Functional and physical interaction between the histone methyl transferase Suv39H1 and histone deacetylases

Abstract

The histone methyl transferase Suv39H1 is involved in silencing by pericentric heterochromatin. It specifically methylates K9 of histone H3, thereby creating a high affinity binding site for HP1 proteins. We and others have shown recently that it is also involved in transcriptional repression by the retinoblastoma protein Rb. Strikingly, both HP1 localisation and repression by Rb also require, at least in part, histone deacetylases. We found here that repression of a heterologous promoter by Suv39H1 is dependent on histone deacetylase activity. However, the enzymatic activity of Suv39H1 is not required, since the N-terminal part is by itself a transcriptional repression domain. Coimmunoprecipitation experiments indicated that Suv39H1 can physically interact with HDAC1, -2 and -3, therefore suggesting that transcriptional repression by Suv39H1 could be the consequence of histone deacetylases recruitment. Consistent with this interpretation, the N-terminal transcriptional repression domain of Suv39H1 bound the so-called 'core histone deacetylase complex', composed of HDAC1, HDAC2 and the Rb-associated proteins RbAp48 and RbAp46. Taken together, our results suggest that a complex containing both the Suv39H1 histone methyl transferase and histone deacetylases could be involved in heterochromatin silencing or transcriptional repression by Rb.

Figure 1

Transcriptional repression by Suv39H1 does not require its enzymatic activity. (A) Schematic representation of Suv39H1. We have indicated the N-terminal chromodomain (hatched box), the SET domain (black box) and the adjacent cysteine-rich region (grey box). Also indicated is the catalytic domain, which includes the cysteine-rich region, the SET domain and the C-terminus of the protein (10). (B) U2OS cells were transiently transfected using 2 µg of GAL4-luciferase reporter vector, 100 ng of pCMV βGAL and 200 ng of an expression vector for the indicated GAL4 fusion protein (expressed from pCMVGT vector). The experiment was performed in duplicate. Luciferase and β-galactosidase activities were measured 24 h after transfection. Results were standardised for transfection efficiency by dividing luciferase activity by β-galactosidase activity. (C) U2OS cells were transiently transfected using 2 µg of GAL4-luciferase reporter vector, 100 ng of pCMV βGAL and 200 ng or 2 µg (as indicated by the height of the open triangle) of an expression vector for the indicated Gal4 fusion protein (expressed from pCMVGT vector). The amount of CMV promoter was kept constant using empty vector. The control sample (with 2 µg of pCMVGT) (Gal4) was performed in duplicate. Luciferase and β-galactosidase activities were measured 24 h after transfection. Results were standardised for transfection efficiency by dividing luciferase activity by β-galactosidase activity. In the lower panel, the expression of Gal4-Suv39H1 fusion proteins was monitored by western blot using an anti-myc antibody (9E10, Roche Diagnostics).

Figure 2

Transcriptional repression by Suv39H1 requires a histone deacetylase. U2OS cells were transiently transfected using 2 µg of GAL4-luciferase reporter vector, 100 ng of pCMV βGAL and 200 ng of the GAL4-Suv39H1 expression vector where indicated. The amount of promoters in the transfection was kept constant using the pCMVGT empty vector. TSA was added, or not, as indicated, 24 h after transfection. Luciferase and β-galactosidase activities were measured 8 h later. Note that we did not divide luciferase by β-galactosidase, because TSA treatment strongly activated the CMV promoter (data not shown, see also the expression of CMV-driven Gal4-Suv39H1 in the lower panel). The expression of Gal4-Suv39H1, which contains three myc tags, was assayed by western blot using the anti-myc antibody (9E10, Roche Diagnostics) (lower panel).

Figure 3

Suv39H1 and histone deacetylases physically interact. (A) HeLa nuclear extracts (400 µl) were immunoprecipitated using the indicated serum (Suv: anti-Suv39H1; PI: preimmune serum). Immunoprecipitates were then assayed for histone deacetylase activity in duplicate. (B) Hela nuclear extracts (400 µl) were immunoprecipitated using the indicated serum. In lane 4, the anti-Suv39H1 serum was preincubated with the peptides (1 µg each) against which the antibody was raised. In lane 1 (Inp), 5 µl of HeLa nuclear extracts were directly loaded. (C) HeLa nuclear extracts (50 µl) were immunoprecipitated using the indicated antibody (HDAC: anti-HDACs; Irr: the anti-HA 12CA5 antibody, Roche Diagnostics). Immunoprecipitates were then assayed for histone methyl transferase activity (HMT activity) using a peptide derived from the histone H3 N-terminal tail as a susbstrate.

Figure 4

The N-terminal transcriptional repression domain of Suv39H1 interacts with histone deacetylases. (A) HeLa nuclear extracts (100 µl) were subjected to a pull-down using beads harbouring ∼1 µg of bacterially produced GST-Suv39H1 4–110 (Suv39H1 4–110) or control GST proteins. After extensive washing, beads were assayed for histone deacetylase activity in duplicate. (B) HeLa nuclear extracts (10 µl) were subjected to a pull-down using beads harbouring ∼1 µg of bacterially produced GST-Suv39H1 4–110 (Suv39H1 4–110) or control GST proteins. After extensive washing, the presence of HDAC1, HDAC2 and HDAC3 on the beads was tested by western blot using the anti-HDACs antibody. The migration of HDAC1, HDAC2 and HDAC3 is indicated by the arrows. In lane 1, 0.5 µl of HeLa nuclear extracts were directly loaded.

Figure 5

Suv39H1 can interact with the ‘histone deacetylase core complex’. The experiment shown in Figure 4B was reprobed using the anti-RbAp48 antibody (anti-RB-BP), which recognises both RbAp48 and the related RbAp46 protein. The migration of RbAp48 and RbAp46 is indicated by the arrows.