doi: 10.1093/emboj/cdg120.
Purification and functional characterization of the human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1
Affiliations
- PMID: 12628926
- PMCID: PMC151047
- DOI: 10.1093/emboj/cdg120
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Purification and functional characterization of the human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1
EMBO J.
.
Abstract
Corepressors N-CoR and SMRT participate in diverse repression pathways and exist in large protein complexes including HDAC3, TBL1 and TBLR1. However, the roles of these proteins in SMRT-N-CoR complex function are largely unknown. Here we report the purification and functional characterization of the human N-CoR complex. The purified N-CoR complex contains 10-12 associated proteins, including previously identified components and a novel actin-binding protein IR10. We show that TBL1/TBLR1 associates with N-CoR through two independent interactions: the N-terminal region and the C-terminal WD-40 repeats interact with the N-CoR RD1 and RD4 region, respectively. In vitro, TBL1/TBLR1 bind histones H2B and H4, and, importantly, repression by TBL1/TBLR1 correlates with their interaction with histones. By using specific small interference RNAs (siRNAs), we demonstrate that HDAC3 is essential, whereas TBL1 and TBLR1 are functionally redundant but essential for repression by unliganded thyroid hormone receptor. Together, our data reveal the roles of HDAC3 and TBL/TBLR1 and provide evidence for the functional importance of histone interaction in repression mediated by SMRT-N-CoR complexes.
Figures
Fig. 1. Purification of the N-CoR complex from HeLa nuclear extracts and identification of N-CoR-associated proteins by mass spectrometry. (A) Diagram illustrating the purification scheme of the N-CoR complex. PC, phosphocellulose p11 resins; DEAE, DEAE–Sepharose Fast Flow resins. (B) The differential fractionation of SMRT and N-CoR proteins by various chromatographic steps. The protein samples from each fraction were analyzed by western blotting using the specific antibodies indicated. (C) Immunoaffinity purification and identification of the N-CoR complex. The pooled fractions of immunoaffinity-purified N-CoR complex were resolved using an 8% NuPAGE (Novex) gel and visualized by Coomassie staining. The peptide sequences derived from p54, p57, p61 and p70 determined by mass spectrometry and the identity of these proteins are shown on the right.
Fig. 2. Characterization of N-CoR-associated proteins. (A) The amino acid sequence of human IR10 protein. The regions identified by mass spectrometry are underlined. The WD-40 repeats are boxed. (B) N-CoR proteins remained in a large protein complex after three chromatographic steps. The PC 0.5 M or HeLa nuclear extract was fractionated with a Superose 6 gel filtration column. Fractions were collected, and analyzed by western blotting using the antibodies indicated on the left. (C) Western blot analyses of N-CoR–SMRT-associated proteins. N-CoR or SMRT complex was immunoprecipitated from HeLa nuclear extracts using N-CoR- or SMRT-specific antibodies, and the presence of various proteins was analyzed by western blotting using antibodies as indicated. Input, 10% HeLa nuclear extract used for immunoprecipitation (IP); control, no antibody; and mock, IP with rabbit anti-mouse IgG. (D) IR10 associates with N-CoR but not SMRT. HeLa nuclear extracts were immunoprecipitated with antibodies against TBL1, HDAC3 and IR10, respectively, and probed by western blotting using N-CoR or SMRT antibody.
Fig. 3. TBL1/TBLR1 associate with N-CoR through two independent, reciprocal interactions. (A) TBL1 and TBLR1 interact with N-CoR1 and N-CoR4 in vitro. The upper panel shows the structure of N-CoR and deletion constructs used for mapping experiments. Each fragment was in vitro translated, [35S]methionine labeled and used for in vitro pull-down assays using GST–TBL1 and GST–TBLR1 fusion proteins. The result of the pull-down assay is summarized in the top right panel. Input was 20% of the sample used for pull-down assay. (B) The TBL1/TBLR1 interaction region in N-CoR4 was mapped to N-CoR 1801–1965. The left hand side shows the N-CoR4 deletion mutants, and experiments were performed as in (A). (C) The interaction with N-CoR4 was mapped to the WD-40 repeat region of TBL1/TBLR1. The deletion constructs of TBL1 and TBLR1 were as indicated. The results of pull-down assays are summarized on the right side of each construct.
Fig. 4. Both N-CoR interaction domains in TBL1 are sufficient for interaction with endogenous N-CoR. (A) HeLa cells were transfected with a GAL4(DBD), GAL4-TBL1-1 or GAL4-TBL1-2 expression construct and, after 24 h, cell extracts were prepared and immunoprecipitated with a GAL4(DBD)-specific antibody. The association with N-CoR and HDAC3 was revealed by western analysis. (B) The WD-40 repeat domain of TBL1 interacts specifically with the N-CoR4 region in HeLa cells. The GAL4-TBL1-2 construct was co-transfected with either a Flag-tagged N-CoR1 or N-CoR4 expression plasmid into HeLa cells. The interaction was assayed by immunoprecipitation using a GAL(DBD)-specific antibody followed by western analysis using a FLAG-specific antibody (M2, Sigma). (C) The N-CoR4 region repressed transcription when tethered to DNA, and this repression could be partially blocked by TBL1/TBLR1 WD-40 repeats. The amount of plasmids used for transfection: 4xUAS/TK-Luc reporter vector, 0.2 µg, and expression vectors, +, 0.3 µg; ++, 1.0 µg. The luciferase data, expressed as RLU, are the mean ± SD of three independent transfection experiments.
Fig. 5. Both TBL1 and TBLR1 interact with core histones H2B and H4. (A) GST pull-down assays with GST–TBL1 or TBLR1 and in vitro translated, [35S]methioine-labeled core histones. Bound core histone was detected by fluorography. GST–RbAp48 was used as positive control. (B) In vitro translated histones H2B or H4 were incubated with the indicated GST fusion proteins and the interaction was determined by GST pull-down. (C) Diagram summarizing the results of GST pull-down assays. (D) TBL1 is capable of binding to in vitro reconstituted nucleosomes. In vitro reconstitution and purification of mononucleosome were essentially as described previously (Wong et al., 1995). The retention of nucleosomes by GST–TBL1 was revealed by western analysis using core histone-specific antibodies.
Fig. 6. The repression activity of TBL1/TBLR1 correlates with histone interaction but not with N-CoR interaction. (A) Repression assay in HeLa cells. HeLa cells were transiently transfected with 0.2 µg of 4xUAS/TK-Luc reporter and GAL4(DBD) fusion plasmids as indicated. Whole-cell extracts were used in the luciferase assay and western blotting employing anti-GAL4(DBD) antibody. The luciferase data, expressed as RLU, are the mean ± SD of three independent transfection experiments. (B) Immunostaining showing that both GAL-TBL-1 and GAL-TBL1-2 were nuclear proteins. (C) Repression assay in Xenopus oocytes. Groups of Xenopus oocytes were injected with mRNAs encoding GAL4(DBD) fusion proteins as indicated at two different concentrations (+, 100 ng/µl, 18.4 nl/oocyte; and ++, 300 ng/µl, 18.4 nl/oocyte) and a 4xUAS-AdML reporter (25 ng/µl, 18.4 nl/oocyte). The injected oocytes were incubated overnight, and the levels of transcription were then analyzed by primer extension assay. Ctrl, the primer extension product from the endogenous storage histone H4 mRNA; Expt, the primer extension product from the injected reporter. The expression of fusion proteins was detected by western analysis using a GAL4(DBD)-specific antibody.
Fig. 7. The roles of HADC3 and TBL1/TBLR1 in SMRT–N-CoR function. (A) ‘Knock-down’ components of SMRT–N-CoR complexes by siRNAs. HeLa cells were cultured with hormone-free medium and transfected with an increasing amount of siRNA (7.5, 15 and 30 nM) and, 2 days after transfection, cells were harvested and the levels of the corresponding target proteins were analyzed by western blotting. The same blots were reprobed with an SRC-3-specific antibody (negative control). (B) Specificity of siRNAs. Western analyses showed that each siRNA used was highly specific for its target and had little, if any, cross-effect or no specific effect. (C) HDAC3 is essential and TBL1 and TBLR1 are functionally redundant in mediating repression by un liganded TR. HeLa cells were first transfected with siRNA as in (A), and 2 days after were transfected again with 1.0 µg of pSG5-TRβ and 0.2 µg of 28TRE/TK-Luc reporter. Cells were harvested after another 2 days. Whole-cell extracts were used in the luciferase assay. The luciferase data, expressed as RLU, are the mean ± SD of three independent transfection experiments. (D) Treatment with siN-CoR and siSMRT had no effect on transcriptional activation in the presence of T3. The experiment was as in (C) for siN-CoR and siSMRT except that two doses of siRNA (15 and 30 nM) were used and T3 was added 12 h before harvesting cells for luciferase assay.
Fig. 8. HDAC3 and TBL1/TBLR1 are also essential for repression of an endogenous TR target gene, deiodinase. (A) RT–PCR analysis confirmed that the deiodinase gene was regulated by TRα in HeLa α2 cells. (B) HeLa α2 cells were transfected with 15 or 30 nM of siRNA as indicated. Two days after transfection, total RNA was prepared from each sample and used for quantitative RT–PCR to measure the expression of the TR target gene deiodinase. As controls, the levels of actin and HDAC1 mRNA were also measured by quantitative RT–PCR using the same batch of RNA samples.
References
-
- Baniahmad A., Steiner,C., Kohne,A.C. and Renkawitz,R. (1990) Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site. Cell, 61, 505–514. - PubMed
-
- Chen J.D. and Evans,R.M. (1995) A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature, 377, 454–457. - PubMed
-
- Edmondson D.G., Smith,M.M. and Roth,S.Y. (1996) Repression domain of the yeast global repressor Tup1 interacts directly with histones H3 and H4. Genes Dev., 10, 1247–1259. - PubMed
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