Amino acid sequence and tissue distribution of BCoR. (A) Deduced amino acid sequence of BCoR. The point of divergence between BCoR and BCoR-S is indicated (▾); the three tandem ankyrin repeats are underlined. (B) Schematic diagram of BCoR and BCoR-S. The ankyrin repeats (codons 1428–1527) and the region corresponding to the yeast two-hybrid-isolated cDNA clone (A3; codons 112–753) are indicated. (▾) The point of divergence between the two proteins. (C) Expression of BCoR mRNA in various human tissues. A Human RNA Master Blot (Clontech) was probed with a cDNA fragment derived from A3. Tissue identification grid, courtesy of Clontech.

Full-length BCL-6 and BCoR interact in vitro and in vivo, the POZ domain of BCL-6 is both necessary and sufficient for interaction with BCoR in vitro, and BCL-6 and BCoR colocalize in the nucleus. (A) Structure of the proteins used in B and C. (Solid oval) The POZ domain; (solid rectangle) zinc fingers. (*) The myc epitope tag. (B) Coimmunoprecipitation of BCL-6 and BCoR in vitro. Pair-wise combinations of [³⁵S]methionine-labeled nontagged BCL-6 derivatives and myc-tagged BCoR were produced by cotranslation and immunoprecipitated with the α-myc antibody. The immune complexes were recovered and analyzed by SDS-PAGE (10% for lanes 1–5; 17% for lanes 6,7). The intensity of myc–BCoR in lanes 6 and 7 appears stronger due to compression on the 17% gel of non-full-length myc-BCoR proteins. Input represents 20% of the total used. (●) The expected migration of the nontagged BCL-6 derivatives. (C) Coimmunoprecipitation of BCL-6 and BCoR in vivo. 293 cells were transfected with 3 μg of BCL-6 expression plasmid alone (lane 2) or cotransfected with 3 μg each of BCL-6 and myc-tagged BCoR expression plasmids (lanes 1,3). Cell lysates were immunoprecipitated with α-myc antibody (lanes 2,3) and the recovered proteins were detected by Western blot analysis using N-3 α-BCL-6 and α-myc antibodies. The input (lane 1) corresponds to 1/50 of the lysate used in the immunoprecipitation (lane 3). (D) Subcellular localization of BCoR and BCoR-S. Immunofluorescence was performed on HeLa cells transfected with 1.2 μg of either myc-tagged BCoR or BCoR-S expression plasmids. Multiple Z series confocal microscopy images of the given fields were compiled for each image of BCoR and BCoR-S. (E) Colocalization of BCL-6 and BCoR. Coimmunofluorescence was performed on HeLa cells cotransfected with 0.6 μg each of BCL-6 and myc-tagged BCoR, and the data collected as in D. The compiled images of BCoR and BCL-6 were merged to detect colocalization.

Self association of the POZ domain of BCL-6 is required for interaction with BCoR, and BCoR interacts selectively with the POZ domain of BCL-6. (A) Structures of the reporter construct, bait, and activator proteins used in B. The bait proteins consist of the GAL4 DBD alone, or fused to wild type or the indicated mutant POZ domains. The activator-tagged protein VB consists of the VP16 activation domain fused to the yeast two-hybrid-isolated BCoR clone (codons 112–753). (B) Interactions of BCoR (codons 112–753) with BCL-6 POZ domain point mutants in the mammalian two-hybrid assay. Luciferase and β-gal assays were conducted on cell extracts from HeLa cells transiently transfected with 500 ng of the GAL4-responsive reporter (pAH205), 50 ng of the indicated bait expression plasmids; 0, 50, or 250 ng of the VB expression plasmid; and 25 ng of the pCMVlacZ plasmid, as control for transfection efficiency. Data displayed represent the average of two experiments with the standard deviations depicted. (C) In vitro coimmunoprecipitation of BCoR and BCoR-S with POZ-containing proteins. The indicated pair-wise combinations of [³⁵S]methionine-labeled nontagged POZ proteins (BCL-6, PLZF, BAZF, ZID, TTK, and GAGA) and either myc-tagged BCoR or BCoR-S were produced by cotranslation and immunoprecipitated with α-myc antibody. The proteins were recovered and analyzed by 8% SDS-PAGE. (●) The expected migration of the nontagged BCL-6 derivatives.

BCoR is a transcriptional corepressor. (A) Structures of the reporter construct and proteins used in B. (B) Luciferase and β-gal assays were performed on cell extracts from HeLa cells transiently transfected with 50 ng of the GAL4-responsive reporter (SV40 Enh G5), along with 0, 5, 10, or 25 ng of the indicated GAL4 fusion constructs [GAL4 DBD alone (DBD) or DBD fused with BCoR or BCoR-S), and 25 ng of pCMVLacZ plasmid. Data displayed represent the average of two experiments, with the standard deviations depicted. Fold repression was determined by normalizing luciferase/β-gal values of all samples to that of the reporter alone.

BCoR can selectively potentiate BCL-6 repression. (A) Structures of the reporter constructs and proteins used in B. (B) Luciferase and β-gal assays were conducted on HeLa cell extracts transiently transfected with 250 ng of either the SV40 Enh or 5× BCL-6 reporter construct, and the indicated combinations of BCL-6 (25 ng), BAZF (25 ng), BCoR (25 and 50 ng), and BCoR-S (25 and 50 ng); pEFLacZ (200 ng) was also included as control for transfection efficiency. Data displayed represent the average of two experiments with the standard deviations depicted. Fold repression was determined by normalizing luciferase/β-gal values (C,D) of all samples to that of the SV40 Enh reporter alone.

BCoR and BCoR-S interact with class I and class II HDACs in vivo. 293 cells were transfected with 4 μg of either myc-tagged BCoR (A) or BCoR-S (B) expression plasmids alone (first lane), or cotransfected with pair-wise combinations of 3 μg of the indicated Flag-tagged HDACs and 3 μg of either myc-tagged BCoR (A) or BCoR-S (B) expression plasmids (remaining lanes). Cell lysates were imunoprecipitated with α-Flag antibody, and the recovered proteins were detected by Western blot analysis using α-myc and α-Flag antibodies (shown in B only). The input corresponds to 1/100 of the lysates used for coimmunoprecipitation.

Interactions between the BCL-6 POZ domain and N-CoR, SMRT, and BCoR are mutually exclusive. (A) Structures of the reporter construct and proteins used in B. (B) Luciferase and β-gal assays were performed on HeLa cell extracts transiently transfected with 250 ng of the GAL4-responsive reporter (pAH205), 2.5 ng of the POZ bait, and the indicated combinations of VP16-tagged (VS, 25 ng; VN, 25 ng; VA, 5 ng) and nontagged (S, N, A, at 100 ng each) corepressor expression plasmids, along with 25 ng of the pCMVlacZ plasmid. Data displayed represent the average of two experiments with the standard deviations depicted.