Properties of a Tobacco DNA Methyltransferase, NtMET1 and Its Involvement
in Chromatin Movement during Cell Division
HYUN-JUNG KIM, AIKO YANO, YUKO WADA and HIROSHI SANO*
Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0192, Japan
Received: 6 November 2006 Returned for revision: 5 December 2006 Accepted: 9 January 2007 Published electronically: 2 March 2007
† Background and Aims Plants possess three types of DNA methyltransferase, among which methyltransferase type
1 (MET1) is considered to play a major role by maintaining the CpG methylation patterns. However, little infor-
mation is available as to its enzymatic activity, interacting proteins and spatial and temporal behaviours during
DNA replication. In the present study, one example, NtMET1 from tobacco plants, was selected and an analysis
was made of its biochemical properties and cellular localization.
† Methods NtMET1 was expressed in Sf9 insect cells, and a purified sample was subjected to a standard in vitro
methylation assay. Intramolecular interaction was examined by the yeast two-hybrid and pull-down assays.
Transgenic tobacco plants (Nicotiana tabacum) over-expressing NtMET1 were constructed via Agrobacterium-
mediated transformation. Cellular localization was examined by fluorescence protein fusion, which was expressed
in tobacco bright yellow 2 cells.
† Key Results In vitro assays showed no detectable methylation activity when both hemimethylated and unmethy-
lated DNA samples were used as the substrate. In planta assays with over-expressing transgenic lines showed no
hypermethylation but rather hypomethylation of genomc DNA. The inability of methylation was conceivably due
to a tight intramolecular interaction between the N- and C-terminal regions with the catalytic domain residing on
the C-terminus being completely masked. Cellular localization analyses indicated that NtMET1 localized to the
nucleus in the resting stage and migrates to the cytoplasm during mitosis, particularly at metaphase. The pattern
observed resembled that of Ran GTPase, and in vitro pull-down assays showed a clear interaction between
NtMET1 and AtRAN3, an Arabidopsis orthologue of tobacco Ran GTPase, NtRan-A1.
† Conclusions The results suggest that enzymatic activity of NtMET1 is well adjusted by its own intra/intermolecu-
lar interaction and perhaps by interactions with other proteins, one of which was found to be Ran GTPase. Results
also revealed that NtMET1 becomes localized to the vicinity of chromatin with the aid of Ran GTPase during cell
division, and may play an important role in progress through mitosis independently of methylation activity.
Key words: DNA methyltransferase, intra/inter molecular interaction, 5-methylcytosine, mitosis, Ran GTPase, Nicotiana
tabacum.
INTRODUCTION
Methylation of cytosine residues is commonly observed in
the DNA of most eukaryotes, this often being called
DNA methylation, and has been considered to play
certain roles in controlling gene expression (Yoder et al.,
1997). Cytosine methylation is enzymatically catalysed by
DNA methyltransferases, which transfer a methyl group
from S-adenosylmethionine to the 5-position of cytosines
in DNA. In plants, three types of DNA methyltransferases
have been identified based on sequence analyses: DNA
methyltransferase type I (MET1), chromomethylase and
domains rearranged methylase (DRM), among which
MET1 is believed to predominantly catalyse methylation
of hemimethylated symmetrical CpG, thereby maintaining
methylation patterns after DNA replication (Finnegan
and Dennis, 1993). In addition, the green alga,
Chlamydomonas reinhardtii was shown to possess a
DNA methyltransferase, whose structure resembles the
MET1, but exhibits catalytic activities similar to DRM
(Nishiyama et al., 2004).
MET1 consists of the N-terminal regulatory domain and
the C-terminal catalytic domain (Fig. 1A). The N-terminal
domain contains several putative signal motifs, including
nuclear localization, bromo-adjacent homology domains,
zinc fingers and serine-rich regions (Fig. 1A). Similar
signal motifs are also found in mammalian DNA methyl-
transferase 1 (Dnmt1), the orthologue of plant MET1 pro-
teins (Fig. 1A). However, despite the similar molecular
size, few common motifs with plant MET1 were found in
the N-terminal of the Chlamydomonas enzyme (Fig. 1A),
suggesting a complicated function of the N-terminal
regions. In contrast, the C-terminal domain contains
highly conserved protein motifs, which are common
among DNA methyltransferases of different types from
different organisms (Fig. 1B).
Curiously, however, biochemical studies on catalytic
properties of MET1 enzymes have been hitherto limited.
As far as is known, only two preliminary reports are avail-
able; one is a study on maize MET1 (ZmMET1,
AF229183), of which N-terminal-truncated protein was
reported to exhibit a low methylation activity in vitro
(Steward et al., 2000). The other case is pea MET (C-5
MTase, AF034419), which showed methylation activity
towards Cp(A/T)pG and poly(dI-dC) synthetic oligomers
(Pradhan et al., 1998). However, lack of enough knowledge
on catalytic properties has made it difficult to fully under-
stand the biological functions of MET1 proteins. Indeed,
* For correspondence. E-mail sano@gtc.naist.jp
# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Annals of Botany 99: 845–856, 2007
doi:10.1093/aob/mcm021, available online at www.aob.oxfordjournals.org

most functional studies, including Arabidopsis enzymes,
have been carried out on genetic bases without substantial
activity assays (Finnegan and Kovac, 2000; Goll and
Bestor, 2005).
In addition to methylation activity, DNA methyltransfer-
ase proteins have been proposed to directly participate
in regulatory function (Rountree et al., 2000). The
N-terminus of mammalian Dnmt1 was shown to possess
transcriptional repressing activity by directly interacting
with histone deacetylase (Rountree et al., 2000), histone
methyltransferase (Fuks et al., 2000) and retinoblastoma
tumour suppressor proteins (Robertson et al., 2000).
These observations suggest that Dnmt1 plays an important
role in the gene regulation network independent of DNA
methylation (Milutinovic et al., 2004). In accordance with
this idea, intracellular localization analyses revealed that
mouse Dnmt1 moved between nuclei and cytoplasm
during embryo development (Mertineit et al., 1998). It
was speculated that Dnmt1 family proteins might be
involved in sister chromatid segregation during the mitotic
phase (Hung et al., 1999). As to plant MET1 proteins, no
such information has so far been available.
In order to understand the general features of MET1
function, a tobacco example, NtMET1, was selected and
was found apparently not to exhibit DNA methylation acti-
tivty in vitro, conceivably due to tight intra/intermolecular
interactions. It is also reported here that NtMET1 interacts
with Ran GTPase, thereby reversibly changing cellular
localization during mitosis in tobacco cells.
MATERIALS AND METHODS
Plant materials
Wild-type and transgenic tobacco plants (Nicotiana
tabacum ‘Xanthi nc’) were grown in a growth cabinet at
23 8C under a 14/10 h light/dark cycle. Tobacco-cultured
bright yellow 2 (BY2) cells, from both wild-type and
B
A
Motif I Motif IV
Motif VI Motif VIII
Motif IX Motif X

NtMET1 1122-TLDIFAGCGGLSEGLQRSGV-1141 NtMET1 1216-VDFINGGPPCQGFSGMNRFNQSTW-1239
Dnmt1 1144-TLDVFSGCGGLSEGFHQAGT-1163 Dnmt1 1217-VEMLCGGPPCQGFSGMNRFNSRTY-1240
CrMET1 731-TLDIFAGCGGLSEGLHQSGV-750 CrMET1 841-VELLVGGPPCQGFSGLNRHPGSEK-864
NtDRM1 474-LKDLFPGGINVLSLFSGIGG-493 NtDRM1 49-GEASSSSVPCQSKFIQQFVVMGFP-72

NtMET1 1258-RPKFFLLENVRNFVSFNQKQ-1277 NtMET1 1298-LEAGAFGVPQSRKRAFIWAA-1258
Dnmt1 1259-RPRFFLLENVRNFVSFKRSM-1278 Dnmt1 1299-LQAGQYGVAQTRRRAIILAA-1318
CrMET1 883-RPRYFILENVMGFTFHKPVQ-902 CrMET1 1006-LNAGNYGVPQSRKRVFIIAA-1025
NtDRM1 287-GPPFFYYENVALAPKGVWDT-306 NtDRM1 402-MRVQKFVLDQCRKWNLVWVG-421

NtMET1 1500-DRIVTVRECARSQGFPD-1516 NtMET1 1512-QGFPDSYQFAGNILHKHRQIGNAV-1535
Dnmt1 1545-HRVVSVRECARSQGFPD-1561 Dnmt1 1557-QGFPDTYRLFGNILDKHRQVGNAV-1580
CrMET1 1258-QRTVSVREVARSQGFPD-1274 CrMET1 1280-GSVADCYKQVGNAVPPPLALALGL-1303
NtDRM1 474-NRIRKAVEDFDGEPPMR-493 NtDRM1 449-ISRTDRYKSLGNSFQVDTVAYHLS-472
NLS
Regulatory region
1556 aa
1620 aa
1344 aa
608 aa
NtMET1
Dnmt1
CrMET1
NtDRM1
100 aa
Catalytic region
Ser-rich ZF BAH BAH I IV VI VIII IX X
NLS
Replication
foci Cys-rich BAH BAH I IV VI VIII IX X
Arg-rich BAH I IV VI VIII IX X
VINLSUBAUBA VIVIII IX X
FIG. 1. Properties of DNA methyltransferase. (A) Schematic illustration of DNA methyltransferases. Structure of NtMET1 (tobacco; accession no.
AB280788), Dnmt1 (murine; NM010066), CrMET1 (Chlamydomonas; AB073989) and NtDRM1 (tobacco; AB087883) are compared. Their in vitro
enzymatic activity was confirmed except for NtMET1. NLS, Nuclear localization signal; Ser-rich, serine-rich region; ZF, zinc-finger region; BAH,
bromo-adjacent homology domains; cys-rich, cysteine-rich region; Arg-rich, arginine-rich region, UBA, ubiquitin association domain. Roman letters indi-
cate conserved domains for catalytic activity. Bar indicates the range of 100 amino acids. (B) Motif alignment. Catalytic motifs illustrated in (A) are
aligned. Amino acids in common among four proteins are boxed, those among three are shaded.
Kim et al. — Type 1 DNA Methyltransferase from Tobacco Plants846