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[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 important 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 underphosphorylated 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: [email protected].

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 midbrain 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 revealed 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 product generated with the oligonucleotide 5'-CCCAAGCTTACCATGGATTA- CAAGGACGACGATGACAAGTTAGAGAAAAATTA-3 ' and a corresponding 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 containing 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 previously (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 osteosarcoma 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 Lipofectamine (Life Technologies) according to the manufacturer's instructions. Typical transfection reactions contained 2 #g of total plasmid DNA and 12 /xl of Lipofectamine. After 48 h, whole-cell lysates were prepared by lysing the cells in 300 ~1 of EBC1 buffer for 30 min on ice. Cell debris was removed by centrifugation at 14,000 rpm for 30 min at 4~ and the supernatant was used for immunoprecipitations. U2-OS cell lysates were prepared as described for COS-7 cells. Nuclear extracts of BJA-B cells were prepared according to Dignam et al. (22).

Purification of Recombinant Proteins from Bacteria. hPax-5 proteins tagged with the FLAG epitope (DYKDDDDK) at the NH 2 terminus were expressed in the E. coli strain BL21-pLysS. GST and the GST-Rb and GST-pl07 fusion proteins were expressed in the E. coli strain DH5a. Over- night cultures of bacteria containing the respective expression plasmid were diluted 1:100 in LB-medium containing 1% glucose and grown at 37~ to an absorbance of A6o 0 n m of 0.6 before expression was induced by the addition of 1 mM isopropyl-/3-D-thiogalactopyranoside for 2 h at 37~ Bacteria were lysed in NETN buffer supplemented with protease inhibitors (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) and 0.1 mg/ml lysozyme. After a 10-rain incubation on ice, the lysate was sonicated for 3 • 20 s. After removal of cellular debris by centrifugation, cell lysates containing FLAG-tagged proteins were incubated with the anti-FLAG M2 affinity gel (Eastman Kodak Co.), and lysates containing GST-fusion proteins were incubated with glutathione-Sepharose beads (Pharmacia) for 2 h at 4~ At the end of the binding reaction, the beads were extensively washed with NETN buffer containing 1% NP40. The anti-FLAG affinity gel was further washed with buffer B [20 mM Tris-C1 (pH 8.0), 0.1 mM EDTA, 5 mM MgCI:, 20% glycerol, and 700 mM KC1]. Beads were resuspended in 3 volumes of TGME buffer [20 mM Tris-C1 (pH 8.0), 100 mM NaC1, 1 mM EDTA, 10% glycerol, and 0.1% NP40] and stored at 4~

Protein-Protein Interaction Assays. For in vitro binding assays, beads coated with 2-5/xg of recombinant proteins were incubated for 2 h at 4~ with 5/xl of 35S-proteins that were synthesized by a coupled in vitro transcription- translation system (TNT, Promega) in the presence of [35S]methionine. Bind- ing assays were performed either in buffer EBC 1 supplemented with 0.5 mg/ml BSA, in buffer BC100 containing 0.2% NP40, 0.5 mg/ml BSA or in buffer A. Where indicated, 100/xg/ml EtBr was included in the binding reaction. After washing with 400 volumes of binding buffer, bound proteins were eluted from the beads by boiling in 2• SDS sample buffer and were applied to SDS- PAGE. 35S-proteins were detected by autoradiography.

Whole-cell lysates from COS-7 cells were mixed with the indicated antibodies and incubated for 2 h at 4~ with constant rotation. If necessary, protein A-Sepharose (Pharmacia) was added for 1 h to collect the immunocomplexes. After stringent washing with binding buffer, the precipitated proteins were resuspended in 2 • SDS sample buffer, separated on SDS-PAGE, and analyzed by Western blotting.

For coprecipitation of endogenous proteins from BJA-B nuclear extract, 1 mg of nuclear extract was dialyzed against the buffer BC100. Dialyzed extracts were supplemented with 0.2% NP40, a variety of protease inhibitors (0.1 mg/ml Pefabloc, 5 /xg/ml pepstatin, 5 /zg/ml leupeptin, 5 /xg/ml aprotinin, 2 /~g/ml antipain, 2/xg/ml chymostatin, and 2 mM benzamidine hydrochloride) and 100/xg/ml EtBr before the affinity-purified polyclonal rabbit anti-hPax-5 antibody or the polyclonal anti-laminin antibody was added for 5 h at 4~ with constant rotation. Immunocomplexes were captured by the addition of 20/xl of

protein A-Sepharose (Pharmacia) for 2 h at 4~ After washing with binding buffer, the immunoprecipitated proteins were resuspended in 2• SDS sample buffer, eluted from the beads by boiling, and separated by SDS-PAGE. Proteins were transferred to an Immobilon-P membrane (Millipore) and monitored for the presence of TBP using the monoclonal anti-TBP antibody 3G3 (19). Antibodies were revealed by an enhanced chemiluminescence (ECL) Western blot detection system (Amersham).

Results

The Transcr ipt ion Factor Pax-5 (BSAP) Binds to T B P in Vivo. Functional analyses (13) and evolu t ionary sequence compar i sons (4,

5) revea led that the transcription factor Pax-5 (BSAP) uses different

conse rved domains to exert its funct ion in gene regulation. Because

some of these domains may media te binding o f Pax-5 to componen t s

of the basal transcription machinery , we have screened for such

interactions by a b iochemica l approach. To this end, a F L A G epi tope

was inserted at the N H a terminus o f Pax-5, w h o s e transcriptional

activity was not altered by this modi f ica t ion (data not shown). Initial

b inding exper iments indicated that at least one componen t of the basal

transcription machinery , the human T B P (23-25) , is capable of bind-

ing in vi tro to the F L A G - t a g g e d Pax-5 protein (see Fig. 2). To

demonst ra te that Pax-5 can also interact with T B P in vivo, we over-

expressed human T B P and the F L A G - t a g g e d Pax-5 protein by tran-

sient t ransfect ion in COS-7 cells (Fig. 1A). Extracts f rom transfected

cells were prepared, and the Pax-5 protein was precipi ta ted with either

the monoc lona l an t i -FLAG M1 or M 2 ant ibodies (Fig. 1A). The

immunoprec ip i ta tes were subsequent ly ana lyzed by Wes te rn blott ing

with a monoc lona l ant ibody specif ic for the NH2-terminal sequence of

T B P (19). A significant propor t ion o f the T B P protein could be

coprecipi ta ted with Pax-5 (Fig. 1A, L a n e 2), which was eff icient ly

deple ted f rom the extract with the an t i -FLAG M 2 ant ibody (data not

shown). The related M I ant ibody is k n o w n to bind to the an t i -FLAG

epi tope only in the presence of Ca 2+ ions (26). In the absence of

calcium, this control M I ant ibody was unable to precipi tate Pax-5

and, hence, T B P (Fig. 1A, L a n e 3), which demonst ra tes that the

A t -

EtBr I ~ L9 t~

- - c~-FLAG ~ "g: .,.., .,.., ~ . 1 ~ (~ c~ t3 ._1 c::: M2 M1 M 2 M 2 ~ ~

I g H - I g H - hTBP -

hTBP - 1 2 3

IgL -

2 3 4 5

C O S - 7 BJA-B Fig. 1. In vivo interaction of Pax-5 and TBP. A, coimmunoprecipitation of Pax-5 and

TBP from COS-7 cell extracts. Expression plasmids coding for human TBP and a FLAG-tagged human Pax-5 protein were transiently transfected into COS-7 cells. Whole- cell lysates prepared from these COS-7 cells were incubated, in the absence of Ca a+ ions, with the anti-FLAG M2 (Lanes 2, 4, and 5) or MI (Lane 3) antibodies that were covalently cross-linked to agarose. Note that the calcium-dependent M1 antibody can only bind to the FLAG epitope in the presence of Ca 2+ (26). Where indicated, EtBr was included in the binding reaction (33 ~g/ml EtBr in Lane 4; 100/xg/ml EtBr in Lane 5). The precipitated proteins were separated by 10% SDS-PAGE, transferred to an lmmobilon-P membrane, and probed with a monoclonal antibody specific for the NH2-terminal sequences of hTBP (19). Lane 1 contained 10% of the total input protein used for immunoprecipitation. The positions of the hTBP and coeluted immunoglobulin heavy (IgH) and light (/gL) chain proteins of the anti-FLAG antibodies are indicated to the left. B, association of endogenous TBP with Pax-5 in human B cells. A nuclear extract (1 rag) prepared from BJA-B cells was incubated with the Pax-5-specific anti-paired domain antibody (Lane 2) or a control anti-laminin antibody (Lane 3) in the presence of 100 /xg/ml EtBr. Antibody complexes were precipitated with protein A-Sepharose and analyzed for the presence of TBP as described above. Lane 1 contained 5% (50 /xg) of the nuclear extract used for precipitation.

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presence of TBP in the immunoprecipitate depends on the specific interaction with Pax-5. The nuclear extracts used as a starting material usually contain some chromosomal DNA, and, therefore, it is conceivable that the observed interaction may be mediated by simulta- neous binding of both proteins to contaminating DNA. To rule out this possibility, the immunoprecipitations with the anti-FLAG M2 antibody were repeated in the presence of increasing amounts of EtBr, which distinguishes between DNA-mediated and bona f ide protein- protein interactions (27). The addition of EtBr to the binding reaction, however, did not influence the interaction between Pax-5 and TBP, which confirms the specificity of protein complex formation (Fig. 1A, Lanes 4 and 5). Comparison of the TBP amount in the starting material (Lane 1; 1/10 of the input protein) and the immunoprecipitate (Lane 2) revealed that --10% of the TBP protein present in the transfected cells was complexed with Pax-5. These data indicate, therefore, that Pax-5 can efficiently bind to the basal transcription factor TBP under the in vivo conditions analyzed.

The high protein expression levels achieved in transfected COS-7 cells could, however, result in spurious protein-protein interactions. Hence, we verified complex formation between endogenous Pax-5 and TBP in the human B cell line BJA-B, which expresses both proteins at physiological concentrations (Fig. 1B). For this purpose, a polyclonal anti-Pax-5 antibody (3) was used to precipitate the endogenous Pax-5 protein from nuclear extracts of BJA-B cells, and the presence of TBP in the precipitate was monitored by immunoblotting. As shown in Fig. 1B (Lane 2), a substantial fraction of the endogenous TBP was coprecipitated with Pax-5 from BJA-B nuclear extracts. This coprecipitation was, however, not mediated by contaminating DNA, because the anti-paired domain antibody used is known to completely block the DNA-binding activity of Pax-5 (3). Furthermore, TBP could not be precipitated with a control antibody directed against laminin (Fig. 1B, Lane 3), thus confirming the specificity of the observed TBP-Pax-5 interaction. Hence, we conclude that Pax-5 forms a stable complex with TBP in BJA-B cells in vivo. It is well established that the majority of the TBP protein is not present in free form in nuclear extracts but instead is part of several multiprotein complexes including TFIID (reviewed in Refs. 28-30). The association of Pax-5 with

TBP in BJA-B nuclear extracts suggests, therefore, that Pax-5 interacts in vivo with the TBP-containing multiprotein complex TFIID.

Pax-5 Interacts with the Conserved Core Domain o f TBP. The domain of TBP that mediates the interaction with Pax-5 was next identified by analyzing COOH-terminally truncated, in vitro synthesized TBP proteins in a Pax-5 interaction assay (Fig. 2). The association of the different mutant proteins with Pax-5 was internally controlled by the inclusion of full-length TBP in the binding reaction. Recombinant Pax-5 prote in-- immobil ized on agarose beads- -was able to efficiently bind full-length TBP as well as the truncation mutant lacking the last 68 amino acids (Fig. 2B, Lanes 7-9) , whereas empty control beads failed to retain any TBP polypeptide (Fig. 2B, Lanes 4 - 6 ) . Interestingly, further deletion of the core domain in TBP (1-167) abolished all of the binding, which indicated that Pax-5 requires the core domain of TBP for stable complex formation (Fig. 2B, Lane 8). The DNA-binding activity of TBP is known to depend on the integrity of the entire core domain (31). The observation that the very COOH-terminal region of TBP can be deleted without affecting Pax-5 binding further demonstrates that the interaction between TBP and Pax-5 takes place in the absence of DNA binding.

The core domain of TBP has been highly conserved in evolution as it is 80% homologous between yeast and man (23, 28). As shown in Fig. 2C, the TBP protein of yeast (32-34) is also able to form specific complexes with Pax-5. Interestingly, the binding efficiency was similar for both the yeast and the human TBP proteins (compare Fig. 2, B and C). We conclude, therefore, that the core domain sequences of TBP, which mediate the interaction with Pax-5, have been conserved throughout evolution.

The Partial H o m e o d o m a i n o f Pax-5 Participates in TBP Bind-

ing. Structure-function analyses demonstrated previously (13) that several distinct domains of Pax-5 contribute to its transcriptional activity (see Fig. 3A). The COOH-terminal sequences of Pax-5 harbor a potent transactivation domain followed by an inhibitory element. In contrast, a weaker transactivation function was assigned to the internal sequences of Pax-5 that encompass the conserved octapeptide motif (3, 35) and a region with sequence similarity to the NH2-terminal part of paired-type homeodomains (2, 3). To delineate the domain of Pax-5

Fig. 2. Pax-5 contacts the highly conserved core domain of TBP. A, schematic diagram of the human TBP protein. The relative positions of the Q-domain and the evolutionary conserved core domain are indicated. Arrows denote the two direct repeat sequences of the core domain. COOH-terminally mm- cated TBP proteins were generated by in vitro translation of a TBP expression vector that was lin- earized at codons 98, 167, and 271 with Pstl, Sspi, and Stul, respectively. The interaction of full-length and truncated TBP proteins with Pax-5 is summarized to the right. B, interaction of the core domain of human TBP with Pax-5. In vitro translated 35S-TBPs were incubated in buffer EBC1 with the anti-FLAG M2 affinity gel, which was either directly used (Con- trol; Lanes 4-6) or first coated with the bacterially expressed FLAG-tagged hPax-5 protein (Pax-5; Lanes 7-9). Protein complexes were collected by centrifugation and separated by SDS-PAGE followed by fluorography of the labeled TBP. Full-length TBP was added as an internal control protein to each binding reaction, and its position is indicated to the left. Lanes 1-3 contained 10% of the labeled input protein (Input) used for the binding assay. C, evolutionary conservation of the Pax-5 interaction domain of TBP. In vitro translated 35S-TBP of yeast (yTBP) was assayed for its ability to bind to immobilized FLAG- tagged hPax-5 (Lane 1) or to the affinity matrix alone (Lane 2). The signal in Lane 3 corresponds to 10% of the total yTBP protein analyzed.

A 55 Q 9.5

B

hTBP -

f Pstl (98)

i i i i

159 Core 339

hTBP

t' t' Pax-5 ssp~ stu~ interaction (167) (271)

u i t q

; I 1-339 1-271 1-167 1-98

Input /

C o n t r o l /

Pax-5 / / / C

yTBP -

1 2 3 4 5 6

1718s

7 8 9

o',

1 2 3



Fig. 3. TBP interacts with the partial homeodomain of Pax-5. A, schematic diagram of Pax-5 deletion mutants. The structural organization of human Pax-5 is shown together with the extent of the various deletions. Numbers refer to the corresponding amino acid positions of hPax-5 (BSAP; Ref. 3). A FLAG epitope was inserted at the NH 2 terminus of all of the hPax-5 proteins to facilitate purification and immobilization on the anti-FLAG M2 affinity gel. For generation of the different constructs, see "Materials and Methods." B, analysis of purified hPax-5 proteins. Wild-type Pax-5 and the COOH- terminal truncation mutants were expressed in E. coIi and purified from the bacterial lysates by binding to the anti-FLAG M2 affinity matrix. The immobilized proteins were eluted and analyzed by 12% SDS-PAGE followed by Coomassie blue staining, which indicated that all of the proteins were purified in similar quantities. The Control Lane contained untreated affinity beads. The size of marker proteins (given in kDa) is indicated to the left. The positions of coeluted immunoglobulin heavy (lgH) and light (IgL) chain proteins are shown. C, the COOH-terminal Pax-5 sequences are dispensable for TBP binding. Similar amounts of the different immobilized proteins were assayed in buffer A for their ability to bind to in vitro synthesized 35S-TBP (Lanes 3-7). Proteins were resolved by SDS-PAGE and detected by autoradiography. Untreated M2 affinity matrix was analyzed in Con- trol Lane 2. The input in Lane 1 con'esponds to 10% of the total 35S-protein used. D, TBP binds to the partial homeodomain of Pax-5. The interaction of fnll-length Pax-5 and indicated deletion mutants with 35S-TBP was analyzed as described in C.


A FLAG paired octa- homeodomain transactivation inhibitory

tag domain peptide homology domain domain

911 NNNNNNN' \NN\'N N N tl

4f

,NNNNx,'xNNNNx,N', I

Pax-5

1-391

1-358

1-268

1-220

1-169

AHD

TBP binding

kO

1oi - .~ :~,~ 83 - : ;~

21 . . . . ; ~

c

hTBP

1 2 3 4 5 6 7

O

1 2 3 4 5

that mediates the interaction with TBP, we expressed full-length

Pax-5 and a series of COOH-terminal truncation mutants (Fig. 3A) in E. coli, affinity-purified these proteins (Fig. 3B), and subsequently used them for in vitro binding assays with 3sS-TBP (Fig. 3C). In

agreement with the result of Fig. 2, TBP was bound by immobi l ized

Pax-5 protein of full length (Lane 3) but not by untreated control beads (Lane 2). Interestingly, the COOH-terminal sequences of Pax-5

could be el iminated up to amino acid 268 without affecting TBP binding (Lanes 4 and 5). Hence, the COOH-terminal transactivating sequences as well as the inhibitory e lement of Pax-5 do not contribute to TBP binding, in contrast to other transcription factors that fre-

quently contact TBP via their transactivation domain (36-41) . Dele- tion of the partial homeodomain in Pax-5 (1-220) reduced, however , TBP binding to background level (Fig. 3C, Lane 6), which was aiso

seen with a polypept ide (1-169) consisting only of the paired domain (Lane 7). Interestingly, precise deletion of the homeodomain in the full-length Pax-5 protein (AHD) was sufficient to prevent the inter-

action with TBP (Fig. 3D, Lane 5). We conclude, therefore, that the partial homeodomain of Pax-5 and not its transactivation region is involved in TBP binding. Hence, these data identify the partial homeodomain as a protein interaction moti f which has been conserved in

different members of the Pax-2/5/8 family throughout vertebrate evolution (5).

The Partial Homeodomain of Pax-5 also Mediates Binding to the Rb Protein. The core domain of TBP is known to share significant sequence homology with the pocket domain of the Rb tumor

suppressor protein (37), which interacts with and, thus, controls a variety of transcription factors (reviewed in Refs. 42-44) . Consistent with this sequence similarity, TBP and Rb frequently associate with

the same interaction domain in a variety o f transcription factors (37,

38, 45, 46). Given the association with TBP, we next investigated whether Pax-5 is also able to interact with Rb. To this end, in vitro

synthesized, radiolabeled Pax-5 protein was incubated with a bacte-

rially expressed GST-Rb (379-928) which consisted of GST linked to the pocket region and COOH-terminal sequences of Rb (see Fig. 6.4). As shown in Fig. 4A, Pax-5 was coprecipitated together with the

GST-Rb fusion protein bound to glutathione-Sepharose beads. In contrast, neither GST nor the empty beads (control) were able to interact with Pax-5 in this pul l -down assay. Al though significant, the binding affinity of Pax-5 was lower than that of the adenovirus E1A

protein (Fig. 4A), which is known to efficiently associate with Rb (47). In conclusion, Pax-5 and Rb are able to form a protein complex under these in vitro binding conditions.

Deletion mutants of Pax-5 (Fig. 4D) were next analyzed in the GST pul l -down assay to identify the interaction domain involved in Rb binding. The Pax5-APD and Pax-5 (1-268) proteins bound Rb as

efficiently as the fllll-length protein, indicating that both the DNA- binding function of the paired domain and the COOH-terminal regulatory sequences of Pax-5 are dispensable for the interaction with Rb (Fig. 4B). In contrast, deletion of the partial h o m e o d o m a i n strongly

interfered with Rb binding (Fig. 4B). Hence, the h o m e o d o m a i n sequences of Pax-5 constitute an interaction mot i f not only for the TBP

but also for the Rb protein. The proteins p 107 and p130 are structurally and functionally related

to Rb but nevertheless differ f rom Rb in their association with distinct

transcription factors (reviewed in Refs. 42 and 44). We, therefore, assessed the specificity of the Pax-5 interaction with Rb family members , using the GST pul l -down assay. Al though bacterially ex-

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Fig. 4. Pax-5 interacts with the Rb protein via its partial homeodomain. A, association of Pax-5 with the Rb protein. Bacterially expressed GST or GST-Rb (379-928) protein (2 /xg each), bound to glntathione-Sepharose, were incubated with in vitro translated 35S-Pax-5 or E1A (13S) protein in buffer BCI00. After stringent washing of the Sepharose beads, the bound proteins were eluted by boiling, analyzed by SDS-PAGE, and detected by autoradiography. Lane 1 contained 10% of the input protein. Untreated control beads were used in Lane 2. B, the homeodomain of Pax-5 is essential for complex formation with Rb. 35S-labeled full-length or mutant Pax-5 proteins were synthesized by in vitro translation and then assayed for binding to GST-Rb (379-928) as described in A. C, selective interaction of Pax-5 with members of the pocket domain protein family. 35S-labeled Pax-5 and E1A proteins was analyzed for binding to GST-Rb (379-928) and GST-p107 (385-1068) as described in A. Sim- ilar amounts of both GST fusion proteins were used in the binding assay, as shown by the equally efficient complex formation of E1A with GST-Rb and GST-pl07. D, schematic diagram of the Pax-5 deletion mutants. The different domains and corresponding amino acid residues of Pax-5 are shown together with the extent of the different deletions.


A c

P a x - 5 - P a x - 5 - P a x - 5 -

E I A -

1 2 3 4

P a x 5 - A P D - E 1 A -

P a x 5 - A H D -

8 9

D P a x - 5

P a x 5 - A P D

P a x 5 - A H D

P a x - 5 ( 1 - 2 6 8 )

P a x - 5 - ( 1 -268 )

5 6 7

homeodomain homology

~ , x ~

pressed GST-pl07 and GST-Rb bound to the control E1A protein with equal efficiency, the p107 fusion protein was incapable of interacting with Pax-5 in contrast to Rb (Fig. 4C). These data indicate, therefore, that Pax-5 is able to selectively associate with Rb and thus to discriminate between different members of the Rb protein family in analogy to the E2F transcription factors (48-50).

Pax-5 Selectively Binds to the Underphosphorylated Form of Rb in Vivo. The activity of Rb is regulated by differential phospho- ryladon during the cell cycle (51-53). Rb is predominantly found in the active, underphosphorylated form in quiescent (Go) cells as well as in the early G 1 phase of the cell cycle. At the transition between G1 and S phase, Rb is rapidly phosphorylated and maintains this hyperphosphorylated, inactive configuration throughout the remainder of the cell cycle. A variety of cellular transcription factors as well as many viral oncoproteins are known to preferentially interact with the underphosphorylated form of Rb (reviewed in Refs. 42 and 44). To investigate the effect of Rb phosphorylation on Pax-5 binding, we tested the endogenous wild-type Rb isoforms of the human osteosarcoma cell line U2-OS (20) for their ability to interact with Pax-5. Extracts of U2-OS cells were incubated and subsequently precipitated with recombinant Pax-5 protein immobilized on affinity beads. As shown in Fig. 5A (Lane 1), cycling U2-OS cells predominantly contained the hyperphosphorylated, more slowly migrating form of Rb. However, the immobilized Pax-5 protein specifically precipitated

the less abundant underphosphorylated form of Rb, and control beads failed to bind any Rb protein (Fig. 5A). These data demonstrate, therefore, that Pax-5 preferentially interacts with the active, underphosphorylated form of Rb.

To verify this protein interaction in vivo, we expressed the FLAG- tagged Pax-5 protein together with wild-type Rb or a phosphorylation- incompetent Rb mutant (Ap34; Ref. 18) in transiently transfected COS-7 cells. Pax-5-Rb protein complexes were precipitated with anti-FLAG antibodies from COS-7 cell extracts and detected by Western blot analysis with an Rb-specific antibody. As shown in Fig. 5B, only the underphosphorylated form of the wild-type Rb protein was coprecipitated by the anti-FLAG M2 antibody (Lane 3), whereas the calcium-dependent control M1 antibody (26) failed to precipitate Pax-5, and thus Rb, in the absence of Ca 2+ (Lane 2). Moreover, a significant increase of coprecipitated Rb protein was obtained with transfected COS-7 cells expressing the Rb mutant Ap34, (Lane 4), which can no longer be phosphorylated by cyclin-dependent kinases (18). We conclude, therefore, that Pax-5 associates specifically in vivo with the underphosphorylated Rb protein, suggesting that in prolifer- ating cells this interaction is regulated by differential phosphorylation of Rb.

Pax-5 Interacts with the Pocket Domain and COOH-Terminal Sequences of Rb. The sequences of Rb that participate in the interaction with Pax-5 were next delineated by analyzing a series

1720s




A

PO 4 - Rt Rt

1 2 3 U2-OS

B o~-FLAG /

PO 4 - Rb RI~

/

1 2 3 4

COS-7

Fig. 5. Selective interaction of Pax-5 with the underphosphorylated form of Rb. A, formation of Rb-Pax-5 complexes in extracts from U2-OS osteosarcoma cells. Whole-cell lysates (100/xg) were incubated with either the untreated anti-FLAG M2 affinity matrix (Lane 2) or the M2 affinity gel coupled to purified FLAG-tagged hPax-5 protein (Lane 3). Bound proteins were precipitated, separated by 6% SDS-PAGE, and analyzed by Western blotting with a polyclonal anti-Rb antibody recognizing the COOH-terminal Rb sequences (C15; Santa Cruz Biotechnology Inc.). Lane 1 contained 10% (10/zg) of the total input protein. The positions of the underphosphorylated and hyperphosphorylated (P04) Rb proteins are indicated to the left. B, in vivo interaction of Pax-5 and Rb proteins. Expression plasmids directing the synthesis of FLAG-tagged hPax-5 (Lanes 1-4) and of the wild-type Rb protein (Lanes 1-3) or the phosphorylation-incompetent Rb mutant Ap34 (Ref. 18; Lane 4) were transiently transfected into COS-7 cells. Cell lysates were immunoprecipitated with the anti-FLAG M2 antibody (Lanes 3 and 4) or the Ca 2+- dependent M1 antibody (Ref. 26; Lane 2), as described in the legend to Fig. 1. The immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting using the Rb-specific antibody C15. The positions of the differentially phosphorylated Rb proteins are indicated to the left. Lane I contained 10% of the input cell lysate.

of mutant Rb proteins (Fig. 6A) in the GST pul l -down assay. Mutant GST-Rb proteins were expressed in E. coli, purif ied on glutathione-Sepharose, and quantitated by SDS-PAGE, as shown in Fig. 6B. These proteins were subsequently analyzed for their potential to bind in vitro synthesized, radiolabeled Pax-5 or control E1A protein. As shown in Fig. 6C (Lane 4), the naturally occurr ing Rb mutat ion C706F (54) significantly reduced the interact ion of Rb with Pax-5 in compar ison with GST-Rb (379-928) containing wild-type pocket domain and COOH-termina l sequences (Lane 3).

Delet ion of the entire exon 21 sequences (55), which code for one third of the subdomain B of the Rb pocket, resulted in complete loss of Pax-5 binding (Lane 5). Interestingly, t runcation of the COOH-terminal sequences in GST-Rb (379-792) also reduced Pax-5 binding to the background level seen with GST alone, indicating that the pocket domain of Rb is not sufficient to mediate the interact ion with Pax-5 (compare Lanes 2 and 6). In contrast, the E1A protein bound with equal affinity to GST-Rb regardless of the presence and absence of the COOH-termina l sequences (Fig. 6C, Lanes 9 and 12), whereas both mutations in the pocket domain severely impaired the interact ion of Rb with E1A (Lanes 10 and 11) as descr ibed previously (54, 56, 57). Hence, the binding of Pax-5 to Rb depends not only on an intact pocket domain but also on the COOH-termina l sequences of Rb in clear contrast to transcription factors such as E1A.

Discussion

Identif ication o f the Partial H o m e o d o m a i n o f Pax-5 as a Protein Interaction Motif. Although the transcription factors of the Pax

family play essential roles in development, organogenesis, and disease (1), little is known about how these developmental regulators exert their function at the transcriptional level. Elucidation of their transcriptional role will depend not only on the identification of critical target genes but also on the characterization of partner proteins that regulate the transcriptional activity of Pax proteins. In this study, we have demonstrated that TBP and Rb directly interact with Pax-5 in

vivo and in vitro, possibly coupling this regulator to the basal transcription machinery and control of cell proliferation, respectively. The partial homeodomain of Pax-5 is essential for the binding of both TBP and Rb and, thus, constitutes a protein interaction motif. In agreement with this finding, the partial homeodomain is dispensable for DNA binding of Pax-5 (3) and is, furthermore, present in different members of the Pax-2/5/8 family from sea urchin to man (4, 5). Hence, this protein interaction motif has been highly conserved in evolution despite the fact that it is homologous only to one third of the entire homeodomain of other Pax proteins (Fig. 7). The homeodomain homology region encompasses helix I but excludes the sequences of the NH2-terminal arm, helix II, and the 'recognition' helix III (Fig. 7). Other transcription factors are also known to use their homeodomain for interacting with TBP or Rb. Oct- l /2 (15) and Msx-1 (58) both associate with TBP through their homeodomain, although in the case of Msx- 1, this interaction appears to be mediated by the NH2-terminal arm sequences that are absent in Pax-5. Recently, another member of the Pax family, Pax-3, has been shown to bind to Rb through helix I and II of its homeodomain (59), which suggests that this protein uses the same interaction motif identified here for Pax-5. It is, therefore, likely that all of the Pax proteins associate with Rb and/or TBP via helix I except for Pax-1 and Pax-9, which lack homeodomain sequences (1). Our mutational analysis of Pax-5 has ruled out a contri- bution of the NH2-terminal paired domain and COOH-terminal transactivation region to the binding of TBP and Rb. These experiments did not, however, address a possible involvement of the conserved octapeptide of Pax-5, which has indeed been implicated in Rb and TBP binding by recent deletion analysis (D. E., data not shown). Hence, the partial homeodomain of Pax-5 is necessary but not sufficient for the interaction with these two proteins.

The Pax-5-binding Domains o f T B P and Rb. TBP and Rb frequently recognize the same or overlapping sequences in different transcription factors such as PU.1 (37), E2F-1 (38, 46), c-Myc (45) and E1A (45). The association with the partial homeodomain of Pax-5 is yet another manifestation of the ability of TBP and Rb to contact the same interaction motif. The reason for the similar binding specificity resides in the significant sequence similarity that is shared between the COOH-terminal core domain of TBP and the pocket region of Rb (37). These homology regions of TBP and Rb are also essential for the interaction with Pax-5. Different transcription factors can, however, be distinguished by their precise requirement for interacting sequences in TBP and Rb. The entire core domain of TBP is necessary for the association of Oct- l /2 (15) and the Zta transactivator of the EBV virus (60), whereas the COOH-terminal 68 amino acids are dispensable for the binding of Pax-5, similar to the interaction with p53 (41), c-Rel (61), and E1A (36). Likewise, the pocket domain (A/B) of the Rb protein is sufficient for binding of most cellular transcription factors and viral oncoproteins that contain the charac- teristic LxCxE sequence as their Rb interaction motif (reviewed in Ref. 43). Consistent with the absence of this sequence element, Pax-5 additionally requires the COOH-terminal Rb sequences for efficient binding in analogy to the E2F transcription factors (62, 63). The consensus Rb recognition sequence of the E2F family members con- sists of 18 amino acids (64, 65) that, interestingly enough, shows some homology with the partial homeodomain of the Pax-2/5/8 proteins (Fig. 7) and the corresponding sequences of Pax-3 and Pax-6 (59).

1721s



I N T E R A C T I N G P R O T E I N S O F T H E T R A N S C R I P T I O N F A C T O R P A X - 5

Fig. 6. Binding of Pax-5 to the pocket domain and COOH-terminal sequences of Rb. A, schematic diagram of the GST-Rb fusion proteins (17). The structural organization of Rb is shown together with the relevant mutations: i.e., the Cys-to-Phe substitution at amino acid 706 (C706F), the deletion of exon 21 (AEx21), and the truncation of COOH-terminal sequences. The interaction of the different GST-Rb proteins with Pax-5 is summarized to the right, B, analysis of the purified GST-Rb proteins. Bacterially expressed GST and GST-Rb fusion proteins were purified on glutathione-Sepharose and analyzed by SDS-PAGE followed by staining with Coomassie blue. Asterisks denote the recombinant proteins of correct size. The migration of molecular mass standards (sizes given in kDa) is shown in Lane M. C. GST pull- down assay. In vitro translated Pax-5 protein was incubated with 2/xg of GST (Lane 2) or GST-Rb fusion proteins (Lanes 3-6) in buffer EBC1 supplemented with 0.1% NP40. After stringent washing, bound proteins were ehited by boiling, separated by SDS-PAGE, and analyzed for the presence of Pax-5 by immunoblotting with a polyclonal anti- Pax-5 antibody. In parallel, the same binding conditions were used for studying the interaction of in vitro translated ssS-E 1A protein (13S isoform) with the GST-Rb fusion proteins (Lanes 9-12). The 35S-E1A protein was detected by auto-radiography. Lanes 1 and 7 contained 10% of the input Pax-5 or E1A protein, respectively.

pocket domain I I

Rbl ~\\\\\\\\~ ~',,\\\\\\\\~ 1 393 573 645 772

GST-Rb (379-928) mr~,.-~i ~,,,\\\\\\\\.~ [~.,,,\\~.\\\\"~

I 928

Pax-5 interaction

+ + +

GST-Rb (C706F)

GST-Rb (AEx21)

GST-Rb (379-792)

706

703 737

~ : ~ " &\\ \ \ \ \ \ \~ ~\\\\\\\\"~1 379 792

GST-Rb 8 / /

~,~ : ....... kD

~ ~ ~'~ ~ ( 101

! .................. - 5 1

C / GST-Rb

Pax-5

1 2 3 4 5 6

- E I A

7 8 9 10 11 12

F u n c t i o n a l S i g n i f i c a n c e o f t h e I n t e r a c t i o n o f P a x - 5 w i t h T B P

a n d R b . The recent character izat ion o f Pax-5 target genes revea led

that this transcription factor can act as an act ivator or repressor

depending on the specif ic regula tory sequence context (6, 12). One of

the identif ied target genes is C D 1 9 which codes for a B-cel l surface

protein and contains, instead of a T A T A box, a high-aff ini ty Pax-5-

Pax-5 (AHD)

Pax-5

Pax-2

Pax-8

Pax-3

Pax-6

E2F-I

E2F-2

E2F-3

E2F-4

E2F-5

Consensus

228 partial homeodomain 254

KQMRGDL ......... deleted ......... EPIKPEQT

KHLRTDA ~ P Q SPSHTKGEQG

, [ I ' ' j J ,

t i ~ :: , ] J

LDYHFG:LE

DDYLWG~I~

GDYLLS~G

HDYIYN~D

DDYNFN~ DY i E .... r. E

IiJ i 'G~IR . . . . . . iD L F ' D

�9 nlE~vc ...... iD, r~ ~.~.'~vc- i i �9 . - .... ~Lr:~

.i'~..- ..... iDLF~

Fig. 7. Sequence conservation of the partial homeodomain in members of the Pax-2/5/8 family. The first 40 amino acids of the homeodomain of Pax-3 (78) and Pax-6 (79) are compared with the respective sequences of the human Pax-2 (80), Pax-5 (BSAP; Ref. 3), and Pax-8 (81) proteins. Gray overlay highlights those amino acids of the Pax-215/8 proteins that are identical to the corresponding residues of the Pax-3 and/or Pax-6 sequences. Brackets denote helix I and II of the paired-type homeodomain as determined by X-ray crystallographic analysis (82). The first 9 amino acids of Pax-3 and Pax-6 constitute the "NH2-terminal arm" of the homeodomain. The extent of sequence deletion in the Pax-5 (AHD) protein is indicated together with the respective amino acid positions. The Rb-binding domain present in the COOH-terminal transactivation region of E2F transcription factors (64, 65) shows some sequence homology with the paired-type homeodomain of Pax proteins, as shown by the sequence alignment according to Wiggan et al. (59). Dashes in the E2F sequence indicate a gap of 6 amino acids that was introduced for optimal alignment.

b inding site in the - 3 0 p romote r region (66). Transcript ion of the

C D 1 9 gene is entirely lost in B - l y m p h o c y t e s o f P a x - 5 ( - / - ) m i c e (8),

which suggests that Pax-5 funct ions to recruit the basal transcription

machinery to the C D 1 9 promoter . The identif icat ion o f an interaction

be tween Pax-5 and T B P now suggests a direct mechanis t ic link,

because T B P is an essential componen t of the basal transcription

factor TFIID, which mediates transcription initiation (29, 30).

Genet ic and funct ional analyses of Pax-5 have thus far fai led to

provide f i rm ev idence that R b is involved in control l ing the activity of

this transcription factor. Hence , it is more diff icult to speculate about

the funct ional s ignif icance o f the Rb-Pax-5 interaction. Rb is known

to p lay a dual role at both the transcriptional and cel lular level, as it

can s imul taneous ly repress the transcription o f genes involved in cell

cycle progress ion and act ivate the express ion of genes promot ing

terminal differentiat ion ( rev iewed in Refs. 4 2 - 4 4 ) . Rb exerts its

repress ion funct ion by different mechanisms. The interaction with Rb

is k n o w n to sequester and inactivate transcription factors such as E2F

by masking their t ransactivation domain and, thus, prevent ing access

of coact ivators and basal transcription factors such as TFI ID (46, 64).

In addition, Rb can act ively repress the activity of several transcrip-

tion factors b o u n d to the same promoter by prevent ing their interac-

tion with the transcription machinery (67). Moreover , the associat ion

o f Rb wi th certain transcription factors results in the recrui tment of

his tone deacetylase , which is able to si lence entire control regions by

p r o m o t i n g the format ion of inact ive chromat in ( 6 8 - 7 0 ) . Rb also

appears to repress the transactivat ion funct ion o f Pax-3, at least in

transiently t ransfected cells (59). By analogy, it is, therefore, conceiv-

able that Rb is involved in si lencing the 3' enhancer o f the immuno-

globl in heavy chain locus as well as in repressing the lymphoid-

specif ic PD-1 and J-chain genes, which are k n o w n to be under

negat ive control by Pax-5 (6, 12). In addit ion to its cell cyc le function,

Rb also plays an important role in deve lopment , as Rb ( - / - ) embryos

exhibi t defec t ive differentiat ion in several t issues including the lens,

1722s




n e r v o u s s y s t e m , a n d h e m a t o p o i e t i c p r e c u r s o r ce l l s ( 7 1 - 7 3 ) . C o n s i s t - 18.

en t w i t h i ts d i f f e r e n t i a t i o n f u n c t i o n , R b a s s o c i a t e s a n d c o o p e r a t e s w i t h

l i n e a g e d e t e r m i n a t i o n f a c t o r s s u c h as M y o D (74, 75) a n d m e m b e r s o f 19.

the C / E B P f a m i l y (76, 77). I n t e r e s t i n g l y , R b is ab le to p o t e n t i a t e the

D N A - b i n d i n g a n d t r a n s a c t i v a t i o n f u n c t i o n o f C / E B P p r o t e i n s d u r i n g 2o.

t e r m i n a l a d i p o c y t e d i f f e r e n t i a t i o n (76, 77) . C o n s e q u e n t l y , it is a l so

p o s s i b l e tha t R b p o s i t i v e l y r e g u l a t e s the a c t i v i t y o f P a x - 5 d u r i n g

B - c e l l a n d / o r C N S d e v e l o p m e n t . F ina l ly , it is i m p o r t a n t to n o t e tha t 21.

P a x - 5 is a c t i v a t e d as an o n c o g e n e b y a s p e c i f i c c h r o m o s o m a l t r ans -

l o c a t i o n in a s u b s e t o f n o n - H o d g k i n ' s l y m p h o m a s ( 9 - 1 1 ) . H e n c e , it

wi l l b e i n t e r e s t i n g to see w h e t h e r the d e r e g u l a t e d e x p r e s s i o n o f the 22.

o n c o p r o t e i n P a x - 5 c o n t r i b u t e s to t u m o r f o r m a t i o n b y i n t e r f e r i n g w i th

the n o r m a l f u n c t i o n o f the t u m o r s u p p r e s s o r p r o t e i n R b in a n a l o g y to 23.

v i ra l o n c o p r o t e i n s (42, 56 , 57).

Acknowledgments 24.

We thank L. Tora (Strasbourg) for providing the anti-TBP antibody, 25.

S. Mittnacht (London) for the GST-Rb and GST-p107 plasmids, T. Wirth

(Wtirzburg) for TBP expression vectors, P. Hamel (Toronto) for the pECE-

Ap34-HA plasmid, H. Stunnenberg (Nijmegen) for E1A expression vectors, 26.

and M. Cotten and P. Pfeffer for critical reading of the manuscript.

27. References

1. Mansouri, A., Hallonet, M., and Gruss, P. Pax genes and their roles in cell differen- 28. tiation and development. Curr. Opin. Cell Biol., 8: 851-857, 1996.

2. Krauss, S., Johansen, T., Korzh, V., and Fjose, A. Expression of the zebrafish paired 29. box gene pax[zf-b] during early neurogenesis. Development (Camb.), 113: 1193- 1206, 1991. 30.

3. Adams, B., Drrfler, P., Aguzzi, A., Kozmik, Z., Urbfinek, P., Maurer-Fogy, I., and Busslinger, M. Pax-5 encodes the transcription factor BSAP and is expressed in B 31. lymphocytes, the developing CNS, and adult testis. Genes Dev., 6:1589-1607, 1992.

4. Czerny, T., Bouchard, M., Kozmik, Z., and Busslinger, M. The characterization of novel Pax genes of the sea urchin and Drosophila reveal an ancient evolutionary 32. origin of the Pax2~5~8 family. Mech. Dev., 67: 179-192, 1997.

5. Pfeffer, P. L., Gerster, T., Lun, K., Brand, M., and Busslinger, M. Characterization of three novel members of the zebrafish Pax2~5~8 family: dependency of Pax5 and Pax8 33. expression on the Pax2.1 (noi) function. Development (Camb.), 125: 3063-3074, 1998. 34.

6. Busslinger, M., and Nutt, S. L. Role of the transcription factor BSAP (Pax-5) in B-cell development. In: 1. G. Monroe and E. V. Rothenberg (eds.), Molecular Biology of B-Cell and T-Cell Development, pp. 83-110. Totowa, NJ: Humana Press Inc., 1998.

7. Urbfinek, P., Wang, Z-Q., Fetka, I., Wagner, E. F., and Busslinger, M. Complete 35. block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell, 79: 901-912, 1994.

8. Nutt, S. L., Urb~nek, P., Rolink, A., and Busslinger, M. Essential functions of Pax5 36. (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoi- esis and reduced Vto-DJ recombination at the IgH locus. Genes Dev., 11: 476-491, 1997. 37.

9. Busslinger, M., Klix, N., Pfeffer, P., Graninger, P. G., and Kozmik, Z. Deregulation of PAX-5 by translocation of the E/x enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma. Proc. Natl. Acad. Sci. USA, 93: 6129-6134, 1996. 38.

10. Iida, S., Rao, P. H., Nallasivam, P., Hibshoosh, H., Butler, M., Louie, D. C., Dyomin, V., Ohno, H., Chaganti, R. S. K., and Dalla-Favera, R. The t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytoid lymphoma involves the 39. PAX-5 gene. Blood, 88: 4110-4117, 1996.

11. Morrison, A. M., JSger, U., Chott, A., Schebesta, M., Haas, O. A., and Busslinger, M. Deregulated PAXk5 transcription from a translocated IgH promoter in marignal zone 40. lymphoma. Blood, 92: 3865-3878, 1998.

12. Nutt, S. L., Morrison, A. M., Drrfler, P., Rolink, A., and Busslinger, M. Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of- function experiments. EMBO J., 17: 2319-2333, 1998. 41.

13. DSrfler, P., and Busslinger, M. C-terminal activating and inhibitory domains deter- mine the transactivation potential of BSAP (Pax-5), Pax-2 and Pax-8. EMBO J., 15: 1971-1982, 1996. 42.

14. Studier, W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol., 185: 60-89, 43. 1990.

15. Zwilling, S., Annweiler, A., and Wil-th, T. The POU domains of the Octl and Oct2 44. transcription factors mediate specific interaction with TBP. Nucleic Acids Res., 22: 1655-1662, 1994. 45.

16. Weigel, R. J., Devoto, S. H., and Nevin, J. R. Adenovirus 12S EIA gene represses differentiation of F9 teratocarcinoma cells. Proc. Natl. Acad. Sci. USA, 87." 9878- 9882, 1990.

17. Kaelin, W. G. J., Pallas, D. C., DeCaprio, J. A., Kaye, F. J., and Livingston, D.M. 46. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell, 64: 521-532, 1991.

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Hamel, P. A., Gill, R. M., Phillips, R. A., and Gallie, B. L. Transcriptional repression of the E2-containing promoters EIIaE, c-rnyc, and RB1 by the product of the RB1 gene. Mol. Cell. Biol., 12: 3431-3438, 1992. Brou, C., Chaudhary, S., Davidson, I., Lutz, Y., Wu, J., Egly, J-M., Tora, L., and Chambon, P. Distinct TFIID complexes mediate the effect of different transcriptional activators. EMBO J., 12: 489-499, 1993. Lee, W-H., Shew, J-Y., Hong, F. D., Sery, T. W., Donoso, L. A., Young, L-J., Bookstein, R., and Lee, E. Y-H. P. The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature (Lond.), 329: 642-645, 1987. Barberis, A., Widenhorn, K., Vitelli, L., and Busslinger, M. A novel B-cell lineage- specific transcription factor present at early but not late stages of differentiation. Genes Dev., 4: 849-859, 1990. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. Accurate transcription initiation by RNA polymerase lI in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res., 11: 1475-1489, 1983. Hoffmann, A., Sinn, E., Yamamoto, T., Wang, J., Roy, A., Horikoshi, M., and Roeder, R. G. Highly conserved core domain and unique N terminus with presump- tive regulatory motifs in a human TATA factor (TFIID). Nature (Lond.), 346: 387-390, 1990. Kao, C. C., Lieberman, P. M., Schmidt, M. C., Zhou, Q., Pei, R., and Berk, A. J. Cloning of a transcriptionally active human TATA binding factor. Science (Wash- ington DC), 248: 1646-1650, 1990. Peterson, M. G., Tanese, N., Pugh, B. F., and Tjian, R. Functional domains and upstream activation properties of a cloned human TATA binding protein. Science (Washington DC), 248: 1625-1630, 1990. Prickett, K. S., Amberg, D. C., and Hoop, T. P. A calcium-dependent antibody for identification and purification of recombinant proteins. Biotechniques, 7: 580-589, t989. Lai, J. S., and Herr, W. Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. Proc. Natl. Acad. Sci. USA, 89: 5658-6962, 1992. Heruandez, N. TBP, a universal eukaryotic transcription factor? Genes Dev., 7." 1291-1308, 1993. Goodrich, J. A., and Tjian, R. TBP-TAF complexes: selectivity factors for eukaryotic transcription. Curr. Opin. Cell Biol., 6: 403-409, 1994. Burley, S. K., and Roeder, R. G. Biochemistry and structural biology of transcription factor IID (TFIID). Annu. Rev. Biochem, 65: 769-799, 1996. Horikoshi, M., Yamamoto, T., Okhuma, Y., Weil, P. A., and Roeder, R. G. Analysis of structure-function relationship of yeast TATA box binding factor TFIID. Cell, 61: 1171-1178, 1990. Horikoshi, M., Wang, C. K., Fujii, H., Cromlish, J. A., Weil, P. A., and Roeder, R. G. Cloning and structure of a yeast gene encoding a general transcription initiation factor TFIID that binds to the TATA box. Nature (Lond.), 341: 299-303, 1989. Schmidt, M. C., Kao, C. C., Pei, R., and Berk, A. J. Yeast TATA-box transcription factor gene. Proc, Natl. Acad. Sci. USA, 86: 7785-7789, 1989. Cavallini, B., Faus, I., Matthes, H., Chipoulet, J. M., Winsor, B., Egly, J.-M., and Chambon, P. Cloning of the gene encoding the yeast protein BTFIY, which can substitute for the human TATA box-binding factor. Proc. Natl. Acad. Sci. USA, 86: 9803-9807, 1989. Buni, M., Tromvoukis, Y., Bopp, D., Frigerio, G., and Noll, M. Conservation of the paired domain in metazoans and its structure in three isolated human genes. EMBO J., 8: 1183-1190, 1989. Lee, W. S., Kao, C. C., Bryant, G. O., Liu, X., and Berk, A. J. Adenovirus EIA activation domain binds the basic repeat in the TATA box transcription factor. Cell, 67: 365-376, 1991. Hagemeier, C., Bannister, A. J., Cook, A., and Kouzarides, T. The activation domain of transcription factor PU. 1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFllB. Proc. Natl. Acad. Sci. USA, 90: 1580-1584, 1993. Hagemeier, C., Cook, A., and Kouzarides, T. The retinoblastoma protein binds E2F residues required for activation in vivo andTBP binding in vitro. Nucleic Acids Res., 21: 4998-5004, 1993. Xu, X., Prorock, C., Ishikawa, H., Maldonado, E., Ito, Y., and Grlinas, C. Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor liB. Mol. Cell. Biol., 13: 6733-6741, 1993. Metz, R., Bannister, A. J., Sntherland, J. A., Hagemeier, C., O'Rourke, E. C., Cook, A., Bravo, R., and Kouzarides, T. c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein. Mol. Cell. Biol., 14: 6021-6029, 1994. Liu, X., Miller, C. W., Koeffler, P. H., and Berk, A. J. The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFI1D, and a neighboring p53 domain inhibits transcription. Mol. Cell. Biol., I3: 3291-3300, 1993. Weinberg, R. A. The retinoblastoma protein and cell cycle control. Cell, 81: 323-330, 1995. Wang, J. Y. J. Retinoblastoma protein in growth suppression and death protection. Curr. Opin. Genet. Dev., 7: 39-45, 1997. Mulligan, G., and Jacks, T. The retinoblastoma gene family: cousins with overlapping interests. Trends Genet., 14: 223-229, 1998. Hoteboer, G., Timmers, H. T. M., Rustgi, A. K., Billaud, M., van 't Veer, L. J., and Bemards, R. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein. Proc. Natl. Acad. Sci. USA, 90: 8489-8493, 1993. Pearson, A., and Greenblatt, J. Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH. Oncogene, 15: 2643-2658, 1997.




47. Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature (Lond.), 334: 124-129, 1988.

48. Dyson, N., Dembski, M., Fattaey, A., Ngwa, C., Ewen, M., and Helin, K. Analysis of pl07-associated proteins: p107 associates with a form of E2f that differs from pRB-associated E2F-1. J. Virol., 67: 7641-7647, 1993.

49. Beijersbergen, R. L., Kerkhoven, R. M., Zhu, L., Cat-lee, L., Voorhoeve, P. M., and Bernards, R. E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo. Genes Dev., 15: 2680-2690, 1994.

50. Ginsberg, D., Vairo, G., Chittenden, T., Xiao, Z-X., Xu, G., Wydner, K. L., DeCaprio, 1. A., Lawrence, J. B., and Livingston, D. M. E2F-4, a new member of the E2F transcription factor family, interacts with p107. Genes Dev., 8: 2665-2679, 1994.

51. Mihara, K., Cat, X-R., Yen, A., Chandler, S., Driscoll, B., Murphree, A. L., T'Ang, A., and Fung, Y-K. T. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science (Washington DC), 246: 1300-1303, 1989.

52. Buchkovich, K., Duffy, L. A., and Harlow, E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell, 58:1097-1105, 1989.

53. Chen, P-L., Scully, P., Shew, J-Y., Wang, J. Y., and Lee, W-H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell, 58: 1193-1198, 1989.

54. Kaye, F. J., Kratzke, R. A., Gerster, J. L., and Horowitz, J. M. A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding. Proc. Natl. Acad. Sci. USA, 87: 6922-6926, 1990.

55. Bookstein, R., Shew, J-Y., Chen, P. L., Scully, P., and Lee, W-H. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science (Washington DC), 247: 712-715, 1990.

56. Hu, Q., Dyson, N., and Harlow, E. The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations. EMBO J., 9: 1147-1155, 1990.

57. Kaelin, W. G. J., Ewen, M. E., and Livingston, D. M. Definition of the minimal simian virus 40 large T antigen- and adenovirus E1A-binding domain in the retinoblastoma gene product. Mol. Cell. Biol., 10: 3761-3769, 1990.

58. Zhang, H., Catron, K. M., and Abate-Shen, C. A role of the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression. Proc. Natl. Acad. Sci. USA, 93: 1764-1769, 1996.

59. Wiggan, O., Taniguchi-Sidle, A., and Hamel, P. A. Interaction of the pRB-family proteins with factors containing paired like homeodomains. Oncogene, 16: 227-236, 1998.

60. Lieberman, P. M., and Berk, A. J. The Zta trans-activator protein stabilizes TFIID association with promoter DNA by direct protein-protein interaction. Genes Dev., 5: 2441-2454, 1991.

61. Kerr, L. D., Ransone, L. J., Wamsley, P., Schmitt, M. J., Boyer, T. G., Zhou, G., Berk, A. J., and Verma, I. M. Association between proto-oncoprotein Rel and TATA- binding protein mediates transcriptional activation of NF-KB. Nature (Lond.), 365: 412-419, 1993.

62. Huang, S., Shin, E., Sheppard, K. A., Chokroverty, L., Shan, B., Quian, Y. W., Lee, E. Y., and Yee, A. S. The retinoblastoma protein region required for interaction with the E2F transcription factor includes the T/EIA binding and carboxy-terminal sequences. DNA Cell Biol., 1l: 539-548, 1992.

63. Hiebert, S. W., Chellappan, S. P., Horowitz, J. M., and Nevins, J. R. The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev., 6: 177-185, 1992.

64. Helin, K., Lees, J. A., Vidal, M., Dyson, N., Halow, E., and Fattaey, A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell,

70: 337-350, 1992. 65. Shah, B., Durfee, T., and Lee, W.-H. Disruption of RB/E2F-1 interaction by single

point mutations in E2F-1 enhances S-phase entry and apoptosis. Proc. Natl. Acad. Sci. USA, 93: 679-684, 1996.

66. Kozmik, Z., Wang, S., Drrfler, P., Adams, B., and Busslinger, M. The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. Mol. Cell. Biol., 12: 2662-2672, 1992.

67. Weintraub, S. J., Chow, K. N. B., Luo, R. X., Zhang, S. H., He, S., and Dean, D. C. Mechanism of active transcriptional repression by the retinoblastoma protein. Nature (Lond.), 375: 812-815, 1995.

68. Luo, R. X., Postigo, A. A., and Dean, D. C. Rb interacts with histone deacetylase to repress transcription. Cell, 92: 463-473, 1998.

69. Magnaghi-Jaulin, L., Groisman, R., Naguibneva, i., Robin, P., Lorain, S., Le Villain, J. P., Troalen, F., Trouche, D., and Harel-Bellan, A. Retinoblastoma protein represses transcription by recruiting a histone deacetylase. Nature (Lond.), 391: 601-605, 1998.

70. Brehm, A., Miska, E. A., McCance, D. J., Reid, J. L., Bannister, A. J., and Kouzar- ides, T. Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature (Lond.), 391: 597-601, 1998.

71. Jacks, T., Fazeli, A., Schmitt, E. M., Bronson, R. T., Goodell, M. A., and Weinberg, R. A. Effects of an Rb mutation in the mouse. Nature (Lond.), 359: 295-300, 1992.

72. Lee, E. Y-H. P., Chang, C-Y., Hu, N., Wang, Y-C. J., Lai, C-C., Herrup, K., Lee, W-H., and Bradley, A. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature (Lond.), 359: 288-294, 1992.

73. Clarke, A. R., Maandag, E. R., van Roon, M., van der Lugt, N. M. T., van der Valk, M., Hooper, M. L., Berns, A., and te Riele, H. Requirement for a functional Rb-1 gene in murine development. Nature (Lond.), 359: 328-330, 1992.

74. Gu, W., Schneider, J. W., Condorelli, G., Kaushal, S., Mahdavi, V., and Nadal- Ginard, B. Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation. Cell, 72: 309-324, 1993.

75. Schneider, J. W., Gu, W., Zhu, L., Mahdavi, V., and Nadal-Ginard, B. Reversal of terminal differentiation mediated by p107 in R b - / - muscle cells. Science (Wash- ington DC), 264: 1467-1471, 1994.

76. Chen, P-L., Riley, D. J., Chen-Kiang, S., and Lee, W-H. Retinoblastoma protein directly interacts with and activates the transcription factor NF-IL6. Proc. Natl. Acad. Sci. USA, 93: 465-469, 1996.

77. Chen, P-L., Riley, D. J., Chen, Y., and Lee, W-H. Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs. Genes Dev., 10: 2794-2804, 1996.

78. Goulding, M. D., Chalepakis, G., Deutsch, U., Erselius, ,l. R., and Gruss, P. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J., 10: 1135-1147, 1991.

79. Ton, C. C. T., Hirvonen, H., Miwa, H., Weil, M. M., Monaghan, P., Jordan, T., van Heyningen, V., Hastie, N. D., Meijers-Heijboer, H., Drechsler, M., Royer-Pokora, B., Collins, F., Swaroop, A., Strong, L. C., and Saunders, G. F. Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region. Cell, 67: 1059-1074, 1991.

80. Eccles, M. R., Wallis, L. J., Fidler, A. E., Spurr, N. K., Goodfellow, P. J., and Reeve, A. E. Expression of the PAX2 gene in human fetal kidney and Wilms' tumor. Cell Growth Diff., 3: 279-289, 1992.

81. Kozmik, Z., Kurzbauer, R., D6rfler, P., and Busslinger, M. Alternative splicing of Pax-8 gene transcripts is developmentally regulated and generates isoforms with different transactivation properties. Mol. Cell. Biol., 13: 6024-6035, t993.

82. Wilson, D. S., Guenther, B., Desplan, C., and Kuriyan, J. High resolution crystal structure of a paired (Pax) class cooperative homeodomain dimer on DNA. Cell, 82: 709-719, 1995.

Discussion

T h e top ic o f Dr . B u s s l i n g e r ' s p r e s e n t a t i o n wi l l be p u b l i s h e d shor t ly , and, h e n c e , the r e a d e r is r e f e r r e d to the f o l l o w i n g m a n u s c r i p t fo r de t a i l ed

i n f o r m a t i o n : Nut t , S. L. , V a m b r i e , S., S te in le in , P,, Ro l ink , A., K o z m i k , Z., We i th , A., and Buss l i nge r , M. I n d e p e n d e n t r e g u l a t i o n o f the t w o

Pax -5 a l le les d u r i n g B - c e l l d e v e l o p m e n t . N a t u r e G e n e t . , in press , 1999.

Dr. Ph i l l ip Sharp: L e t m e ask the f i rs t ques t ion , As fa r as I k n o w ,

the a l l e l e - s p e c i f i c r e g u l a t i o n o f P a x - 5 is an u n p r e c e d e n t e d m e c h a n i s m

o f g e n e r e g u l a t i o n in m a m m a l i a n cells . As y o u o u t l i n e d in the last

s l ide, this r e g u l a t o r y m e c h a n i s m is d i s t i n g u i s h a b l e f r o m i m p r i n t i n g b y

b e i n g r e v e r s i b l e in t e rms o f sw i t ch ing w i t h i n the ce l l l ineage . C o u l d

y o u c o m m e n t o n th is?

Dr. B u s s l i n g e r : Yes , as far as I can tell , this s e e m s to be the f irs t

e x a m p l e o f this k ind o f t r ansc r ip t i ona l r egu l a t i on . It is, h o w e v e r ,

i m p o r t a n t to r ea l i ze tha t it is qu i te d i f f i cu l t to d e m o n s t r a t e this

p h e n o m e n o n f o r a g i v e n gene . W e w e r e e x t r e m e l y l u c k y to h a v e

i den t i f i ed a t a rge t g e n e w h o s e e x p r e s s i o n is to ta l ly d e p e n d e n t on the

t r an sc r i p t i on f a c t o r Pax-5 . S u c h t a rge t g e n e s are n o r m a l l y no t avai l -

ab le f o r o t h e r t r an sc r i p t i on fac tors . H e n c e , m i c e h e t e r o z y g o u s fo r

m u t a t i o n s in o t h e r t r ansc r ip t i on f ac to r g e n e s c a n n o t be u s e d fo r

a n a l y s e s s im i l a r to t hose o f the P a x - 5 m u t a n t m o u s e .

Dr. Sharp: B u t do y o u th ink that the a l l e l e - spec i f i c r e g u l a t i o n

d e s c r i b e d by y o u m i g h t be m o r e c o m m o n ?

Dr. Bus s l i nger : I am cer ta in that this is a m o r e gene ra l p h e n o m e -

non . I w o u l d no t be surpr i sed , if, fo r e x a m p l e , the g e n e c o d i n g fo r the

o s t e o b l a s t - s p e c i f i c t r an sc r i p t i on f ac to r Cbfa -1 (Osf -2 ) s h o w e d a s im-

i lar m o n o a l l e l i c t r ansc r ip t ion pa t te rn . H o w e v e r , I h a v e to r e m i n d y o u

h o w d i f f i cu l t it is to d e m o n s t r a t e a l l e l e - spec i f i c r e g u l a t i o n in gene t i -

ca l ly u n m a n i p u l a t e d cel ls , e v e n i f the two a l le les can be d i s t i n g u i s h e d

b y a n u c l e o t i d e p o l y m o r p h i s m in the t r a n s c r i b e d reg ion . D u e to the

r eve r s ib i l i t y o f the p h e n o m e n o n , it is i m p o s s i b l e to g r o w a s ing le ce l l

in to a c o l o n y to d e m o n s t r a t e a l l e l e - spec i f i c t r ansc r ip t i on by r e v e r s e

t r a n s c r i p t i o n - P C R ana lys i s . B y the t ime the c o l o n y has b e e n ex-

p a n d e d , the e x p r e s s i o n f r o m b o t h a l le les has b e e n r a n d o m i z e d . Th i s is

the m a i n r e a s o n w h y all o u r ana lyses had to be p e r f o r m e d at the

s ing le -ce l l level ,

1724s




Speaker: You have partly answered my question already. I was just wondering if you felt that this regulation might apply to many genes which are expressed at a low level. In other words, a low level of transcription might always tend to be monoallelic by chance.

Dr. Busslinger: Let me rephrase your question. Essentially you would like to know the mechanism underlying the allele-specific transcriptional regulation. At present, I think that we do not have to invoke a grand novel mechanism to explain this phenomenon. Low- level transcription may indeed mean that only one allele is selected by stochastic choice to be transcribed at an early point in development. This allele then continues to be expressed, unless switching of expression to the second allele occurs. One could argue that the revers- ibility of allele-specific transcription may be responsible for the haploinsufficient phenotypes of Pax genes. It is conceivable that the switch of expression from the initially transcribed wild-type allele to the mutant allele may adversely affect the development of a tissue. A cell which undergoes such a switch past the lineage commitment stage may not be able to further participate in normal development. In the case of Pax-5, we know from conditional gene inactivation experiments that this transcription factor is indeed required from the begin- ning to the end of B cell development. A similar situation may also apply to other Pax genes.

Speaker." I am a bit surprised about your findings. Many genes are known to be involved in lineage determination of cells, but only few of them seem to be haploinsufficient. One could argue that there is an evolutionary strategy to avoid haploinsufficiency. What are your thoughts on this issue?

Dr. Busslinger: Let me get the following point straight. Pax-5 is haploinsufficient at the cellular level, as I presented it in my talk. However, Pax-5 does not cause a clinical syndrome, probably due to a peculiarity of the B-lymphoid system. B cells are either selected for expansion or are deleted depending on whether they have undergone in-frame or out-of-frame rearrangements of immunoglobulin genes. In such a system, a 2-fold difference in cell number will not result in a phenotypic manifestation. However, this situation may be different for lineage determination genes expressed in solid tissues.

Speaker: Well, the lymphoid system is not the only one where lineage decisions are taking place. So the issue still is that very few genes involved in lineage determination seem to be haploinsufficient.

Dr. Busslinger: Yes, but then again it is known even for the Pax gene family that haploinsufficiency does not affect every tissue where a given Pax gene is expressed. As I have shown in my presentation, the Pax-5 gene proceeds from a monallelic to a biallelic transcription mode during B cell development. In analogy it is, therefore, possible that other Pax genes may also be biallelically transcribed in some

tissues where no haploinsufficient phenotype is seen. A similar situation may also hold true for other lineage determination genes.

Dr. Nicholas Hastie: A couple of questions: First, as you men- tioned, Pax-5 does not necessarily show a haploinsufficient phenotype, while other Pax genes do. What do you know about monoallelic expression of other Pax genes?

Dr. Busslinger: We did not perform any experiments with other Pax genes. The groups of Dr. Howard Cedar and Dr. Andrew Chess apparently have experimental data indicating that the Pax-3 and Pax-6 genes are also subject to monoallelic transcriptional regulation. As I have never seen these data, I cannot further comment on this issue.

Dr. Nicholas Hastie: The other question relates to the point made earlier about whether many genes, which are expressed at a low level, go through some stochastic program of transcriptional regulation. This observation may be relevant also for cancer, the actual topic of this meeting. For instance, a tumor suppressor gene could be transcribed from the inherited mutant allele during a window, where the wild-type allele is not expressed. There may be considerable selective advantage for such monoallelically transcribing cells to lose the second allele. From this point of view, the phenomenon of allele-specific regulation could have broader significance.

Dr. Busslinger: I agree that this is a possibility. Dr. Suzanne Cory: I was fascinated by your talk and am coming

back to the mechanism, following on from your answer to Jerry Adams. You have observed a progression from monoallelic to biallelic to monoallelic Pax-5 expression during B cell development. Do these transitions correlate with the concentration of Pax-5 during B cell development. In other words, does the level of Pax-5 start off low, then increase and go back down low again?

Dr. Busslinger: This is a difficult question to answer, as it is not easy to distinguish a 2-fold difference in transcription factor concentration. Interestingly, however, the expression of the CD19 gene, which is strictly dependent on Pax-5 function, shows an inverse correlation relative to Pax-5. Immature B cells express less CD19 on the surface compared to the early pro-B and mature B cells. In other words, higher Pax-5 expression levels correlate with lower CD19 expression. In this context I would like to mention the elegant experiments performed in Nick Hastie's laboratory that clearly demonstrated a narrow dosage sensitivity of Pax-6. Moderate overexpression of the Pax-6 gene due to extra copies on a wild-type background resulted in as similar Small eye phenotype in the mouse as mutation of one of the two endogenous alleles. It is, therefore, a possibility that the dosage sensitivity and allele-specific regulation of Pax genes may be related phenomena.

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1999;59:1716s-1725s. Cancer Res Dirk Eberhard and Meinrad Busslinger TATA-binding Proteins(BSAP) Is an Interaction Motif for the Retinoblastoma and The Partial Homeodomain of the Transcription Factor Pax-5

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