G9a (SET Domain-containing Protein, G9a Histone Methyltransferase, G9a HMTases)

The covalent modification of histone tails has regulatory roles in various nuclear processes, such as control of transcription and mitotic chromosome condensation. Among the different groups of enzymes known to catalyze the covalent modification, the most recent additions are the histone methyltransferases (HMTases), whose functions are now being characterized. Here we show that a SET domain-containing protein, G9a, is a novel mammalian lysine-preferring HMTase. Like Suv39 h1, the first identified lysine-preferring mammalian HMTase, G9a transfers methyl groups to the lysine residues of histone H3, but with a 10-20-fold higher activity. It was reported that lysines 4, 9, and 27 in H3 are methylated in mammalian cells. G9a was able to add methyl groups to lysine 27 as well as 9 in H3, compared with Suv39 h1, which was only able to methylate lysine 9. Our data clearly demonstrated that G9a has an enzymatic nature distinct from Suv39 h1 and its homologue h2. Finally, fluorescent protein-labeled G9a was shown to be localized in the nucleus but not in the repressive chromatin domains of centromeric loci, in which Suv39 h1 family proteins were localized. This finding indicates that G9a may contribute to the organization of the higher order chromatin structure of non-centromeric loci. \n\nApplications:\nSuitable for use in Immunoblot. Other applications not tested.\n\nRecommended Dilution: \nWestern Blot: 0.2-2ug/ml detected G9a in HeLa nuclear extract cell lysate. HeLa nuclear extract was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-G9a (0.5ug/ml). Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.\n\nOptimal dilutions to be determined by the researcher.\n\nIncluded Positive Antigen Control:\nHeLa nuclear extract. Add an equal volume of Laemmli reducing sample buffer to 10ul of extract and boil for 5 minutes to reduce the preparation. Load 20ug of reduced extract per lane for minigels.\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. For long-term storage, store at -20 degrees C. Aliquots are stable for at least 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

G1009

Size

200ug

Applications

WB

Hosts

Rabbit

Reactivities

Hum

Form

Supplied as a liquid in 0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide before the addition of glycerol to 30%.

P Type

Pab

Purity

Purified by Protein A affinity chromatography.

Isotype

IgG

References

1. Tachibana, M., et al., J. Biol. Chem. 276: 25309-25317, 2001. 2. Tachibana, M., et al., Genes Dev. 16: 1779-1791, 2002. 3. Gyory, I., et al., Nat. Immunol. 5: 299-308, 2004. 4. Xin, Z. et al., J. Biol. Chem. 278: 14996-15000, 2003.

Additional Info

Recognizes human G9a protein, Mr ~140kD. Species Crossreactivity: Human. Predicted to crossreact with mouse based on sequence homology.