Methylation of lysine 9 (K9) in the N-terminus tail of histone H3 (H3) in 
chromatin is associated with transcriptionally silenced genes and is mediated by 
histone methyltransferases. Murine G9a is a 1263 amino acid H3-K9 
methyltransferase that possesses characteristic SET domain and ANK repeats. In 
this paper, we have used a series of green fluorescent protein-tagged deletion 
constructs to identify two nuclear localization signals (NLS), the first NLS 
embedded between amino acids 24 and 109 and the second between amino acids 394 
and 401 of murine G9a. Our data show that both long and short G9a isoforms were 
capable of entering the nucleus to methylate chromatin. Full-length or 
N-terminus-deleted G9a isoforms were also catalytically active enzymes that 
methylated recombinant H3 or synthetic peptides representing the N-terminus tail 
of H3. In vitro methylation reactions using N-terminus tail peptides resulted in 
tri-methylation of K9 that remained processive, even in G9a enzymes that lacked 
an N-terminus region by deletion. Co-expression of G9a and H3 resulted in di- 
and tri-methylation of H3-K9, while siRNA-mediated knockdown of G9a in HeLa 
cells resulted in reduction of global H3-K9 di- and tri-methylation. A 
recombinant deletion mutant enzyme fused with maltose-binding protein 
(MBP-G9aDelta634) was used for steady-state kinetic analysis with various 
substrates and was compared with full-length G9a (G9aFL). Turnover numbers of 
MBP-G9aDelta634 for various substrates was approximately 3-fold less compared 
with G9aFL, while their Michaelis constants (K(m)) for recombinant H3 were 
similar. The K(AdoMet)m for MBP-G9aDelta634 was approximately 2.3-2.65 microM 
with various substrates. Catalytic efficiencies (kcat/K(m)) for both 
MBP-G9aDelta634 and G9aFL were similar, suggesting that the N-terminus is not 
essential for catalysis. Furthermore, mutation of conserved amino acids R1097A, 
W1103A, Y1120A, Y1138A and R1162A, or the metal binding C1168A in the catalytic 
region, resulted in catalytically impaired enzymes, thereby confirming the 
involvement of the C-terminus of G9a in catalysis. Thus, distinct domains 
modulate nuclear targeting and catalytic functions of G9a.