Properties of a tobacco DNA methyltransferase, NtMET1 and its involvement in chromatin movement during cell division.
OBJECTIVE
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 information 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.


RESULTS
In vitro assays showed no detectable methylation activity when both hemimethylated and unmethylated 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/intermolecular 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.
Kim
Hyun-Jung
HJ
Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0192, Japan.
Yano
Aiko
A
Wada
Yuko
Y
Sano
Hiroshi
H
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D010940:Plant Proteins; C516651:RAN3 protein, Arabidopsis; D011993:Recombinant Fusion Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases