Characterization of CoREST. (a) Predicted amino acid sequence of human CoREST (hCoREST) (accession no. AF155595). The underlined amino acids represent the protein fragment isolated in the yeast two-hybrid screen. The two SANT domains (15) are shaded. (b) Alignment of the two repeated SANT domains in CoREST and NCoR. For comparison, the SANT consensus and predicted structure are shown below. h, hydrophobic residues; + and −, positively and negatively charged residues. Amino acids conforming to the SANT consensus are in boldface. Additional homologies between CoREST and NCoR are shaded. (c) Alignment of hCoREST (h) with a similar Drosophila protein (d) (accession no. AA392295). Conserved amino acids are shaded. Boldface type indicates the SANT domain. (d) Western blot of nuclear extracts from HEK293 cells transfected with pcDNA or with a myc-tagged CoREST cDNA. The probes were a polyclonal CoREST antibody (αCoREST) and a monoclonal myc antibody (αmyc). The positions of migration of endogenous CoREST (66 kDa) and myc-CoREST (70 kDa) are indicated.

CoREST interacts with the C-terminal repressor domain of REST. (a) The family of REST cDNAs used in in vitro and in vivo experiments with CoREST. The larger solid rectangle depicts the eight zinc finger motifs in the DBD; the smaller rectangle depicts the single zinc finger in the C terminus. REST (amino acids 1–1097) represents full-length protein. (b) Yeast two-hybrid interactions between pGADCoREST (amino acids 109–293) and four different REST-LexA fusion proteins. (c) REST/CoREST interactions in GST pull-down assay. Indicated REST constructs were transcribed and translated in vitro to yield ³⁵S-labeled products and were incubated with immobilized GST or GSTCoREST (amino acids 109–293). Input (3% of total protein) and proteins bound to GST were analyzed by SDS/PAGE.

(a) CoREST is expressed in neuronal and nonneuronal cells. Western blot of nuclear extracts (50 μg) prepared from neuronal (PC12, GH3) and nonneuronal (HeLa, COS-1, L6) cell lines. The blot was probed with αCoREST antibody. Endogenous CoREST protein migrates at 66 kDa. (b) Demonstration of REST/CoREST interaction in vivo by coimmunoprecipitation analysis. Whole-cell lysates prepared from L6 skeletal muscle cells were immunoprecipitated with the indicated antibodies and then subjected to Western blotting. The membrane was probed with αREST antibody (1). The arrow depicts migration of REST protein. Input corresponds to 50 μg of extract. αCoREST and αGal4DBD refer to antibodies against CoREST and against the DBD of Gal4 protein, respectively.

CoREST exhibits repressor activity when fused to a Gal4- DBD and mediates repressor activity of REST. (a) Gal4-DBD (Gal4; amino acids 1–147) or Gal4CoREST expression vectors were transfected into HEK293 and PC12 cells, with a UAS type II CAT reporter gene (molar ratio of 1:1). (b Upper) Gal4REST lacking the N-terminal repressor domain (Gal4RESTΔN) was transfected into HEK293 cells along with a UAS type II CAT reporter gene. The role of CoREST was tested by addition of a cDNA coding for a REST competitor peptide containing the CoREST-binding site (Gal4RESTΔN + C3). Specificity of derepression was tested by over-expressing CoREST (Gal4RESTΔN + C3 + CoREST). (Lower) REST lacking the N-terminal repressor domain (RESTΔN) was transfected into PC12 cells along with an RE1 type II reporter gene. The role of CoREST was tested by addition of a cDNA coding for a REST competitor peptide containing the CoREST-binding site (RESTΔN + C3). Specificity of derepression was tested by overexpressing CoREST (RESTΔN + C3 + CoREST). CAT activity was monitored by TLC and autoradiography. Statistics were performed by using the nonparametric Mann–Whitney U test. ∗, P < 0.056 for RESTΔN + C3 +CoREST compared with RESTΔN + C3 and Gal4RESTΔN + C3 + CoREST compared with Gal4RESTΔN + C3; ∗∗, P < 0.0028 for RESTΔN + C3 compared with RESTΔN; ∗∗∗, P < 0.0008 for Gal4RESTΔN + C3 compared with Gal4RESTΔN. (c) Western blot of nuclear extracts of HEK293 cells transfected as in b. The probe was a monoclonal αGal4 antibody. (d) Western blot from nuclear (N) and cytoplasmic (C) extracts of HEK293 cells transfected with an HA-tagged C3. The probe was a monoclonal αHA antibody.