Volume 6, Issue 4, Pages 757-768 (October 2000) Sequence-Specific Transcriptional Corepressor Function for BRCA1 through a Novel Zinc Finger Protein, ZBRK1  Lei Zheng, Hongyi Pan, Shang Li, Andrea Flesken-Nikitin, Phang-Lang Chen, Thomas G Boyer, Wen-Hwa Lee  Molecular Cell  Volume 6, Issue 4, Pages 757-768 (October 2000) DOI: 10.1016/S1097-2765(00)00075-7

Figure 1 ZBRK1 cDNA Encodes a Novel Protein with a KRAB Domain and a Zinc Finger Domain (A) The ZBRK1 protein sequence. The KRAB domain is underlined, and the Cys and His residues of the C2H2-type zinc fingers are shadowed. (B) Amino acid sequence alignment of the ZBRK1 KRAB domain with KRAB domains of other zinc finger proteins (Friedman et al. 1996). Conserved residues are indicated by bold letters. Molecular Cell 2000 6, 757-768DOI: (10.1016/S1097-2765(00)00075-7)

Figure 2 Specific Interaction between ZBRK1 and BRCA1 (A) Schematic diagram of the full-length BRCA1 protein and different BRCA1 polypeptide fragments fused to GST or the Gal4 DNA-binding domain. (B) Purified GST-BRCA1 fusion proteins were visualized by SDS–PAGE and Coomassie blue staining. Comparable amounts of each fusion protein were used for binding reactions shown in (C). (C) Specific binding of 35S-methionine-labeled in vitro–translated ZBRK1 to GST-BRCA1Bgl (lane 5) but not to other BRCA1 polypeptide fragments, as detected by SDS–PAGE and subsequent autoradiography. Lane 1 represents the total input of in vitro–translated ZBRK1 used in each binding reaction. (D) The indicated fragments of BRCA1, expressed as Gal4 DNA-binding domain fusion proteins, were tested for interaction with ZBRK1 fused to the Gal4 transactivation domain in yeast two-hybrid assays. β-galactosidase activities were quantified as described (Durfee et al. 1993). (E) Schematic diagram of the full-length ZBRK1 and different polypeptide fragments of ZBRK1 fused to GST. (F) Comparable amounts of each fusion protein were used for binding reactions shown in (G). (G) Specific binding of in vitro–translated BRCA1 Bgl fragment to the full-length ZBRK1 (lane 4) and the ZBRK1C1 region (lane 7). Lane 1 represents the total input of in vitro–translated BRCA1 Bgl fragment used in each binding reaction. Molecular Cell 2000 6, 757-768DOI: (10.1016/S1097-2765(00)00075-7)

Figure 3 Detection of Cellular ZBRK1 Protein and In Vivo Interaction of ZBRK1 and BRCA1 (A) Detection of cellular ZBRK1 protein. Lysate from 35S-methionine-labeled T24 cells (1.6 × 107) was subjected to immunoprecipitation once by preimmune sera (lane 2) or twice by anti-ZBRK1 antibodies (lane 3). In vitro translated ZBRK1 is indicated in lane 1. (B) In vivo interaction between ZBRK1 and BRCA1. T24 cell (1.6 × 107) lysate was immunoprecipitated by rabbit IgG (lane 1), anti-ZBRK1 antibodies (lane 2), or anti-BRCA1 mAb 6B4 (lane 3). Immunoprecipitated proteins were eluted by boiling in SDS sample buffer and separated by SDS-7.5% PAGE followed by immunoblot analysis with 6B4 to detect BRCA1 (upper panel) or with anti-ZBRK1 antibodies to detect ZBRK1 (lower panel). (C) Specific coimmunoprecipitation of ZBRK1 and BRCA1. Myc-GFP-ZBRK1 was cotransfected with either HA-tagged wild-type BRCA1 (lanes 1, 4, and 7), or BRCA1 mutants, A1708E (lanes 2, 5, and 8), or Q356R (lanes 3, 6, and 9). Lysates were immunoprecipitated with anti-GST mAb 8G11 (lanes 1–3) or anti-HA (Santa Cruz) polyclonal antibodies (lanes 4–6). The immune complexes were separated by SDS-7.5% PAGE followed by immunoblot analysis with anti-BRCA1 mAb 6B4 to detect BRCA1 (a), anti-CtIP mAb to detect CtIP (b), and anti-GFP mAb (Clontech) to detect GFP-tagged ZBRK1 (c). Equivalent amount of CtIP or GFP-tagged ZBRK1 was present in the lysates (lanes 7–9) as determined by immunoblot analysis with anti-CtIP mAb (d) and anti-GFP mAb (e). (D) Nuclear distribution patterns of ZBRK1 and BRCA1. Three representative U2OS cells stably transfected with GFP-ZBRK1 shown in I, II, and III, respectively, were stained with DAPI (row a) and anti-BRCA1 mAb Ab-1 (Oncogene Research Products) followed by Texas red–conjugated secondary antibody (row c), as described elsewhere (Zhong et al. 1999). Row b shows the images of GFP signals, and row d shows the merged images of GFP signals and BRCA1 staining. Molecular Cell 2000 6, 757-768DOI: (10.1016/S1097-2765(00)00075-7)

Figure 4 ZBRK1 Binds to a Specific DNA Sequence (A) Selection and amplification of a ZBRK1-binding site. Equivalent amounts of PCR-amplified DNA derived from each of the second, fourth, and sixth rounds of the SAAB assay were subjected to EMSA using 50, 100, or 200 ng of recombinant GST-ZBRK1Zn protein . (B) Alignment of individual DNA sequences selected by SAAB assay. The deduced consensus ZBRK1 DNA-binding sequence is indicated below the individually aligned sequences. (C) Competition EMSA assays. EMSA was performed with Wt. probe, corresponding to the consensus ZBRK1 binding sequence, and either no added protein (lane 1), GST alone (lane 2), or GST-ZBRK1Zn (lanes 3–6 and lanes 8-10). Mut probe, corresponding to a double-stranded oligonucleotide identical in length but differing in sequence from the Wt. probe, was incubated with GST-ZBRK1Zn (lane 7). A 100-, 200-, or 400-fold molar excess of unlabeled Wt. probe (lanes 4–6) or Mut. Probe (lanes 8–10) was added to binding reactions as indicated. (D) Nuclear complex formation on Wt. probe. EMSA was performed with the indicated amounts of T24 cell nuclear extract and radioactively labeled Wt. probe (lanes 1–5 and 11–16) or Mut Probe (lanes 6–10). A molar excess of unlabeled Wt. probe (lanes 11–13) or Mut probe (lanes 14–16) was added to binding reactions as indicated. DNA–protein complexes I and II are indicated. Molecular Cell 2000 6, 757-768DOI: (10.1016/S1097-2765(00)00075-7)

Figure 5 The ZBRK1 Recognition Sequence in GADD45 Intron 3 Binds to a Nuclear Complex Containing Both ZBRK1 and BRCA1 (A) Schematic diagram of the GADD45 intron 3 gene region. The p53 responsive element is indicated by a small black box, and the putative ZBRK1 recognition sequence is indicated by a large black box. The consensus ZBRK1 recognition sequence is aligned with GADD45 intron 3 sequences. (B) GST-ZBRK1Zn binds to the GADD45 intron 3–derived ZBRK1 recognition sequence. EMSA performed with 50 ng GST-ZBRK1Zn (lanes 1–3) or GST (lane 4) and radioactively labeled Wt (lane 1), WIN3 (lanes 2 and 4–8), or MP1 (lane 3) probes. Binding reactions included either no specific competitor DNA (lanes 1–4) or the indicated amounts of unlabeled Wt (lanes 5 and 6), or Mut (lanes 7 and 8) probes. (C) Nuclear complex formation on the GADD45 intron 3–derived ZBRK1 recognition sequence. EMSA was performed with the indicated amounts (μg) of T24 nuclear extract and radioactively labeled Wt (lane 1), WIN3 (lanes 2–4), or MP1 (lanes 5–7) probes. (D) Competitive EMSA assays. EMSA was performed with 5 μg T24 nuclear extract and radioactively labeled WIN3 probe. Binding reactions included either no specific competitor DNA (lane 1) or the indicated amounts of unlabeled Wt (lanes 2–4), WIN3 (lanes 5–7), or MP1 (lanes 8–10) probes. (E) WIN3 probe binds a nuclear complex containing ZBRK1 and BRCA1. Fifty micrograms of nuclear extract in each of lanes 1–18 was incubated with no probe (lanes 1, 7, and 13), the Wt. probe (lanes 2, 8, and 14), indicated amounts of the WIN3 probe (lanes 3–5, 9–11, and 15–17), or the MP1 probe (lanes 6, 12, and 18). Binding reactions were resolved by electrophoresis on a native polyacrylamide gel, and DNA–protein complexes were subsequently analyzed by Western transfer and immunoblotting with anti-ZBRK1 antibodies (lanes 7–12). The blot was stripped of antibodies and sequentially immunoblotted with mouse preimmune serum (lanes 1–6) and anti-BRCA1 mAb 6B4 (lanes 13–18). Control binding reactions using 2 or 50 μg of nuclear extract and radioactively labeled WIN3 probe were run on the same gel to mark the electrophoretic positions of DNA–protein complexes I and II (lanes 19 and 20). (F) Coimmunoprecipitation of the WIN3 probe with ZBRK1 and BRCA1. Equivalent amounts of nuclear extract were incubated with radioactively labeled WIN3 (lanes 1–4) or MP1 (lanes 5–8) probes, followed by immunoprecipitation with anti-GST mAb 8G11 (lanes 2 and 6), anti-ZBRK1 antibodies (lanes 3 and 7), or anti-BRCA1 mAb 6B4 (lanes 4 and 8). Lanes 1 and 5 indicate 2% of the total amount of each probe used in binding reactions. Molecular Cell 2000 6, 757-768DOI: (10.1016/S1097-2765(00)00075-7)

Figure 6 ZBRK1 Is a Transcriptional Repressor through GADD45 Intron 3 (A) pBLcat or pBLcat-E (containing four copies of the ZBRK1 recognition sequence) was transfected into Saos2 cells either with (+) or without (−) pCHPL-ZBRK1. Resultant CAT activities are expressed relative to the level of CAT activity observed with pBLcat vector–transfected cells. (B–E) U2OS cells were transfected as indicated with 0.5 μg of pGL3p-IN3AB (B and E), pGL3p-IN3A (C and E), pGL3p-IN3ABM (D and E), or pGL3p (E) along with the indicated amounts (micrograms of DNA) of pCEPF-ZBRK1. (F) U2OS cells were transfected with pGL3p-IN3AB (0.5 μg) along with the indicated amounts (micrograms of DNA) of either pCNF-ZBRK1, pCNF-ZBRK1ΔK, or pCNF-ZBRKΔC. In (A)–(F), relative CAT or luciferase activities are calculated as the fold increases in CAT or luciferase activities relative to those observed in cells transfected by reporter and CMV vector alone (first lane in each panel). CAT or luciferase activities were normalized to β-galactosidase activity obtained by cotransfection of 1 μg of SV40-β-gal vector as described previously (Li et al. 1999). (G) Expression of Flag-tagged wild-type ZBRK1 and ZBRK1 mutant derivatives in U2OS cells transiently transfected with pCNF, pCNF-ZBRK1, pCNF-ZBRK1ΔC, and pCNF-ZBRK1ΔK. Flag-tagged proteins were detected by immunoblot analysis using anti-Flag mAb M2 (Sigma). (H) Semiquantitative RT–PCR analysis of endogenous GADD45 (upper panel) or GADPH (lower panel) mRNAs in U2OS cells stable-expressing GFP (lanes 1 and 2) or GFP-ZBRK1 (lanes 3 and 4). RT–PCR reactions were run in duplicate. (I) Expression of endogenous GADD45 protein in U2OS cells stable-expressing GFP (lane 1) or GFP-ZBRK1 (lane 2), or transiently transfected with either pCNF, pCNF-ZBRK1, pCNF-ZBRK1ΔC, or pCNF-ZBRK1ΔK (lanes 3–6, respectively). GADD45 (lower panel) or nuclear matrix protein p84 (upper panel, included as an internal loading control [Li et al. 1999]) were detected by immunoblot analysis using rabbit anti-GADD45 antibody H-165 (Santa Crutz), and anti-p84 mAb 5E10, respectively. Molecular Cell 2000 6, 757-768DOI: (10.1016/S1097-2765(00)00075-7)

Figure 7 BRCA1 Functions as a ZBRK1 Corepressor through GADD45 Intron 3 (A) Brca1-deficient (Brca1−/−; p53−/−) MEFs were transfected with 2.5 μg of pGL3p-IN3AB or pGL3p-IN3ABM along with the indicated amounts (micrograms of DNA) of pCNF-ZBRK1. (B) Brca1-proficient (p53−/−) MEFs were transfected with 2.5 μg of pGL3p-IN3AB along with the indicated amounts (micrograms of DNA) of pCNF-ZBRK1. (C–E) Brca1-deficient (Brca1−/−; p53−/−) MEFs were transfected as indicated with 2.5 μg of pGL3p-IN3AB (C and D) or pGL3p-IN3ABM (E) along with the indicated amounts (micrograms of DNA) of pCNF-ZBRK, pCNF-ZBRK1ΔC, pcDNA3.1-BRCA1, pcDNA3.1-BRCA1A1708E, pcDNA3.1-BRCA1Q356R, or pcDNA3.1-BRCA1C64G. (F) U2OS cells were transfected with pGL3p-IN3AB (0.5 μg) along with the indicated amounts (micrograms of DNA) of pCNF-ZBRK, pcDNA3.1-BRCA1, pcDNA3.1-BRCA1A1708E, pcDNA3.1-HA-Bgl expressing the ZBRK1-binding region in BRCA1, or pVP16 expressing the VP16 transactivation domain (Li et al. 1999). (G) p53−/− MEFs were transfected with 2.5 μg of pGL3p-IN3AB along with the indicated amounts (micrograms of DNA) of pcDNA3.1-BRCA1 and pCNF-ZBRK1. In (A)–(G), relative luciferase activities are calculated as described in the previous figure. Molecular Cell 2000 6, 757-768DOI: (10.1016/S1097-2765(00)00075-7)