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The Partial Homeodomain of the Transcription Factor Pax-5 (BSAP) Is an Interaction Motif for the Retinoblastoma and TATA-Binding Proteins 1

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The Partial Homeodomain of the Transcription Factor Pax-5 (BSAP) Is an Interaction Motif for the Retinoblastoma and TATA-Binding Proteins 1 [CANCER RESEARCH (SUPPL,) 59, 1716s 1725s, April 1, 1999] The Partial Homeodomain of the Transcription Factor Pax-5 (BSAP) Is an Interaction Motif for the Retinoblastoma and TATA-binding Proteins 1 Dirk Eberhard and Meinrad Busslinger 2 Research Institute of Molecular Pathology, A-t030 Vienna, Austria Abstract expression pattern, targeted inactivation of Pax-5 in the mouse germ- line revealed essential functions of this transcription factor in mid- Pax-5 codes for the transcription factor BSAP, which plays an impor- brain and B-cell development (7, 8). Interestingly, the human PAX-5 tant role in midbrain patterning, B cell development, and lymphoma gene is involved together with the immunoglobulin heavy chain locus formation. Pax-5 is known to control gene expression by recognizing its in a recurring t(9;14)(p13;q32) translocation associated with a subset target genes via the NH2-terminal paired domain and by regulating transcription through a COOH-terminal regulatory module consisting of of non-Hodgkin's lymphomas (9-11). Hence, PAX-5 can be activated activating and inhibitory sequences. The central region of Pax-5 contains by gain-of-function mutations to participate as an oncogene in tumor- a sequence with significant homology to the first a-helix of the paired-type igenesis. The recent genetic identification of Pax-5 target genes re- homeodomain. This partial homeodomain has been highly conserved vealed that Pax-5 controls their transcription either as an activator or throughout vertebrate evolution because it is found not only in Pax-5 but repressor depending on the specific regulatory sequence context (6, also in the related Pax-2 and Pax-8 members of the same Pax subfamily. 12). Structure-function analysis, furthermore, demonstrated that Pax-5 Here we report that the partial homeodomain binds the TATA-binding recognizes its target genes via the NH2-terminal paired domain and protein (TBP) and retinoblastoma (Rb) gene product. Both TBP and Rb controls u'anscription through a COOH-terminal regulatory module were shown by coimmunoprecipitation experiments to directly associate consisting of activating and inhibitory sequences (13). In addition, the with Pax-5 in vivo. The conserved core domain of TBP and the pocket central sequences containing the partial homeodomain were also region as well as COOH-terminal sequences of Rb are required for interaction with the partial homeodomain of Pax-5 in in vitro binding shown to contribute to the transcriptional activity of Pax-5 (13). assays. Furthermore, Pax-5 was specifically bound only by the underphos- Here we demonstrate by different protein binding assays that the phorylated form of Rb. These data indicate that Pax-5 is able to contact TBP and the Rb protein directly interact with the transcription factor the basal transcription machinery through the TBP-containing initiation Pax-5 in vivo and in vitro. Deletion analysis, furthermore, identified factor TFIID, and that its activity can be controlled by the cell cycle- the partial homeodomain of Pax-5 as an essential recognition motif for regulated association with Rb. both TBP and Rb. These data suggest therefore that the partial homeodomain controls the activity of Pax-5 by linking it either Introduction through TBP to the basal transcription machinery or through Rb to the The Pax gene family codes for transcription factors that play control of cell proliferation. important roles in embryonic development, cell differentiation, and Materials and Methods human disease. A hallmark of these developmental regulators is their conserved DNA-binding motif, the so-called paired domain. The Expression Constructs. The expression plasmids coding for lull-length mammalian genome contains nine Pax genes that can be grouped into Pax-5 (pKW2T-hBSAP) and Pax-5 (1-268) have been described previously four distinct classes based on their similarity in sequence and expres- (13). Pax5-APD was constructed by subcloning a HindlIi-BamHI fragment sion (reviewed in Ref. 1). Two of these subclasses contain, in addition from pKW-ABSAP-ER (12) into pKW2T (t3). The plasmid pKW2T-Pax5- AHD contains a 25-amino-acid deletion (amino acids 229-253) that was to the paired domain, also a homeodomain as a second DNA-binding generated in pKW2T-hBSAP via PCR-mediated mutagenesis by introducing region. A sequence motif, which is homologous only to the first an XhoI site at the deletion site without affecting amino acids 228 and 254. The a-helix of the homeodomain (2), has been identified in the subfamily FLAG epitope was added at the NH2 terminus of Pax-5 by replacing a 260-bp consisting of Pax-2, Pax-5, and Pax-8 (3). This partial homeodomain HindlII-BamHI fragment of pKW2T-hBSAP with a corresponding PCR prod- has no DNA-binding activity and yet is conserved in members of the uct generated with the oligonucleotide 5'-CCCAAGCTTACCATGGATTA- Pax-2/5/8 family from sea urchin to man (4, 5), which suggests that it CAAGGACGACGATGACAAGTTAGAGAAAAATTA-3' and a corre- constitutes a protein interaction motif. sponding downstream Pax-5 primer. The HindlII-EcoRI insert containing the The Pax-5 gene codes for the transcription factor BSAP, 3 which is full-length Pax-5 cDNA was subsequently recloned in the expression plasmid expressed in the developing midbrain, all of the lymphoid tissues, and pRK7 (3) to generate pRK7-FLAGhBSAP, which was used for transient adult testis of the mouse (reviewed in Ref. 6). Consistent with this transfection in COS-7 cells. The HindlII site in the above oligonucleotide was converted into a BamHI site, and the frill-length FLAG-tagged Pax-5 cDNA was assembled in the prokaryotic expression plasmid pET2a (14) by ligating a Received 9/18/98; accepted 2/1/99. 260-bp BamHI PCR fragment (5' end) together with a 900-bp BamHI-EcoRI 1 Contributed as part of the April 1, 1999 Supplement to Cancer Research, "General Motors Cancer Research Foundation Twentieth Annual Scientific Conference: Develop- cDNA fragment (3' end). The COOH-terminal Pax-5 deletion mutants con- mental Biology and Cancer." This work was supported in part by a Grant from the taining the FLAG epitope were generated by using the corresponding BamHI- Austrian industrial Research Promotion Fund. D. E. was the recipient of a fellowship from EcoRI cDNA fragments of the previously described deletion clones (13) in the the Deutsche Forschungsgemeinschaftand European Community. above ligation reaction. The expression plasmid pET-FLAGhPax5-AHD was 2 To whom requests for reprints should be addressed, at Research Institute of Molec- ular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. Phone: 43-I-797-30-884; Fax: similarly generated by cloning the corresponding BamHI-EcoRI fragment from 43-1-798-71-53; E-mail: [email protected]. pKW2T-Pax5-AHD. The TBP expression constructs were described previ- 3 The abbreviations used are: BASP, B-cell-specific activator protein; TBP, TATA- ously (15), and the E1A (13S) expression vector was obtained by subcloning binding protein; Rb, retinoblastoma; GST, glutathione S-transferase; EtBr, ethidium the respective HindIII-BamHI fragment from pH/3APr-I-Neo-13S (16) into bromide; TFIID, transcription factor IID; EBC1 buffer, 150 mM NaC1, 50 mM Tris-C1 (pH 8.0), l rnM EDTA, 0.2% NP40, I mM DTT, 400 /.ZMNa3VO4, 10 mM NaF, 0.1 mg/ml pKW2T. The GST-Rb (379-928), GST-Rb (C706F), GST-Rb (3,21), and Pefabloc, 5 /xg/ml pepstatin, 5 /xg/ml leupeptin, 5 /xg/ml aprotinin, 2 /xg/ml antipain, 2 GST-Rb (379-792) fusion constructs have been described previously (17). /xg/ml chymostatin, and 2 mM benzamidine hydrochloride; NETN buffer, 20 mM Tris-Cl The GST-p107 (385-1068) plasmid was provided by S. Mittnacht (London). (pH 7.9), 100 mM NaC1, 1 mM EDTA, 0.5% NP40, and 1 mM DTT; buffer BCI00, 20 mM The human Rb expression plasmid was generated by cloning full-length Rb Tris-C1 (pH 8.0), 100 rnM KC1, 0.1 mM EDTA, 5 mM MgC12, 20% glycerol, 1 mM DTE, 0.1 mg/ml Pefabloc; buffer A, 50 mM Tris-C1 (pH 8.0), 400 mM NaC1, 1 rnN EDTA, 0.2% cDNA into the BamHI site of pcDNA3 (Invitrogen). The expression plasmid NP40, 0.5 mg/ml BSA, and 1 mM DTT. pECE-Ap34-HA has been described previously (18). 1716s Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1999 American Association for Cancer Research. INTERACTING PROTEINS OF THE TRANSCRIPTION FACTOR PAX-5 Antibodies. A polyclonal rabbit anti-hPax-5 antibody, which was directed protein A-Sepharose (Pharmacia) for 2 h at 4~ After washing with binding against the paired domain of human Pax-5 (amino acids 17-145; Ref. 3), was buffer, the immunoprecipitated proteins were resuspended in 2• SDS sample affinity-purified. The polyclonal rabbit anti-laminin serum was purchased from buffer, eluted from the beads by boiling, and separated by SDS-PAGE. Serotec Ltd. (Oxford, England) and the anti-FLAG M1 and M2 affinity gels Proteins were transferred to an Immobilon-P membrane (Millipore) and mon- from Eastman Kodak Co. (New Haven, CT). The mouse monoclonal anti-TBP itored for the presence of TBP using the monoclonal anti-TBP antibody 3G3 antibody, which recognizes the NH2-terminal region of hTBP, was described (19). Antibodies were revealed by an enhanced chemiluminescence (ECL) previously (19). The polyclonal anti-Rb antibody (C15), which is directed Western blot detection system (Amersham). against the COOH terminus of human Rb, was obtained from Santa Cruz Results Biotechnology Inc. (Santa Cruz, CA). Cell Lines. The monkey kidney cell line COS-7 and the human osteosar- The Transcription Factor Pax-5 (BSAP) Binds to TBP in Vivo. coma cell line U2-OS (20) were grown in high glucose DMEM supplemented Functional analyses (13) and evolutionary sequence comparisons (4, with 10% FCS.
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