Published online 6 June 2005
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Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase
New England Biolabs 240 Country Road, Ipswich, MA 01938-2723, USA 1Department of Pathology, Molecular Biology Institute and Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA Los Angeles, CA 90095, USA
*To whom correspondence should be addressed. Tel: +1 978 927 5054; Fax: +1 978 921 1350; Email: pradhan{at}neb.com
Received April 14, 2005. Revised May 16, 2005. Accepted May 16, 2005.
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-G9a
634) was used for steady-state kinetic analysis with various substrates and was compared with full-length G9a (G9aFL). Turnover numbers of MBP-G9a634 for various substrates was 
3-fold less compared with G9aFL, while their Michaelis constants (Km) for recombinant H3 were similar. The 
for MBP-G9a634 was 2.3–2.65 µM with various substrates. Catalytic efficiencies (kcat/Km) for both MBP-G9a634 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.
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