The BRCT domain binds to Rb-binding proteins in vitro. (A) Schematic representation of BRCA1 gene. The region used as a probe in the library screen is indicated. (B) GST pull-down assays demonstrating that BRCT domain interacts with partial polypeptide of RbAp46 and full-length RbAp46 and RbAp48 proteins. In vitro-translated, ³⁵S-labeled BRCT (10 μl) was incubated with 20 μl of glutathione-Sepharose beads and an equal amount of GST or GST-fusion proteins, as indicated. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE, and visualized by using autoradiography. A portion of the in vitro-translated, ³⁵S-labeled BRCT, corresponding to ≈20% of the labeled protein in the binding reaction, was loaded as “Input”. (C) Schematic representation of the BRCT repeats. Mutations analyzed for in vitro binding are indicated. (D) Mutations in BRCT domain interfere with interaction between RbAp46 and BRCT. GST pull-down assays were performed as in A with the wild type or mutation-containing BRCT polypeptides as indicated. Approximately 5% of each in vitro-translated protein in the binding reactions were loaded as Input.

In vivo association of BRCA1 with Rb-binding proteins. (A) Coimmunoprecipitation of BRCA1 and RbAp46/48. Endogenous BRCA1 protein was immunoprecipitated by anti-BRCA1 mAb SG11 (lane 2), anti-RbAp46/48 mAbs: RbBp (lane 3); RbAp48–3-255 (lane 4) and controls: normal mouse IgG (NMIgG, lane 1) and anti-c-jun mAb KM-1 (lane 5) from 1.5 mg of HeLa cell extract. Total cell lysate (100 μg) was run in lane 6. Protein extract (1.5 mg) from the BRCA1-null cell line, HCC1937 (42), were immunoprecipitated with anti-BRCA1 mAb SG11 (lane 7). Samples were separated on SDS/6% PAGE and immunoblotted with the mAb anti-BRCA1 antibody (Ab-1, Oncogene Science). (B) Colocalization of BRCA1 and RbAp46/48 in HeLa cells. Cells were prepared as described in Materials and Methods, stained with a mouse mAb against RbAp48/46, 3–225, (green in a), a rabbit polyclonal Ab against BRCA1, I-20, (red in b), and visualized by using microscope and charge-coupled device camera. The regions of overlap between red and green signals appear as yellow (c), indicating colocalization of BRCA1 and RbAps. The nucleus of each cell are shown by 4′,6-diamidino-2-phenylindole (DAPI) staining (d). White arrowheads indicate some regions of overlap of BRCA1 and RbAps staining. (C) Colocalization of BRCA1 and RbAp46/48 in Saos2 cells. Cells were prepared as described in Materials and Methods and stained as in B.

Rb interacts with the BRCT domain. (A) GST pull-down experiments show that Rb interacts with BRCT domain in the presence or absence of partial RbAp46 polypeptide. In vitro-translated, ³⁵S-labeled BRCT (12.5 μl) and partial RbAp46 polypeptides were incubated individually or in combination with 20 μl of glutathione-Sepharose beads with equal amount of GST-Rb or GST-Rb pocket mutation fusion proteins as indicated. GST-coated beads were incubated with each polypeptide separately. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE and visualized by using autoradiography. Approximately 20% of labeled protein in the binding reaction were loaded as Input. (B) Colocalization of BRCA1 and Rb. HeLa cells were prepared as described in Material and Methods, stained with a mouse mAb against Rb, IF8, (green in a), a rabbit polyclonal antibody against BRCA1, I-20, (red in b). The regions of overlap between red and green signals appear as yellow (c), indicating colocalization of BRCA1 and Rb. The nucleus of each cells are shown by DAPI staining (d).

The BRCT domain associates with HDAC1 and HDAC2. Western blot analysis of GST pull-down assays using antibodies against HDAC1 and HDAC2. HeLa whole-cell lysates (300 μg) were incubated with 20 μl of glutathione-Sepharose beads covered with equal amounts of GST, GST-BRCT (amino acids 1,536–1,863), or GST-NH₂-BRCA1 (amino acids 1–304) fusion proteins. Bound proteins were resolved on SDS/10% PAGE, transferred to nitrocellulose membranes, and blotted with HDAC1 or HDAC2 Abs.