The N-terminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity.
In most eukaryotes, the histone methyltransferase SU(VAR)3-9 and its orthologues play a major role in the function of centromeric heterochromatin. Although the methyltransferase domain is required for the formation of a fully functional centromere, mutations within other regions of the gene such as the N-terminus also have a strong impact on its in vivo function. To analyze the contribution of the N-terminus on the methyltransferase activity, we have expressed the full-length Drosophila SU(VAR)3-9 (dSU(VAR)3-9) together with various N-terminal deletions in Escherichia coli and analyzed the structural and enzymatic properties of the purified recombinant enzymes. Full-length dSU(VAR)3-9 specifically methylates lysine 9 within histone H3 on peptides, on intact histones, and, to a lesser extent, on nucleosomes. A detailed analysis of the reaction products shows that dSU(VAR)3-9 adds two methyl groups to an unmethylated H3 tail peptide in a nonprocessive manner. The full-length enzyme elutes with an apparent molecular weight of 160 kDa from a gel filtration column, which indicates the formation of a dimer. This property is dependent on an intact N-terminus. In contrast to the full-length enzymes, proteins lacking the N-terminus fail to dimerize, and show a 10-fold lower specific activity and a linear dependence of methyltransferase activity on enzyme concentration. A N-terminal peptide containing amino acids 1-152 of dSU(VAR)3-9 is sufficient to mediate this interaction in vitro. The dimerization of dSU(VAR)3-9 and the subsequent increase of its methyltransferase activity provide a starting point to understand the molecular details of the formation of heterochromatic structures in vivo.
Eskeland
Ragnhild
R
Adolf-Butenandt Institute, Department of Molecular Biology, Ludwig-Maximillians University of Munich, Schillerstrasse 44, 80336 Munich, Germany.
Czermin
Birgit
B
Boeke
Jörn
J
Bonaldi
Tiziana
T
Regula
Jörg T
JT
Imhof
Axel
A
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D019281:Dimerization; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011496:Protein Methyltransferases; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion / Q000235:genetics
D029721:Drosophila Proteins; D006657:Histones; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000076983:Histone Methyltransferases; D008780:Methyltransferases; D011496:Protein Methyltransferases; C445404:SU(VAR)3-9; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine