FIG. 4.
Gfi1 complex recruits G9a-type histone methyltransferase activity. (A) Immunoprecipitated Gfi1-containing complex possesses histone methyltransferase activity. Vectors expressing either Gfi1 or β-Gal, as a negative control, were transfected into HeLa cells, and cell extracts were immunoprecipitated with anti-Gfi1 or nonspecific antibody, then incubated with histone mixtures (H1 and core histones) and S-[3H]adenosylmethionine, and resolved by SDS-PAGE with Coomassie blue staining (bottom panel). Autoradiography (top panel) demonstrates transfer of tritiated methyl to histones. (B) Coexpression of G9a in HeLa cells, but not Suv39H1, increases histone methyltransferase activity of the immunoprecipitated Gfi1 complex. G9a was HA tagged, Suv39 was Myc tagged, and immunoprecipitation was performed with anti-Gfi1. (Western blotting with the indicated antibodies was performed on the cell extract to confirm expected expression [bottom three panels].) (C) Histone methyltransferase activity recruitment by Gfi1 requires interaction with G9a. Immunoprecipitates of full-length Gfi1, but not the Gfi1(N160) fragment (which does not interact with G9a), demonstrate histone methyltransferase activity. [Gfi1 was Myc tagged, and anti-Myc was used for immunoprecipitation, because the Gfi1 antibody does not recognize the Gfi1(N160) fragment.] (D) Endogenous Gfi1 complex immunoprecipitated from HL-60 cells possesses histone methyltransferase activity, and the Gfi1 immunoprecipitate was similarly assayed. (E) The Gfi1 complex predominantly methylates histone H3, as demonstrated by comparing a mixture of histone substrates to those enriched for H3. (F) The Gfi1 complex methylates H3 but not H4. (G) The Gfi1 complex methylates H3-K9, but not H3-K4. A synthetic peptide corresponding to the amino-terminal 20 residues of H3 and one in which the K4 was predimethylated are both methylated, but predimethylation of K9 diminishes its acceptance as a substrate. (H) Gfi1 complex demonstrates the same site specificity as purified G9a on a series of recombinant histone H3 substrates. The first 84 amino acids of H3, in which lysines were each mutated to an arginine, were purified as GST fusion proteins and tested as substrates. Immunoprecipitates were prepared from HeLa cells transfected with the indicated vector, and their activity was compared to that of purified G9a. Cotransfection with a G9a expression vector does not add significant activity to the Gfi1 immunoprecipitate. Asterisks mark positions corresponding between Coomassie blue-stained and autoradiogram-detected proteins on SDS-polyacrylamide gels.