The N-Terminus of Drosophila SU(VAR)3−9 Mediates Dimerization and Regulates Its Methyltransferase Activity

Abstract

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.

Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila

“…In agreement with its in vitro HMTase activity (Eskeland et al 2004), Su(var)3-9 regulates H3-K9 di-and trimethylation at pericentric heterochromatin. In addition, other H3-K9-specific HMTases must exist that mediate H3-K9 dimethylation at the fourth chromosome and in the chromocenter core of Su(var)3-9 mutants.…”

Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9

“…vSET from Paramecium bursaria chlorella virus, which can dimethylate H3-K27, forms dimers via a Domain II-Domain II interaction between SET domains (Manzur et al 2003). The Drosophila Suv39h homolog dSU(VAR)3-9 was also shown to form dimers via its N terminus (Eskeland et al 2004). It is possible that more SET domain proteins form dimers to exert HMTase activity in vivo.…”

Increased expression ofDrosophilaSu(var)3-7 triggers Su(var)3-9-dependent heterochromatin formation

“…Moreover, methylation of H3-K9 has also been associated with the silencing of euchromatic genes (Nielsen et al, 2001;Ayyanathan et al, 2003). In Drosophila, most of the dimethylation of H3K9 is catalysed by the product of the modifier of PEV Su(var) 3-9 (Tschiersch et al, 1994;Schotta et al, 2002;Czermin et al, 2001;Eskeland et al, 2004). Su(var)3-9 and HP1 physically interact and their localisation The Su(var)3-7 protein is essential for fly viability, and several lines of evidence support its key importance in heterochromatin formation: it binds to pericentric heterochromatin, it potently suppresses variegation and it interacts with HP1.…”