzymes (Supplementary Fig. S2a), under
the saturating conditions of high enzyme concentration ([E] = 2 μM) where CcrM completed
reactions on its known substrates (oligos #4 and #6), we observed no activities of
MettL3-14 on the double-stranded oligos examined (Fig. 1b). However, we did observe
strong activity of MettL3-14 on single-stranded 6 T (containing GGACT), much reduced
activities on 4 T (containing GGAGT) and 4B (AGACT), and no activity on oligos having
two substitutions within the recognition, e.g. 6B (AGAGT) or 5 T and 5B (GGATC) (Fig.
1c, d). Furthermore, we confirmed that MettL3-14 has no activity on an oligo-containing
G and C only (control 1), and no activity on A-containing oligos without matching
consensus sequence (controls 2 and 3) (Supplementary Fig. S2b). Importantly, single
A-to-G substitution in oligo 6 T abolished the activity (Supplementary Fig. S2c),
clearly demonstrating the A within the GGACT recognition sequence is the site of methylation
by MettL3-14 complex.
Fig. 1
Human MettL3-14 complex is active on ssDNA and mismatched DNA adenine.
a Olignonuceotides (14-mer) used as substrates. b MettL3-14 is not active on dsDNA.
The enzyme concentration [E] is noted in each panel. c, d MettL3-14 is active on ssDNA
by two independent assays: incorporation of tritium from 3H-SAM into DNA substrate
(panel c) and formation of byproduct SAH in a bioluminescence assay (panel d). Inserted
box listed ssDNA sequence alignment. e Substitution of conserved cytosine within GGACT
diminished Mettl3-14 activity. f Replacement of the second guanine-to-adenine (G
A
ACT, red) or the last thymine-to-cytosine (GGAC
C
, blue) retained comparable activity. The two lines fell on top to each other, giving
rise to purple. g Comparison of CcrM (GAnTC) and MettL3-14 (GGACT) on ssDNA oligo
6 T and its derivatives. h Comparison of MettL3-14 on oligo 6 T and its corresponding
RNA. i Comparison of the kinetic parameters on ssDNA and ssRNA, derived from Supplementary
Fig. S2d. j Quantitative measurement of N6mA by mass spectrometry, derived from Supplementary
Fig. 3. k, l Comparison of CcrM and MettL3-14 on oligo 6 T. m, n MettL3-14 is active
on 28-bp dsDNA-containing mismatched pairs. Data represent the mean ± SD of three
independent determinations (N = 3) performed in duplicate.
We tested the sequence specificity for the conserved cytosine after the target adenine
(AC) by substituting the C to the other three nucleotides (T, A and G) in the context
of oligo 6 T (GGAnT). We used lower enzyme concentration ([E] = 0.2 μM) and shorter
reaction time to perform the assays in the linear range. We found that MettL3-14 has
no activity on the C-to-T or C-to-A substitutions, and residual activity on the C-to-G
substitution (GGAGT) (Fig. 1e). The outer guanine-to-adenine substitution (AGACT)
had minor activity, while combining both replacements resulted in total loss of activity
(AGAGT) (Fig. 1e). However, replacement of the second guanine-to-adenine (G
A
ACT) or the last thymine-to-cytosine (GGAC
C
) retained comparable activity (Fig. 1f). The preferred sequence (G-g/a-AC-t/c) is
consistent with the consensus RNA sequence (RRACH)
2
, particularly the requirement of a cytosine following the target adenine (Supplementary
Fig. S1c). As noted above, the oligo 6 T also contains an overlapping CcrM consensus
sequence with the cytosine after target adenine corresponds to the variable position
(GAnTC). As expected, CcrM methylates oligo 6 T and its derivatives with the N variations
(Fig. 1g).
More strikingly, MettL3-14 acts on ssDNA (6 T) faster than that on the corresponding
ssRNA under these conditions; but the heterocomplex is inactive on dsDNA or RNA/DNA
hybrid (Fig. 1h). Under stead-state kinetics conditions, MettL3-14 methylates the
6 T ssDNA with k
cat = 1.98 min−1 and Km = 7.7 µM (Fig. 1i and Supplementary Fig. S2d). On ssRNA of
the same sequence and length, the MettL3-14 methylation rate is ~5-fold slower (k
cat = 0.42 min−1) with ~3-fold weaker affinity for the ssRNA substrate (Km = 23 µM).
In other words, MettL3-14 shows 13-fold weaker catalytic efficiency of methylation
on an RNA substrate (comparing k
cat/Km value of 0.02 µM min−1 for RNA and 0.26 µM min−1 for DNA). In addition, we
applied quantitative mass spectrometry to monitor the product formation using the
single-A-containing ssDNA oligo (6 T) as substrate. We detected ~95% conversion of
A-to-N6mA in the presence of SAM, but not for the DNA alone or with Sinefungin (an
analog of SAM and a pan-inhibitor against SAM-dependent MTases) (Fig. 1j and Supplementary
Fig. S3). Furthermore, the in vitro activity of MettL3-14 on ssDNA is comparable (~2×
slower) to that of the well-characterized CcrM on the same substrate (Fig. 1k, l).
We note that the preference of MettL3-14 for ssDNA over ssRNA is analogous to that
of E. coli AlkB, which removes methyl lesions in ssDNA more efficiently than RNA in
vitro (Supplementary Ref. S17).
While DNA sequences are base paired in canonical double helix, transient local unwinding
of dsDNA does occur during processes of transcription, replication, recombination
and DNA repair, such as a transcriptional bubble (Supplementary Ref. S18). The feature
of ssDNA in E. coli could be induced by stress-induced DNA duplex destabilization
(SIDD) enriched in promoter regions
13
and ssDNA is too a common feature of mammalian genome potentially involved in gene
regulation
14
. In Caulobacter crescentus, CcrM methylates the adenine of hemimethylated GAnTC sites
following replication (Supplementary Ref. S19). Recently we show that CcrM binds DNA
by strand-separation of dsDNA and creates a bubble at its recognition site
15
(Supplementary Fig. S4a), in agreement with CcrM being active on both dsDNA and ssDNA
in vitro (Supplementary Ref. S16). In addition, CcrM can accommodate mismatch within
or immediate outside of the recognition sequence (Supplementary Ref. S20).
As noted above, MettL3-14 is rapidly recruited to the UV-induced DNA damage sites
6
. We thus asked whether MettL3-14 also has the capacity to methylate dsDNA with mismatches
or unpaired region within the recognition sequence. We synthesized a longer DNA molecule
(28-bp) with one-to-six unpaired bases flanked by at least eleven base pairs on either
side to assure the formation of one complete helical turn of dsDNA (Supplementary
Fig. S4b). We first validated that MettL3-14 complex methylates the 28-nt and 14-nt
ssDNA about equally (Supplementary Fig. S4c), and that it is completely inactive on
the fully paired duplex (as expected). However, partial activity was observed on the
28-bp dsDNA-containing unpaired bases, either 1, 3, 5 or 6 mismatched bases (U1 to
U6) centered on the target adenine, while no activity was detected when the mismatched
pairs do not include the target A (U2) (Fig. 1m). Interestingly, the level of activity
on dsDNA correlates positively with the number of mismatched bases (Fig. 1n) and is
not affected by the excess of bottom strand which contains no recognition sequence—added
to assure that the top, target strand is fully annealed (Supplementary Fig. S4d).
In summary, we characterized for the first time the in vitro enzymatic activity of
mammalian MettL3-14 as a sequence-specific DNA adenine–N6 MTase complex. The complex
specifically methylates single-strand DNA and unpaired region (with reduced activity)
in the context of double-strand DNA. Additional study will be required to address
whether MettL3-14 mediates DNA adenine methylation in vivo and its impact on chromatin
organization. Finally, there are ancillary factors, such as Wilms tumor suppressor-associated
protein (WTAP), that ultimately determine the cellular functions of MettL3-14. WTAP
is required for MettL3-14 localization (Supplementary Ref. S21) and in vivo methylation
activity on mRNA (Supplementary Ref. S21.1-3). In addition, WTAP plays a role in both
transcriptional (perhaps acting on DNA) and post-transcriptional (perhaps acting on
mRNA) regulation of certain cellular genes (Supplementary Ref. S22). How WTAP affects
the activity of MettL3-14 on RNA vs DNA requires further study. We note that MettL3
and MettL14 (but not WTAP) are recruited to the damaged sites upon ultraviolet irradiation
6
.
A potential correlation might exist between markedly upregulated N6mA levels in glioblastoma
5
and stress-induced DNA duplex destabilization
14
. The DNA strand-separation event might facilitate the access to the target base by
the MTase complex studied here (as well as other β-class MT-A70 family members). Furthermore,
the methyl group covalently attached to the adenine N6 atom—which is directly involved
in Watson–Crick A:T base pairing—might in turn compromise DNA stability locally (Supplementary
Ref. S23). The destabilized N6mA-containing region might also facilitate the removal
of the methyl group of N6mA by the AlkB family of repair enzymes.
Supplementary information
Supplementary Information