Fig. 1. SMRT and N-CoR proteins exist in large protein complexes with estimated sizes of 1.5–2 MDa. HeLa nuclear extract was fractionated with a Superose 6 gel filtration column. The indicated fractions were analyzed by western blotting using the antibodies indicated. The αN-CoR(C) antibody detected both SMRT and N-CoR proteins. The arrows at the bottom show the elution positions of calibration proteins of known molecular weights.

Fig. 2. Purification of a SMRT protein complex. (A) A diagram of the purification scheme for the SMRT complex. PC, phosphocellulose p11 resins; DEAE, DEAE–Sepharose Fast Flow resins. (B) Differential fractionation of SMRT and N-CoR proteins by a PC column. Note that αSMRT and αN-CoR(N) antibodies are specific for SMRT and N-CoR, respectively, whereas αN-CoR(C) antibody recognizes both SMRT and N-CoR. (C) A Coomassie blue-stained SDS–polyacrylamide gel showing the purified SMRT complex. The identity of the indicated subunits was determined by mass spectrometry and confirmed by western analyses as shown in (D). The Hsc70 protein was a contaminant because it also bound to the control IgG beads; 10% of the input (0.2 M DEAE fraction) was used in lane 1 in (D).

Fig. 3. Confirmation of the association of TBL1 and HDAC3 with SMRT complex. (A) HDAC3 and TBL1 co-fractionated with SMRT in gel filtration chromatography. HeLa nuclear extract was fractionated by using a Superose 6 gel filtration column as in Figure 1 and the indicated fractions were analyzed by western blotting using the antibodies indicated. (B) SMRT protein in HeLa nuclear extracts was co-immunoprecipitated by antibodies specific for HDAC3 and TBL1, respectively; 5% input HeLa nuclear extract was used in lanes 1 and 3.

Fig. 4. Both TBL1 and HDAC3 are also associated with the N-CoR protein complex(es). (A) N-CoR complex(es) was affinity-purified by both αN-CoR(N) and αN-CoR(C) antibody affinity columns and the associated proteins were analyzed by western blotting; 5% input HeLa nuclear extract was used in lanes 1 and 3. (B) The N-CoR protein in HeLa nuclear extracts was also co-immunoprecipitated by antibodies specific for HDAC3 and TBL1, respectively; 5% input HeLa nuclear extract was used in lanes 1 and 3.

Fig. 5. The majority of the N-CoR and SMRT complexes are likely to contain HDAC3. (A) HeLa nuclear extracts were incubated with an increasing amount of HDAC3 antibody in an attempt to deplete HDAC3 from HeLa nuclear extracts. The levels of SMRT and N-CoR proteins in the supernatants or IP fractions were analyzed by western blotting using the αN-CoR(C) antibody, which detected both N-CoR and SMRT. The levels of HDAC3 in the supernatants are also shown, whereas the HDAC3 in IP fractions could not be determined by western analysis due to the overlapping of the signal from antibody heavy chain with that of HDAC3. (B) The immunopurified N-CoR complexes exhibited histone deacetylase activity. The N-CoR complex(es) or a mixture of SMRT and N-CoR complexes were pull-downed from HeLa nuclear extracts by using αN-CoR (N) and αN-CoR(C) affinity columns. The rabbit anti-rat IgG (lane 2) was used as negative control for IP, whereas αHDAC3 antibody served as a positive control. The purified calf thymus core histones were labeled with [³H]acetyl-CoA in vitro by using the purified recombinant p300 HAT domain and then used for measuring HDAC assay. In lane 6, TSA was added to a final concentration of 0.5 µM in the deacetylation assay.

Fig. 6. HDAC3 may interact directly with N-CoR protein. (A) The schematic diagram illustrating the known functional domains of N-CoR protein and the five different N-CoR constructs. (B) In vitro translated N-CoR co-immunoprecipitated with the in vitro translated HDCA3. (C) The region between amino acids 1496 and 1965 of N-CoR interacted with MBP–HDAC3 in the in vitro pull-down assay. The indicated N-CoR fragments were translated and tested for binding to MBP–HDAC3; 5% input. (D) Yeast two-hybrid assay of the interactions between HDAC3 and different regions of the N-CoR protein. Yeast were transformed with the expression constructs for Gal4-HDAC3 and each of the N-CoR fragments fused with Gal4AD, and cell lysates were measured for activity of a β-galactosidase reporter.

Fig. 7. The HDAC3-containing SMRT and N-CoR complexes interact with unliganded TR. HeLa nuclear extracts were incubated with the immobilized GST control or GST–TR fusion protein. The super natants and bound fractions were then fractionated by SDS–PAGE and analyzed by western blotting using the antibodies indicated. The HeLa NE (nuclear extract) in lane 1 and FT (supernatant) in lanes 2 and 4 were equivalent to 20% of the HeLa nuclear extracts used for pull-down.

Fig. 8. The HDAC3-containing SMRT and N-CoR complexes are conserved during evolution and involved in the repression exerted by unliganded TR/RXR. (A) Gel filtration analysis of Xenopus oocyte extracts indicated that Xenopus HDAC3 also exists in large protein complexes with an estimated size of 1.5–2 MDa. (B) The association of Xenopus HDAC3 with Xenopus homologs of SMRT and N-CoR as revealed by co-immunoprecipitation experiments. Xenopus oocyte extracts were immunoprecipitated with a control rabbit anti-mouse IgG antibody (IgG), αSMRT, αN-CoR(N) and αHDAC3 antibodies as indicated, and the IP fractions were analyzed by western blotting. (C) Partial relief of the repression by unliganded TR/RXR through microinjection of antibodies against HDAC3 and SMRT/N-CoR. The reporter (pTRβA) was microinjected into Xenopus oocytes and the specific transcripts from this reporter were measured by primer extension analysis (Expt). The repression by unliganded TR/RXR was established by expression of TR and RXR in Xenopus oocytes by microinjection of their corresponding mRNAs. All antibodies used here were affinity-purified and injected at a concentration of 60 ng/µl (18.4 nl/oocyte). The control (Ctrl) was the primer extension product from the endogenous histone H4 mRNA using an H4 primer and served as a loading control.