The histone deacetylase HDAC3 targets RbAp48 to the retinoblastoma protein - PubMed (original) (raw)

The histone deacetylase HDAC3 targets RbAp48 to the retinoblastoma protein

E Nicolas et al. Nucleic Acids Res. 2001.

Abstract

The product of the retinoblastoma susceptibility gene, the Rb protein, functions partly through transcriptional repression of E2F-regulated genes. Repression by Rb is mediated, at least in part, by a histone deacetylase complex, whose enzymatic activity relies on HDAC1, HDAC2 or HDAC3. Recently, we have shown that the Rb-associated histone deacetylase complex contains RbAp48 protein, which interacts with HDAC1 and HDAC2. RbAp48 could favour the deacetylation of histones since it binds directly to histone H4. In agreement with that, we show that transcriptional repression of E2F activity requires the presence of RbAp48. HDAC3 was thought not to interact with RbAp48. However, we found that it shared with HDAC1 the ability to favour the recruitment of RbAp48 to Rb. This latter effect was unlikely to be due to activation of Rb function, since HDAC3 did not increase Rb-E2F1 interaction. Rather, we found, surprisingly, that HDAC3 could physically interact with RbAp48 both in vitro and in living cells. Taken together, our data suggest a model in which Rb mediates the recruitment to E2F-regulating promoters of a repressive complex containing either HDAC1, HDAC2 or HDAC3 and the histone-binding protein RbAp48.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Exogenous HDAC3 increases the Rb–RbAp48 interaction. SAOS-2 cells were transfected with 5 µg of the indicated expression vectors [the Rb expression vector encodes the Rb pocket domain (379–928)] by the calcium phosphate co-precipitation method. The amount of promoter in the transfection was kept constant using empty vectors. Twenty-four hours after transfection, total cell lysates were prepared and immunoprecipitated as described (16), using 1 µg of the indicated antibody [anti-Rb, antibody C15G (Santa Cruz Biotechnologies); anti-HA, antibody 12CA5 (Roche Diagnostics)]. Immunoprecipitates were subjected to western blot analysis using the anti-HA antibody (top) or an anti-Rb antibody (antibody XZ55; Pharmingen) (bottom) using standard procedures. The arrows indicate the position of the exogenous proteins. Note that exogenous Rb migrates at ∼60 kDa, because the expression vector used in these experiments expressed a version of Rb deleted for the first 378 amino acids of the molecule.

Figure 2

Figure 2

Exogenous HDAC3 does not increase the E2F1–Rb interaction. SAOS-2 cells were transiently transfected by calcium phosphate co-precipitation with the indicated expression vectors. Total cell lysates were immunoprecipitated using the anti-Rb antibody (top and middle) or an anti-E2F1 antibody (antibody KH95; Santa Cruz Biotechnologies) (bottom). Immunoprecipitates were subjected to western blot analysis using the anti-E2F1 antibody (top and bottom) or the anti-Rb antibody (middle).

Figure 3

Figure 3

Physical association between HDAC3 and RbAp48. (A) 35S-labelled in vitro translated HDAC1 (lanes 5 and 6) or HDAC3 (lanes 3–4 and 7–10) was subjected to GST pull down analysis using beads harbouring 1 µg GST–RbAp48 fusion protein (lanes 3, 5 and 8), control GST (lanes 4, 6 and 7), GST–E2F1 AD (lane 9) or GST–CREB AD (lane 10), as indicated. After extensive washing, bound proteins were analysed by SDS–PAGE followed by autoradiography. In lanes 1 and 2, 10% of the amount of in vitro translated HDAC3 or HDAC1 used in the pull down reaction was directly loaded. (B) SAOS-2 cells were transiently transfected by calcium phosphate co-precipitation with the indicated expression vector and total cell extracts were immunoprecipitated with the indicated antibody (anti-Flag M2 antibody, antibody F; Sigma). Immunoprecipitates were subjected to western blot analysis using the anti-HA antibody. The arrows indicate the position of the two RbAp48 bands. (C) Hela nuclear extracts [50 (lanes 1, 2, 6 and 7) or 200 µl (lanes 11–12)] were immunoprecipitated with 1 µg of either an anti-RbAp48 antibody [lane 2, antibody RBBP (Transduction Laboratories); lane 6, antibody N19 (Santa-Cruz); lane 11, antibody 11G10 (Genetex)] or control anti-HA antibody [lanes 1, 7 and 12, antibody 12CA5 (Roche Diagnostics)]. In lanes 4, 8 and 10, 4 µl of HeLa nuclear extracts were directly loaded. In lanes 3, 5 and 9, 1 µg RBBP, N19 and 11G10 antibodies, respectively, were loaded, to monitor the migration of immunoglobulins. Immunoprecipitates were tested for the presence of HDAC1, HDAC2 and HDAC3 by western blotting using an anti-HDAC antibody (Transduction Laboratories). The stars indicate bands due to the immunoglobulin heavy chains from the anti-RbAp48 antibody (lanes 9 and 11) or the anti-HA antibody (lanes 7 and 12). Note that at longer exposures HDAC1 and HDAC2 could be detected in RBBP immunoprecipitates (lane 2). Also, the amount of HDAC3 in N-19 immunoprecipitates (lane 6) is likely to be overestimated due to co-migration with the immunoglobulin heavy chains, which were weakly detected (lane 5).

Figure 4

Figure 4

Model of transcriptional repression by Rb through the recruitment of histone deacetylases. In G0 or during the beginning of the G1 phase of the cell cycle Rb protein (or one of its cousins, ‘Pocket protein’ in the figure) binds to E2F sites (15). It recruits a histone deacetylase (‘HDAC’) through a direct (HDAC1 or HDAC2) or indirect (HDAC3, through RBP1) interaction. These three deacetylases share the ability to recruit the histone-binding protein RbAp48, leading to deacetylation of histones present on the promoter.

Similar articles

Cited by

References

    1. Muller H. and Helin,K. (2000) The E2F transcription factors: key regulators of cell proliferation. Biochim. Biophys. Acta, 1470, M1–M12. - PubMed
    1. Black A.R. and Azizkhan-Clifford,J. (1999) Regulation of E2F: a family of transcription factors involved in proliferation control. Gene, 237, 281–302. - PubMed
    1. Kaelin W.G.Jr (1999) Functions of the retinoblastoma protein. Bioessays, 21, 950–958. - PubMed
    1. Krek W., Xu,G. and Livingston,D.M. (1995) Cyclin A-kinase regulation of E2F-1 DNA binding function underlies suppression of an S phase checkpoint. Cell, 83, 1149–1158. - PubMed
    1. Zhang H.S., Postigo,A.A. and Dean,D.C. (1999) Active transcriptional repression by the Rb-E2F complex mediates G1 arrest triggered by p16INK4a, TGFbeta and contact inhibition. Cell, 97, 53–61. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources