Transcriptional repression by the retinoblastoma protein through the recruitment of a histone methyltransferase

Abstract

The E2F transcription factor controls the cell cycle-dependent expression of many S-phase-specific genes. Transcriptional repression of these genes in G(0) and at the beginning of G(1) by the retinoblasma protein Rb is crucial for the proper control of cell proliferation. Rb has been proposed to function, at least in part, through the recruitment of histone deacetylases. However, recent results indicate that other chromatin-modifying enzymes are likely to be involved. Here, we show that Rb also interacts with a histone methyltransferase, which specifically methylates K9 of histone H3. The results of coimmunoprecipitation experiments of endogenous or transfected proteins indicate that this histone methyltransferase is the recently described heterochromatin-associated protein Suv39H1. Interestingly, phosphorylation of Rb in vitro as well as in vivo abolished the Rb-Suv39H1 interaction. We also found that Suv39H1 and Rb cooperate to repress E2F activity and that Suv39H1 could be recruited to E2F1 through its interaction with Rb. Taken together, these data indicate that Suv39H1 is involved in transcriptional repression by Rb and suggest an unexpected link between E2F regulation and heterochromatin.

FIG. 1

Physical interaction between Rb and an HMT. (A) Glutathione-agarose beads containing 2 μg of recombinant bacterially expressed GST Rb 379–928 (Rb) or control GST (GST) were incubated with 200 μl of Jurkat cell nuclear extracts. After extensive washing, beads were subjected to an HMT assay using 2 μg of purified histones. Histones were then separated by SDS-PAGE (18% polyacrylamide) and were detected by Coomassie blue staining or fluorography. (B) Beads containing the indicated GST fusion proteins were incubated with 25 μl of Jurkat cell nuclear extracts and, after extensive washing, were subjected to an HMT assay using the histone H3 peptide [wt(1–24)] as a substrate (final concentration, 30 μM). Methylation was quantified using the filter binding assay. (C) Jurkat cell nuclear extracts (150 μl) were subjected to immunoprecipitation (IP) using 5 μg of either a control anti-HA antibody (Irr) (Santa Cruz) or an anti-Rb antibody. Immunoprecipitates were then assayed for HMT activity as described above for panel B.

FIG. 2

Binding of the HMT correlates with Rb antiproliferative activity. (A) Beads containing bacterially expressed GST Rb 379–928 (GST-Rb), GST Rb 379–928 706C-F (GST-Rb Mut), or control GST were incubated with 25 μl of Jurkat cell nuclear extracts and, after extensive washing, were subjected to an HMT assay using the histone H3 peptide [wt(1–24)] as a substrate (final concentration, 30 μM). Methylation was quantified using the filter binding assay. (B) Beads containing either fusion protein GST-Rb or GST-RbMut were used in pulldown reactions as described above for panel A, except that, prior to the addition of Jurkat cell nuclear extracts, beads were incubated with or without (−) 5 or 10 μg (amount indicated by the height of the white triangle) of an SV40 T-antigen-derived peptide or an irrelevant peptide (Irr), as indicated. Bound HMT activity was measured as described in the legend to Fig. 1B.

FIG. 3

The Rb-associated HMT specifically methylates K9 from histone H3. (A) Beads containing either GST-Rb or control GST fusion protein were incubated with 25 μl of Jurkat cell nuclear extracts and were assayed for bound HMT activity using peptides containing either the first 24 amino acids [wt(1–24)] or the first 17 amino acids [wt(1–17)] of histone H3. (B) Beads containing either GST-Rb or control GST fusion protein were incubated with 25 μl of Jurkat cell nuclear extracts and were assayed for bound HMT activity using either the wild-type H3 peptide [wt(1–24)] or the same peptide with the indicated lysine mutated.

FIG. 4

Suv39H1 physically associates with Rb. (A) U2OS cells were transfected with 10 μg of pCMV myc-Suv39H1 and/or pCMV-Rb 379–928 as indicated. Total cell extracts were then immunoprecipitated using an anti-Rb antibody, and immunoprecipitates (IP) were tested for the presence of myc-Suv39H1 by Western blotting (WB) (top gel). The position of the immunoglobulin heavy chain of the immunoprecipitating antibody is indicated by an asterisk. The expression levels of transfected Rb or myc-Suv39H1 are shown in the lower gels. (B) Jurkat cell nuclear extracts (200 μl) were immunoprecipitated with the indicated antibody (anti-Suv39H1 [Suv] or preimmune serum [PI]), and immunoprecipitates were tested for the presence of endogenous Rb by Western blotting using the G3-245 antibody (Pharmingen). In lane 1, 2 μl of Jurkat nuclear extracts was directly loaded as input (inp). (C) Beads containing 0.2 μg of GST, GST-Rb Mut, or GST-Rb fusion protein (middle gel) or 2 μg of GST-Rb (Rb) or control GST (GST) fusion protein (right gel) were incubated with whole extracts (80 μl) from cells transfected with 2.7 μg of myc-Suv39H1 expression vector. Competitor peptides (10 μg) were added where indicated (right gel). After extensive washing, the amount of myc-Suv39H1 pulled down was tested by Western blotting. In lane 1, whole-cell extracts (13 μl) were directly loaded as input.

FIG. 5

Phosphorylation of Rb abolishes its interaction with Suv39H1. (A) Beads containing GST-Rb or GST proteins were phosphorylated in vitro using purified cyclin E-cdk2 kinase complex. The extent of Rb phosphorylation was assessed by Coomassie blue staining. Note the slight change in the migration velocity of GST-Rb upon cyclin E-cdk2 treatment. These beads were used in GST pulldown experiments with whole extracts from cells transfected with the myc-Suv39H1 expression vector, as described in the legend to Fig. 4C (left gel). Bound proteins were detected by Western blotting (WB) using the anti-myc antibody. In lane 4, whole-cell extracts (13 μl) were loaded directly as input (inp). (B) U2OS cells were transfected as described in the legend to Fig. 3B with 10 μg of the indicated expression vectors. Total cell extracts were immunoprecipitated with the anti-myc antibody, and immunoprecipitates (IP) were tested for the presence of Rb by Western blotting (WB) (top gel). In the middle and bottom gels, expression levels of transfected Rb and myc-Suv39H1 are shown. Exogenous Rb migrates at about 60 kDa because the N-terminal part of the protein was deleted (see Materials and Methods). Note that addition of cyclin-cdk expression vectors led to a shift in the migration of transfected Rb (phosphorylated Rb [phospho-Rb] in the middle gel).

FIG. 6

Suv39H1 represses E2F activity. (A) HeLa cells were transiently transfected with 2 μg of E2F-TK luc or control TK-luc reporter vectors, 20 ng of pCMV NeoBam, and 100 ng of pCMV lacZ to monitor transfection efficiency, and with increasing amounts of myc-Suv39H1 expression vector (0, 0.5, 1, and 2 μg [amount indicated by the height of the white triangle]). Luciferase and β-galactosidase activities were measured 48 h later. E2F activity (normalized to that of empty reporter construct) was calculated relative to 100% in the absence (−) of exogenous Suv39H1. The means of four independent experiments are shown. (B) HeLa cells were transiently transfected with 2 μg of GAL4-luc reporter construct, with the indicated amount of SV40 promoter-driven Gal4 E2F1-AD (pHKGal4 E2F1-AD) and/or Rb (pSG5 Rb) expression vectors and with either 0, 1, or 2 μg of pCMV myc-Suv39H1 expression vector. Luciferase activity (in relative light units [RLU]) was measured 48 h later. The result of a typical experiment is shown. Note that transcriptional repression by Rb is more efficient in the presence than in the absence (−) of exogenous Suv39H1. (C) HeLa cells were transiently transfected with 2 μg of GAL4-luc reporter construct, 2 μg of pHKGal4 E2F1-AD, the indicated amount of pSG5 Rb, and various amounts (0, 1, or 2 μg) of either pCMV 2N3T Suv39H1 (HA-Suv39H1) or pCMV 2N3T Suv39H1 1–332 (HA-Suv39H1 1–332), as indicated. Luciferase activity was measured 48 h later. The result of a typical experiment is shown. In the lower panels, the expression levels of HA-tagged Suv39H1 fl or Suv39H1 1–332 were assayed by anti-HA Western blotting.

FIG. 7

Ternary complex formation between E2F1, Rb, and Suv39H1. (A) Beads containing 10 μg of either GST-E2F1 359–437 (E2F1 AD) or control GST fusion proteins were incubated with 200 μl of Jurkat cell nuclear extract, and bound proteins were assayed for HMT activity. (B) U2OS cells were transfected as described in the legend to Fig. 3B with 5 to 15 μg of the indicated expression vectors. Note that the Gal4-Suv39H1 fusion protein is tagged with the myc tag. Total cell extracts were immunoprecipitated with the anti-E2F1 antibody, and immunoprecipitates were tested for the presence of Gal4-Suv39H1 by Western blotting (WB) (top gel). The position of the immunoglobulin heavy chain of the immunoprecipitating antibody is indicated by an asterisk. In the lower gels, expression levels of transfected Rb, Gal4-Suv39H1, and E2F1 are shown.

FIG. 8

Transcriptional repression through artificial recruitment of Suv39H1. (A) U2OS cells or Rb-negative SAOS2 cells were transiently transfected with 2 μg of the GAL4-luciferase reporter construct and the indicated doses of pCMVGal4-Suv39H1. Fold repression by Gal4-Suv39H1 is calculated relative to luciferase activity in the absence of Gal4-Suv39H1 expression vector. (B) Model for transcriptional repression of E2F-regulated promoters by Suv39H1. Rb recruits Suv39H1 through an indirect interaction (protein X, which contains an LXCXE motif) to E2F1 and E2F-regulated promoters. Once on the promoter, Suv39H1 represses transcription.