The Partial Homeodomain of the Transcription Factor Pax-5 consisting of Pax-2, Pax-5, and Pax-8 (3)

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Text of The Partial Homeodomain of the Transcription Factor Pax-5 consisting of Pax-2, Pax-5, and Pax-8 (3)

  • [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

    D i r k E b e r h a r d a n d M e i n r a d B u s s l i n g e r 2

    Research Institute of Molecular Pathology, A-t030 Vienna, Austria

    Abstract

    Pax-5 codes for the transcription factor BSAP, which plays an impor- tant role in midbrain patterning, B cell development, and lymphoma formation. Pax-5 is known to control gene expression by recognizing its target genes via the NH2-terminal paired domain and by regulating transcription through a COOH-terminal regulatory module consisting of activating and inhibitory sequences. The central region of Pax-5 contains a sequence with significant homology to the first a-helix of the paired-type homeodomain. This partial homeodomain has been highly conserved throughout vertebrate evolution because it is found not only in Pax-5 but also in the related Pax-2 and Pax-8 members of the same Pax subfamily. Here we report that the partial homeodomain binds the TATA-binding protein (TBP) and retinoblastoma (Rb) gene product. Both TBP and Rb were shown by coimmunoprecipitation experiments to directly associate with Pax-5 in vivo. The conserved core domain of TBP and the pocket region as well as COOH-terminal sequences of Rb are required for interaction with the partial homeodomain of Pax-5 in in vitro binding assays. Furthermore, Pax-5 was specifically bound only by the underphos- phorylated form of Rb. These data indicate that Pax-5 is able to contact the basal transcription machinery through the TBP-containing initiation factor TFIID, and that its activity can be controlled by the cell cycle- regulated association with Rb.

    Introduction

    The Pax gene family codes for transcription factors that play important roles in embryonic development , cell differentiation, and human disease. A hal lmark of these developmenta l regulators is their

    conserved DNA-binding motif, the so-called paired domain. The mammal ian genome contains nine Pax genes that can be grouped into four distinct classes based on their similarity in sequence and expres-

    sion (reviewed in Ref. 1). Two of these subclasses contain, in addition to the paired domain, also a h o m e o d o m a i n as a second DNA-binding region. A sequence motif, which is homologous only to the first

    a-hel ix of the homeodomain (2), has been identified in the subfamily consist ing of Pax-2, Pax-5, and Pax-8 (3). This partial h o meodoma i n has no DNA-binding activity and yet is conserved in members of the Pax-2/5/8 family from sea urchin to man (4, 5), which suggests that it

    constitutes a protein interaction motif. The Pax-5 gene codes for the transcription factor BSAP, 3 which is

    expressed in the developing midbrain, all of the lymphoid tissues, and

    adult testis of the mouse (reviewed in Ref. 6). Consistent with this

    Received 9/18/98; accepted 2/1/99. 1 Contributed as part of the April 1, 1999 Supplement to Cancer Research, "General

    Motors Cancer Research Foundation Twentieth Annual Scientific Conference: Develop- mental Biology and Cancer." This work was supported in part by a Grant from the Austrian industrial Research Promotion Fund. D. E. was the recipient of a fellowship from the Deutsche Forschungsgemeinschaft and European Community.

    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: 43-1-798-71-53; E-mail: busslinger@nt.imp.univie.ac.at.

    3 The abbreviations used are: BASP, B-cell-specific activator protein; TBP, TATA- binding protein; Rb, retinoblastoma; GST, glutathione S-transferase; EtBr, ethidium 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 /.ZM Na3VO4, 10 mM NaF, 0.1 mg/ml Pefabloc, 5 /xg/ml pepstatin, 5 /xg/ml leupeptin, 5 /xg/ml aprotinin, 2 /xg/ml antipain, 2 /xg/ml chymostatin, and 2 mM benzamidine hydrochloride; NETN buffer, 20 mM Tris-Cl (pH 7.9), 100 mM NaC1, 1 mM EDTA, 0.5% NP40, and 1 mM DTT; buffer BCI00, 20 mM 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% NP40, 0.5 mg/ml BSA, and 1 mM DTT.

    expression pattern, targeted inactivation of Pax-5 in the mouse germ-

    line revealed essential functions of this transcription factor in mid- brain and B-cell deve lopment (7, 8). Interestingly, the human PAX-5

    gene is involved together with the immunoglobul in heavy chain locus in a recurring t(9;14)(p13;q32) translocation associated with a subset of non-Hodgkin ' s lymphomas (9-11) . Hence, PAX-5 can be activated by gain-of-function mutat ions to participate as an oncogene in tumor-

    igenesis. The recent genetic identification of Pax-5 target genes re- vealed that Pax-5 controls their transcription either as an activator or repressor depending on the specific regulatory sequence context (6,

    12). Structure-function analysis, furthermore, demonstrated that Pax-5 recognizes its target genes via the NH2-terminal paired domain and controls u'anscription through a COOH-terminal regulatory module

    consist ing of activating and inhibitory sequences (13). In addition, the central sequences containing the partial homeodomain were also shown to contribute to the transcriptional activity of Pax-5 (13).

    Here we demonstrate by different protein binding assays that the

    TBP and the Rb protein directly interact with the transcription factor Pax-5 in vivo and in vitro. Deletion analysis, furthermore, identified

    the partial homeodomain of Pax-5 as an essential recognit ion moti f for both TBP and Rb. These data suggest therefore that the partial home odoma i n controls the activity of Pax-5 by linking it either

    through TBP to the basal transcription machinery or through Rb to the control of cell proliferation.

    Materials and Methods

    Expression Constructs. The expression plasmids coding for lull-length Pax-5 (pKW2T-hBSAP) and Pax-5 (1-268) have been described previously (13). Pax5-APD was constructed by subcloning a HindlIi-BamHI fragment 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 generated in pKW2T-hBSAP via PCR-mediated mutagenesis by introducing an XhoI site at the deletion site without affecting amino acids 228 and 254. The FLAG epitope was added at the NH2 terminus of Pax-5 by replacing a 260-bp HindlII-BamHI fragment of pKW2T-hBSAP with a corresponding PCR prod- uct generated with the oligonucleotide 5'-CCCAAGCTTACCATGGATTA- CAAGGACGACGATGACAAGTTAGAGAAAAATTA-3 ' and a corre- sponding downstream Pax-5 primer. The HindlII-EcoRI insert containing the full-length Pax-5 cDNA was subsequently recloned in the expression plasmid pRK7 (3) to generate pRK7-FLAGhBSAP, which was used for transient 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 260-bp BamHI PCR fragment (5' end) together with a 900-bp BamHI-EcoRI cDNA fragment (3' end). The COOH-terminal Pax-5 deletion mutants con- taining the FLAG epitope were generated by using the corresponding BamHI- EcoRI cDNA fragments of the previously described deletion clones (13) in the above ligation reaction. The expression plasmid pET-FLAGhPax5-AHD was similarly generated by cloning the corresponding BamHI-EcoRI fragment from pKW2T-Pax5-AHD. The TBP expression constructs were described previ- ously (15), and the E1A (13S) expression vector was obtained by subcloning the respective HindIII-BamHI fragment from pH/3APr-I-Neo-13S (16) into pKW2T. The GST-Rb (379-928), GST-Rb (C706F), GST-Rb (3,21), and GST-Rb (379-792) fusion constructs have been described previously (17). The GST-p107 (385-1068) plasmid was provided by S. Mittnacht (London). The human Rb expression plasmid was generated by cloning full-length Rb cDNA into the BamHI site of pcDNA3 (Invitrogen). The expression plasmid pECE-Ap34-HA has been described previously (18).

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  • INTERACTING PROTEINS OF THE TRANSCRIPTION FACTOR PAX-5

    Antibodies. A polyclonal rabbit anti-hPax-5 antibody, which was directed against the paired domain of human Pax-5 (amino acids 17-145; Ref. 3), was affinity-purified. The polyclonal rabbit anti-laminin serum was purchased from Serotec Ltd. (Oxford, England) and the anti-FLAG M1 and M2 affinity gels from Eastman Kodak Co. (New Haven, CT). The mouse monoclonal anti-TBP antibody, which recognizes the NH2-terminal region of hTBP, was described previously (19). The polyclonal anti-Rb antibody (C15), which is directed against the COOH terminus of human Rb, was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Cell Lines. The monkey kidney cell line COS-7 and the human osteosar- coma cell line U2-OS (20) were grown in high glucose DMEM supplemented with 10% FCS. The human B cell line BJA-B expressing Pax-5 (21) was cultured in RPMI 1640 containing 10% FCS.

    Cell Transfection and Extract Preparation. COS-7 cells were grown on 6-cm dishes to 80% confluency and then were transfected by Lipofectami