DNA Binding and Transcriptional Activation by a PDX1·PBX1b·MEIS2b Trimer and Cooperation with a Pancreas-specific Basic Helix-Loop-Helix Complex*

Ying Liu, Raymond J. MacDonald, and Galvin H. SwiftDagger

From the Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148

Received for publication, January 24, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In pancreatic acinar cells, the HOX-like factor PDX1 acts as part of a trimeric complex with two TALE class homeodomain factors, PBX1b and MEIS2b. The complex binds to overlapping half-sites for PDX1 and PBX. The trimeric complex activates transcription in cells to a level about an order of magnitude greater than PDX1 alone. The N-terminal PDX1 activation domain is required for detectable transcriptional activity of the complex, even though PDX1 truncations bearing only the PDX1 C-terminal homeodomain and pentapeptide motifs can still participate in forming the trimeric complex. The conserved N-terminal PBC-B domain of PBX, as well as its homeodomain, is required for both complex formation and transcriptional activity. Only the N-terminal region of MEIS2, including the conserved MEIS domains, is required for formation of a trimer on DNA and transcriptional activity: the MEIS homeodomain is dispensable. The activity of the pancreas-specific ELA1 enhancer requires the cooperation of the trimer-binding element and a nearby element that binds the pancreatic transcription factor PTF1. We show that the PDX1· PBX1b·MEIS2b complex cooperates with the PTF1 basic helix-loop-helix complex to activate an ELA1 minienhancer in HeLa cells and that this cooperation requires all three homeoprotein subunits, including the PDX1 activation domain.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The HOX transcription factors are key mediators for establishing the embryonic body plan and guiding organogenesis (1-3). The many members of this large gene family have distinct roles in development, despite having very similar DNA binding specificities and overlapping patterns of expression. At least part of the increased binding specificity required to distinguish the roles of the various HOX proteins comes from their interaction with members of the TALE class of homeodomain proteins (for review see Ref. 4). The TALE proteins are characterized by the presence of a 3-amino acid loop extension between alpha -helices 1 and 2 of the homeodomain (5) and include both the PBC class (mammalian PBX proteins, Drosophila Extradenticle, and Caenorhabditis elegans Ceh-20 (6)) and the MEIS-like TALE factors (mammalian MEIS and PREP1 proteins, Drosophila Homothorax (HTH), C. elegans Ceh-25 (5, 7)). Other homeodomain proteins important in development also form complexes with the TALE proteins, including the pancreas/duodenal homeobox-1 protein (PDX1) (8-10).

Pdx1 is a member of the PARAHOX cluster of homeobox genes implicated in the development of endoderm-derived organs (11). Pdx1 is essential to the development of both the endocrine and exocrine compartments of the pancreas (12-14). The interaction of PDX1 with PBX is required for the expansion of the various endocrine and exocrine cell types during development (15). Although Pdx1 is expressed throughout the pancreas during embryonic development, its expression is predominantly localized to the beta -cells of the islets of Langerhans of adult mammals (16, 17). PDX1 has been implicated in the transcriptional control of a number of beta -cell-specific genes, including insulin (16, 18), glucokinase (19), islet amyloid polypeptide (20, 21), and glucose transporter type 2 (22). PDX1 also participates in the activation of an acinar cell-specific gene, elastase 1 (ELA1); in this instance it acts as part of a trimeric complex with PBX1b and MEIS2 (10).

Our previous studies identified the PDX1·PBX1b·MEIS2 trimer as one of two pancreatic transcription factor complexes that cooperate in the transcriptional activation of the ELA1 enhancer in acinar cells (23). The ELA1 enhancer comprises three functional elements (A, B, and C) within 100 bp1 (24). The proper acinar activity of the enhancer requires the synergistic cooperation between the A and B elements (25, 26). The PDX1·PBX1b·MEIS2 complex binds and mediates the acinar activity of the B element (10). The other factor, PTF1, is a bHLH acinar cell-specific complex that binds and mediates the activity of the A element (24, 27).2 PTF1 is essential for the formation of the exocrine pancreas (28) as well as for the transcription of the digestive enzyme genes (24, 29).

In this report, we show which domains of PDX1, PBX1b, and MEIS2b are essential for the formation of the trimeric complex, for binding of the complex to DNA, for transcriptional activity, and for cooperative activation with PTF1. PDX1 contributes the only transcriptional activation domain, even though the activity of the trimer is an order of magnitude greater than that of PDX1 alone. PDX1 recruits PBX1b, and they bind in tandem to adjacent PDX1 and PBX half-sites. PBX1b recruits MEIS2b to the DNA-bound complex. MEIS2b is required for both the transcriptional activity of the PDX1·PBX1b·MEIS2 trimer and its cooperation with PTF1.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oligonucleotides-- The ELAI B element has been described previously (10); the top strand sequences of the B element and its derivatives B2 and B7 are shown in Fig. 1A. The CRS1 element from the bovine CYP17 gene has the top strand sequence of CCTCGAGACGTTGATGGACAGTGAGCAAGG, and the bottom strand sequence of TCGACCTTGCTCACTGTCCATCAACGTCTCGAGGAGCT; the adjoining PBX and MEIS binding sites are underlined (30).

Antibodies-- Polyclonal rabbit antibodies against PDX1, PBX, PBX1, and MEIS2 have been described previously (10). The polyclonal rabbit antiserum specific for the a and b isoforms of MEIS2 proteins were raised against an 18-amino acid peptide, PMSGMGMNMGMDGQWHYM, corresponding to amino acids 377-394 of MEIS2b. Similarly, a 16-amino acid peptide, SVDPNVGGQVMDIHAQ, spanning amino acids 455-470 of MEIS2d, was used to produce the anti-MEIS2c/d. A 7-amino acid insert, GFLLDPS, corresponding to amino acids 346-352 of MEIS2a and MEIS2c, is specifically present in these two isoforms. The antibody alpha MEIS2a/c was raised against a 14-amino acid peptide containing a tandem repeat of the insert. All of the MEIS isoform-specific antibodies were raised against peptide sequences common to mouse and human orthologues.

DNA Constructs-- The B element reporter construct (5B.EIp.hGH) has five head-to-tail copies of the B element (Fig. 1A) linked to the Ela1 minimal promoter from -92 to +8 and fused to an hGH reporter gene (10). In the B2 and B7 multimer constructs, sense strand and antisense strand oligonucleotides containing five copies of the B2 or B7 elements (Fig. 1A) were annealed and cloned into the Ela1 minimal promoter/hGH reporter plasmid. The mini-enhancer construct 3(BA).EIp.hGH (26) has three head-to-tail copies of adjacent B and A elements (PDX1·PBX1b·MEIS2b and PTF1 binding sites, respectively) from the Ela1 gene inserted in front of the Ela1 promoter. The Pdx1.luciferase reporter plasmid, used as an internal control in RIN1046-38 cell transfections, has 4.5 kb of 5'-flanking sequence from the Mus musculus Pdx1 gene (GenBankTM accession number AF192495; a gift of Dr. Chris Wright, Nashville, TN) inserted upstream of the luciferase gene in pGL3basic (Promega, Madison, WI).

The deletion series of PDX1, PBX1b, and MEIS2b were created by polymerase chain reaction amplification of the corresponding coding regions and cloning of the fragments into expression vectors. The full-length and PDX1 deletion cDNAs were cloned into pcDNA3.1/V5/His TOPO (Invitrogen Co., Carlsbad, CA). The encoded PDX1 proteins contain V5 and His epitope tags at their C termini. To synthesize the PBX1b deletion series by in vitro transcription and translation (IVT), the cDNAs were cloned into FLAG-pSP65, thereby adding a FLAG-tag at the N terminus of each protein. Each PBX1b cDNA, together with the N-terminal FLAG-tag, was transferred into pcDNA1.1/Amp (Invitrogen) for expression in transfected cells. The MEIS2b deletion series was cloned into pET-28b (Novagen, Madison, WI) under the control of the T7 promoter for IVT. Tandem His and T7 epitope tags were included at the N terminus of each MEIS2b protein. For expression in transfected cells, the MEIS2b cDNAs, together with the His and T7 tags, were transferred into pcDNA1.1/Amp. An IVT expression plasmid for MEIS2d was generated by polymerase chain reaction amplification of Meis2d cDNA using Meis2d/pBS.SK- (a gift of Professor Pierre Chambon, CNRS-INSERM, Strasbourg, France) as template, and the insert was cloned into pET-28b. The cDNA for the mouse p48 subunit of PTF1 (a gift of Dr. Scott Rose, University of Texas Southwestern, Dallas) and the cDNA for HEB (a gift of Dr. Richard Baer, New York, NY) were transferred to pcDNA1.1/Amp for transfection experiments.

Electophoretic Mobility Shift Assays-- Procedures for preparing nuclear extracts, in vitro translation (IVT), EMSA, and antibody supershift assays have been described previously (10). In all EMSA reactions containing nuclear extract from 266-6 cells, a GATA binding site oligonucleotide beta G1 (31) was added to compete with GATA4 binding to the B or mutant B elements. IVT proteins were incubated at 37 °C for 15 min prior to the standard EMSA protocol.

Immunological Techniques-- Western blot analyses were conducted as previously described (10). 30 µg of nuclear extracts, or 0.25 µl of the rabbit reticulocyte lysates containing the IVT proteins were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and electrophoretically transferred onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford MA). Immune complexes were detected by enhanced chemiluminescence with Supersignal West (Pierce, Inc., Rockford, IL). The exocrine cell lines were: 266-6 (ATCC CRL-2151) (32), C5-2E, (10), ARIP (ATCC CRL-1674) (33), and AR4-2J (ATCC CRL-1492) (33). The beta -cell lines were: beta TC3 (34), Ins1 (35), and RIN1046-38 (36).

Co-immunoprecipitation of the PBX and MEIS proteins was performed as follows. 10 µl of reticulocyte lysate containing equimolar amounts of 35S-labeled IVT protein and the unlabeled IVT partner was incubated at 37 °C for 40 min. 5 µl of polyclonal antibody specific for the non-labeled protein was added, and the reaction volume was increased to 300 µl by adding buffer containing 10 mM HEPES (pH 7.6), 250 mM NaCl, 0.1% Nonidet P-40, and 5 mM EDTA. After gentle mixing by rotation at 4 °C for 1 h, 20 µl of protein A beads (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was added, and the mixing was continued overnight. Following the incubation, the beads were collected and washed, then resuspended in SDS-gel loading buffer. Samples were separated by SDS-10% polyacrylamide gel electrophoresis, and the 35S-labeled IVT proteins were detected with a PhosphorImager (Molecular Dynamics, Santa Clara, CA).

Cell Transfection-- Transfections of the RIN1046-38 beta -cell line (RIN-38) (36) and HeLa (ATCC CCL-2) were performed with FuGene (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions. All of the plasmids for the expression of transcription factors, as well as the beta -galactosidase internal control plasmid (pCMVbeta , CLONTECH, Palo Alto, CA) utilized the CMV enhancer/promoter. All transfections contained the same total amount of CMV enhancer/promoter, balanced as necessary by the addition of insertless CMV vector (e.g. pcDNA1.1/Amp). The activity of the hGH reporter gene was assayed by radioimmunoassay (Nichols Institute, San Juan Capistrano, CA). Transfections of HeLa cells were corrected for relative efficiency based on the activity of the cotransfected beta -galactosidase reporter assayed with a Galacto-Light Plus kit (Tropix Inc., Bedford, MA). Transfections of RIN1046-38 cells were corrected for relative efficiency based on the activity of a cotransfected Pdx1.luciferase reporter, in which the luciferase reporter gene was under the transcriptional control of the Pdx1 gene promoter sequence. Luciferase activity was assayed with a kit from Promega (Madison, WI). Transfection results reported are the mean and standard error of a minimum of four transfections, except as noted. For those deletion plasmids of PDX1, PBX1b, and MEIS2b that had significantly less than wild type activity in transfections, we confirmed the production of protein and its nuclear localization by immunohistochemical staining using monoclonal antibodies recognizing their peptide tags (PDX1 with anti-V5, Invitrogen, Carlsbad, CA; PBX1b with anti-FLAG, Sigma Chemical Co., St Louis, MO; MEIS2b with anti-T7, Novagen, Madison, WI). All were expressed and localized to the nucleus at approximately wild type levels, except for PDX116-206, which was found only in the cytoplasm (data not shown).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Trimeric Complex Binds Only to Sites Containing Both PDX1 and PBX Half-sites-- We previously identified a trimeric homeoprotein complex that binds to and mediates the activity of the B element of the elastase I (ELA1) enhancer in pancreatic acinar cell lines (10). The complex consists of a pancreatic HOX-like protein, PDX1, and two TALE-class homeodomain proteins, PBX1b and an isoform of MEIS2/MRG1. The trimeric complex binds an 11-bp region of the B element containing overlapping half-sites for PDX1 and PBX but no recognizable site for MEIS (Fig. 1A). Mutations in either the PDX1 or PBX half-site eliminate trimer binding (10). The central role of PDX1 in trimer binding was demonstrated by mutation of a single nucleotide in the PDX1 half-site of the B element (the A at position 7 of the PBX-HOX consensus): neither PDX1 nor the PDX1-containing trimeric complex from 266-6 acinar cell nuclear extracts bound detectably to the oligonucleotide containing this A to C transversion (B7, Fig. 1B) and this B7 oligonucleotide was unable to compete for trimer binding in EMSA competition assays (Fig. 1C). These results confirmed the binding of PDX1 to its half-site and demonstrated that the formation of the trimeric complex is dependent on an intact PDX1 half-site.


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Fig. 1.   An acinar homeoprotein complex contains PDX1, PBX1b, and MEIS2 and requires both PDX1 and PBX half-sites for DNA binding. A, sequences of the ELA1 B element and derivatives B2 and B7. The PBX and HOX/PDX1 binding half-sites are in boldface and numbered, with the consensus binding sequence shown on top. Dots indicate identical residues. B, equal amounts of 266-6 nuclear extract were analyzed for B, B2, and B7 binding by EMSA. The PDX1 and trimer complexes are indicated. C, binding of the trimer complex can be effectively competed by B2 and B oligonucleotides, but not by B7 oligonucleotides. 30- or 150-fold excess unlabeled oligonucleotides were added as competitors in the EMSA reactions as indicated. D, antisera against PDX1, PBX, and MEIS2 eliminated the trimer complex bound to the B or B2 elements. Anti-PDX1 also eliminated the PDX1 complexes with the B and B2 elements.

To be able to study the structure of the trimeric complex, it was necessary to increase the PBX binding affinity of the B element without altering the qualitative nature of the activity of the element in cells. The binding affinity is low, because nucleotides at positions 1, 2, and 6 differ from the consensus binding sequence for PBX/HOX heterodimers (37, 38). Changing the A at position 2 to T matches the consensus PBX half-site (B2, Fig. 1A) and increased the binding of the trimeric complex from acinar cell nuclear extracts without affecting the binding of the PDX1 monomer (Fig. 1B). In competition assays the B2 element was ~5-fold more effective than the unmodified B element (Fig. 1C). Antisera specific for PDX1, PBX, and MEIS2 eliminated the B and B2 complexes with equal effectiveness (Fig. 1D), confirming that the homeodomain protein composition is the same for the two complexes. The transcriptional activities of B and B2 in transfected cells were also qualitatively the same (see Fig. 4, below). We subsequently used the more effective B2 element to test the ability of various forms of the three transcription factors to form complexes on DNA and activate transcription.

The Trimeric Complex Is Composed of PDX1, PBX1b, and MEIS2b/MEIS2d-- To determine which of four MEIS2 isoforms are present in the trimeric complex, we developed antisera against distinguishing peptide regions of the isoforms. Four alternatively spliced variants of Meis2 mRNA encode four distinct protein products: 2a, 2b, 2c, and 2d (39). MEIS2a and 2b have a different C-terminal tail (18 amino acids) than MEIS2c and 2d (94 amino acids); MEIS2a and 2c share a common seven amino acid insert, GFLLDPS, which MEIS2b and 2d lack. The antiserum specific for MEIS2a/b supershifted most of the trimeric complex from acinar cell extracts, leaving behind a residual complex of slightly slower electrophoretic mobility (Fig. 2A, lane 3). The antiserum specific for MEIS2c/d decreased the amount of the trimeric complex slightly (lane 5 versus lane 4), although a supershifted band was not observed. Antiserum against the MEIS2a/c-specific heptapeptide did not reduce the amount of the trimeric complex more than the preimmune serum (lanes 6 and 7). These results indicate that MEIS2b and possibly MEIS2d are present in the complex, but MEIS2a and 2c are not. To determine whether the residual slower mobility complex remaining after treatment with alpha MEIS2a/b antiserum contained MEIS2d, nuclear extract was treated with a mixture of alpha MEIS2a/b and alpha MEIS2c/d antisera (Fig. 2B, lane 4). The combination eliminated all of the trimeric complex. In contrast, combining alpha MEIS2a/b and alpha MEIS2a/c did not eliminate the slower migrating complex (lane 6), further showing that MEIS2c is not in the trimeric complex.


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Fig. 2.   The MEIS2 isoforms in the acinar trimeric complex are MEIS2b and MEIS2d. A, supershift EMSA using isoform-specific MEIS2 antisera or the corresponding preimmune sera (PI) showed that MEIS2b and possibly MEIS2d are in the trimeric complex from 266-6 nuclear extract. B, supershift EMSA with mixtures of MEIS2 isoform-specific antisera confirmed that both MEIS2b and MEIS2d were in the 266-6 complex (lanes 1-6). Reconstituted trimeric complexes containing IVT PDX1, PBX1b, and MEIS2b (lane 7) or MEIS2d (lane 8) had electrophoretic mobilities similar to the 266-6 acinar trimeric complex. Mobilities of the trimeric complexes containing MEIS2b or MEIS2d are indicated by the short lines. The band of intermediate mobility in the reconstituted complexes (lanes 7 and 8) is the PBX1·PDX1 heterodimer.

PDX1, PBX1b, and MEIS2 proteins synthesized by translation in vitro can form trimeric complexes on DNA (Fig. 2B, lanes 7 and 8). The complex formed with recombinant PDX1·PBX1b·MEIS2b comigrates with the predominant trimer from 266-6 acinar cells (Fig. 2B, lane 7), whereas the PDX1·PBX1b·MEIS2d complex (lane 8) comigrates with the slower mobility complex that remains after alpha MEIS2b treatment. Thus, we conclude that the major molecular form of the acinar trimeric complex comprises PDX1, PBX1b, and MEIS2b and that MEIS2d replaces MEIS2b in a small fraction of the complexes.

The presence of MEIS2b and 2d in acinar cells was confirmed by Western blot analysis (Fig. 3). Unlike PBX1b, which is present in pancreatic acinar but not beta -cell lines (10), the MEIS2 proteins were detected in nuclear extracts from both. The levels of the MEIS2 proteins were lower in beta -cells than in acinar cells.


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Fig. 3.   MEIS2 proteins are present in pancreatic exocrine and beta -cell lines. Western blots of nuclear extracts from pancreatic cell lines were probed with antiserum that detects all four isoforms of MEIS2. The two left lanes contained IVT MEIS2b and MEIS2d, respectively.

The Trimeric Complex Is More Transcriptionally Activate Than PDX1-- Multimers of the B element are transcriptionally active in the beta -cell line RIN1046-38 (RIN-38), due to the presence of endogenous PDX1 in these cells (10). PBX1 is absent in these beta -cells (10), and MEIS2 is present at extremely low levels (Fig. 3). Adding either PBX1b or MEIS2b to the beta -cell line by transfection had little or no effect on the activity of the B multimer or on the B element with the augmented PBX1b binding site (B2) (Fig. 4A). Co-expression of PBX1b and MEIS2b in RIN-38 beta -cells increased the activity of either the B or B2 multimer severalfold over the activity directed by the endogenous PDX1 (7-fold for the B element; 4-fold for the B2 element). These results indicate that the trimer complex is more transcriptionally active in RIN-38 cells than PDX1 alone. The B7 mutation which eliminated PDX1 and trimer binding in EMSA had no activity in the beta -cell line, whether in the presence or absence of cotransfected PBX1b and MEIS2b (Fig. 4A), confirming the central importance of PDX1 binding to the activity of this element.


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Fig. 4.   The trimeric complex acts as a transcriptional activator. A, RIN-38 cells were transfected with plasmids bearing five repeats of B, B2, or B7 elements driving an hGH reporter gene. The relative activity of the hGH reporter was assayed with or without cotransfected PBX1b and/or MEIS2b. Transfections were normalized based on the activity of a cotransfected Pdx1.luciferase reporter plasmid. B, the relative activity of the B2 multimer in HeLa cells was assayed with or without cotransfected PDX1, PBX1b, and MEIS2b individually or in combination. Transfections were normalized based on the activity of a cotransfected CMV-beta -gal reporter plasmid. All values are the average of four or more transfections. Error bars represent standard errors of the mean.

Domains of PDX1 Required for Trimer Binding and Activity-- PDX1 contains three important functional domains (Fig. 5A): an activation domain (AD) at the N terminus (amino acid residues 1-79), a pentapeptide motif (PP; residues 121-125) that directly contacts PBX, and the homeodomain (HD) (residues 145-206) (9, 40, 41). A cryptic C-terminal activation domain also functions in the transcriptional activation of the somatostatin gene by PDX1 (40). We constructed a series of PDX1 deletion mutants to test the function of each domain in the context of the trimer (Fig. 5A). The mutant proteins were synthesized by in vitro translation (IVT), and their ability to bind DNA was assayed by EMSA.


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Fig. 5.   Domains of PDX1 required for trimer binding and activity. A, schematic representation of the PDX1 deletion series. The activation domain (AD), pentapeptide motif (PP), and the homeodomain (HD) are indicated. B, binding of the PDX1 mutants as a monomer (upper panel), a heterodimer with PBX1b (middle panel), and a heterotrimer with PBX1b and MEIS2b (lower panel) to the B2 element. Monomers, dimers, and trimers are labeled as 1, 2, and 3, respectively. Lane 1, reticulocyte lysate without added template. Lanes 2-5, PDX1 test constructs as noted at the top of the panel. Note that full-length PDX1 has approximately the same mobility as the major complex endogenous to the reticulocyte lysate. C, activation of the B2 multimer in HeLa cells by co-transfected PDX1 mutants, alone (black bars) or in combination with PBX1b and MEIS2b (gray bars). All activities are expressed relative to the activity of wild type PDX1 (PDX1-284) alone. Because the PDX116-206 does not accumulate in the nucleus of transfected cells (data not shown), its inherent transcriptional activity could not be assessed.

Full-length PDX1 binds to the B2 element as a monomer, a heterodimer with PBX1b, and a heterotrimer with PBX1b and MEIS2b (Fig. 5B, lane 2). PDX1 mutants missing the regions C-terminal to the HD or N-terminal to the PP motif had similar DNA binding activities as the wild type PDX1 (lanes 3, 4, and 6). When the PP motif was also deleted, however, the protein was unable to form heterodimeric and heterotrimeric complexes on DNA (lane 5). Therefore, only the DNA-binding domain and the PBX-interaction motif are required for PDX1 to participate fully in forming the trimeric complex on DNA. These are the same domains required for dimer formation with PBX1 (Fig. 5B and Ref. 9).

To investigate which PDX1 domains are essential for trimer function, we tested the activity of the PDX1 deletion series by cotransfection with PBX1b and MEIS2b into a non-pancreatic cell line (HeLa). HeLa has no endogenous PDX1, although it contains both PBX1b (42) and MEIS2 (data not shown). Multimers of the B2 element were inactive in HeLa in the absence of cotransfected transcription factors (Fig. 4B). Addition of PDX1 activated the B2 element at least 50-fold, whereas addition of either PBX1b or MEIS2b alone or together had a barely detectable activating effect. Addition of either PBX1b or MEIS2b in cotransfections with PDX1 augmented the PDX1 activity 2- to 4-fold, whereas cotransfection of all three transcription factors increased activity 9-fold. The synergistic activity of the three proteins is consistent with the cooperative binding of PBX1b and MEIS2b with PDX1. As seen for the RIN-38 beta  cell line, the activity of the B2 element in the presence of the various combinations of transcription factors paralleled the activity of the natural B element (data not shown). The increased PDX1 activity by cotransfection of MEIS2b, which was not observed with transfected RIN-38 cells, is likely due to the presence of endogenous PBX1b, which is absent in RIN-38 cells.

Reporter gene activation by the trimer, as well as by PDX1 alone, required the N-terminal activation domain of PDX1 (Fig. 5C). Both the PDX116-284 and PDX126-284 mutants missing the activation domain were inactive, either in the presence or absence of cotransfected PBX1b and MEIS2, even though the PDX116-284 could form DNA-binding trimers (Fig. 5B). These results imply that neither PBX1b nor MEIS2b contribute an activation domain to the trimer.

Domains Important for the Function of PBX1b-- Three polypeptide domains are uniquely conserved among the PBC class of homeodomain proteins (mammalian PBX proteins, Drosophila Extradenticle, and C. elegans Ceh-20 (6)). The PBC-A domain near the N terminus of the proteins (Fig. 6A) mediates the interaction of PBX with MEIS and the closely related PREP1 protein (7, 43, 44). The function of the PBC-B domain, located between the PBC-A and the HD, has not been delineated. In addition, the C terminus of the HD is extended by a fourth alpha -helix, which forms part of the hydrophobic pocket that binds the pentapeptide motif of HOX-like proteins (45, 46). We tested a series of PBX1b deletion mutants to determine which of the PBX1b-conserved domains are critical for trimer formation, DNA binding, and transcriptional activation (Fig. 6).


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Fig. 6.   Domains important for the function of PBX1b in the trimer. A, diagram of the PBX1b deletion series. The relative positions of PBC-A, PBC-B, the HD and helix 4 (H4) are shown. B, binding of the PBX1b mutants alone (upper panel; labeled as 1), in combination with PDX1 (middle panel; dimers labeled 2), or with PDX1 and MEIS2b (lower panel; trimers labeled 3) to the B2 element by EMSA. Lane 1: reticulocyte lysate without added template. Lanes 2-6: PBX1b test constructs as noted at the top of the panel. C, interaction of PBX1b mutants with MEIS2b in solution and bound to DNA. Upper panel: 35S-labeled IVT PBX1b proteins were incubated with unlabeled IVT MEIS2b, precipitated with anti-MEIS2, and the precipitated protein resolved by SDS-10%-polyacrylamide gel electrophoresis. Lower panel: binding of PBX1b·MEIS2b dimers to the CRS1 element. The PBX1b monomers and PBX1b·MEIS2b dimers are labeled as 1 and 2, respectively. Lane 1: reticulocyte lysate without added template. Lanes 2-6: PBX1b test constructs as noted at the top of the panel. D, activation of the B2 multimer in HeLa cells by PBX1b mutants in co-transfections with PDX1 and MEIS2b. All activities are expressed relative to the activity of PDX1 plus MEIS2b in the absence of co-transfected PBX1b.

In vitro synthesized, full-length PBX1b does not bind the B2 element, but does bind as a heterodimer with PDX1 and as a trimeric complex with PDX1 and MEIS2b (Fig. 6B, lane 2). Illustrating the critical role that helix 4 plays in complex formation, deletion of sequences C-terminal to it had no effect (PBX1b1-318, lane 3), whereas further deletion to remove helix 4 abolished both dimer and trimer formation (PBX1b1-295, lane 4). Deletion of both PBC domains abolished the formation of the trimer on DNA without affecting the interaction between PBX1b and PDX1 (PBX1b233-347, lane 5). However, the PBC-A domain alone is not sufficient to bring MEIS into the trimeric complex, because deletion of just the PBC-B domain also abolished the trimer formation (PBX1bDelta B, lane 6). Thus, formation of the trimer on DNA requires helix 4 of the PBX1b HD to recruit PDX1 and the PBC domains to recruit MEIS2b.

PBX and MEIS can form stable dimers, even in the absence of DNA (42, 44), and the PBX·MEIS heterodimer binds a consensus sequence of adjacent PBX and MEIS half-sites (7, 44). To confirm that the failure of PBX1b233-347 and PBX1bDelta B to form trimeric complexes was due to their inability to interact with MEIS2b, we examined the formation of PBX1b·MEIS2b dimers by coimmunoprecipitation and their ability to bind a composite PBX·MEIS site (Fig. 6C). In the absence of DNA, deletion of both PBC domains (PBX1b233-347) eliminated dimerization with MEIS2b, whereas deletion of PBC-B alone (PBX1bDelta B) reduced, but did not eliminate, dimer formation. To analyze the binding of PBX·MEIS dimers on DNA, we used the CRS1 element from the bovine CYP17 gene, which contains adjacent PBX and MEIS half-sites (30). PBX1bDelta B and MEIS2b formed a very small amount of bound complex, but PBX1b233-347 did not, paralleling the results in solution. The dimer of PBX1b1-295 and MEIS2b, although fairly stable in solution, did not bind DNA efficiently (lane 4). These results agree with previous findings that the region immediately C-terminal to the HD (i.e. helix 4) is important for stabilizing PBX binding to DNA and for interacting with HOX-like proteins (44, 45). Deletion of the entire region N-terminal to the HD (PBX1b233-347) or just the PBC-B domain (PBX1bDelta B) permits binding to both the B2 and CRS1 elements as a monomer (Fig. 6B, lanes 5 and 6; Fig. 6C, lanes 5 and 6). Relief of inhibition of DNA binding by deletion of the PBC N-terminal region has also been observed by Neuteboom and Murre (47).

We analyzed the ability of each PBX1b mutant to participate in the transcriptionally active trimer complex with PDX1 and MEIS2b in transfected cells (Fig. 6D). The PBX1b1-318 mutant is only slightly less active than the full-length PBX1b, indicating that no crucial domain is present C-terminal to the HD helix 4. Surprisingly, the PBX1b1-295 protein, although lacking helix 4 and unable to form a trimer with PDX1 and MEIS2b in EMSA, is active in transfections. The two PBC domain mutants, PBX1b233-347 and PBX1bDelta B, had no detectable activity in transfections, indicating that a principal role for PBX1b in trimer activity is the recruitment of MEIS2b.

Trimer Formation and Activity Requires the M1 and M2 Domains but Not the HD of MEIS2b-- The HD and two N-terminal MEIS domains (M1 and M2) are conserved among mammalian MEIS proteins, Drosophila Homothorax, C. elegans Ceh-25, and a closely related protein pKNOX/PREP1 (5, 7). The functional significance of these regions in DNA binding, protein interactions, and transcriptional activation was evaluated with a series of deletion mutants (Fig. 7).


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Fig. 7.   Domains important for the function of MEIS2b in the trimer. A, schematic of the MEIS2b deletion series. The positions of the MEIS domains, M1 and M2, as well as the HD are indicated. B, formation of the trimeric complexes with the MEIS2b mutants on the B2 element. 3 denotes the trimers and 2 in lane 2 indicates the PDX1·PBX1b dimer. Lane 1: reticulocyte lysate without added template. Lanes 2-7: MEIS2b test constructs as noted at the top of the panel. C, analyses of the interaction of MEIS2b mutants with PBX1b. Upper panel: 35S-labeled IVT MEIS2b proteins were incubated with unlabeled IVT PBX1b, precipitated with anti-PBX1N, and the precipitated protein resolved by SDS-10%-polyacrylamide gel electrophoresis. Lower panel: binding of the PBX1b·MEIS2b heterodimers to the CRS1 element. Lane 1: reticulocyte lysate without added template. Lanes 2-7: MEIS2b test constructs as noted at the top of the panel. D, activation of the B2 multimer in HeLa cells by MEIS2b mutants in co-transfections with PDX1 and PBX1b. All activities are expressed relative to the activity of PDX1 plus PBX1b in the absence of co-transfected MEIS2b.

MEIS2b does not bind the B2 element as a monomer or as a heterodimer with either PDX1 or PBX1b (data not shown). Deletion of regions C-terminal to the HD and N-terminal to the M1 domain had no impact on formation of a trimer (MEIS2b1-338 and MEIS2b71-394, Fig. 7B, lanes 3 and 4). Deletion of the M1 and M2 domains in MEIS2b192-394 eliminated the formation of a trimeric complex (lane 5). The MEIS2b HD has an inhibitory effect on trimer formation: removing the HD in MEIS2bDelta HD, and MEIS2b1-275 enhanced the formation of the trimers (lanes 6 and 7). These results showed that the MEIS2b HD is dispensable for the formation of a heterotrimer on DNA. This is in agreement with the absence of a MEIS consensus site in the B element and with the absence of any sequence requirements beyond the PBX-PDX1 binding site for trimer binding and transcriptional activity (10).

Consistent with its inability to form a trimer, MEIS2b192-394 failed to dimerize with PBX1b both in solution and on CRS1 (Fig. 7C, lane 5). The MEIS2b HD was not required for the PBX·MEIS dimers to form in solution, but was essential for these dimers to bind DNA (MEIS2bDelta HD and MEIS2b1-275, lanes 6 and 7). PREP1 can also dimerize with PBX in the absence of its HD, but, as for MEIS, the HD is required for DNA binding (43). Therefore, the N-terminal region containing the M1 and M2 domains is sufficient for trimer formation. The M2 domain is known to interact with PBX (7). Our deletion analysis did not unveil any direct interaction between MEIS2b and PDX1.

The region containing M1 and M2 was also sufficient for a fully active trimer in cotransfection experiments (Fig. 7D). All of the deletion mutants that contained the MEIS domains, including the minimal construct MEIS2b1-275, had approximately the same activity as the full-length MEIS2b. The MEIS2b192-294, which lacks both MEIS domains, had no activity.

The PDX1·PBX1b·MEIS2b Trimer Cooperates with a bHLH Complex to Activate Transcription-- The synergistic interaction between the A and B elements of the ELA1 enhancer (26) suggests that the DNA-binding factor complexes that mediate their activities cooperate within the context of the ELA1 enhancer. PTF1, which binds the A element, comprises a bHLH heteromultimer that minimally contains the acinar cell-specific p48 protein (29) and a ubiquitous E-box binding protein (REB in rats, HEB in humans, ALF1 in mice).3,4 A mini-enhancer construct bearing three head-to-tail copies of tandem A and B elements (Fig. 8A) was used to test possible cooperativity between the PDX1·PBX1B·MEIS2b trimer and the p48·HEB dimer.


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Fig. 8.   The PDX1·PBX1b·MEIS2b trimer and the p48·HEB dimer of PTF1 cooperate in activating transcription from an ELA1 mini-enhancer in HeLa cells. A, schematic of the 3(BA).EIp.hGH mini-enhancer. B, the relative activity of the 3(BA).EIp.hGH reporter gene in HeLa cells was assayed with or without cotransfected PDX1, PBX1b, MEIS2b, and p48·HEB individually or in various combinations as indicated. The gray line indicates the activity of the 3(BA).EIp.hGH in the absence of added transcription factors. This level of background activity was the same as that of a promoter-only EIp.hGH construct (data not shown), which was unaffected by added transcription factors in any combination. All values are the mean of six or more transfections, except for the pairwise combinations of PDX1 and PBX1b, PDX1 and MEIS2b, PBX1b and MEIS2b, and p48 and HEB, which represent the mean of two transfections. Error bars represent standard errors of the mean. C, the relative activity of PDX1, PBX1b, and MEIS2b deletion proteins in the cooperative activation of the BA mini-enhancer by the homeodomain heterotrimer and PTF1. The presence of full-length PDX1, PBX1b, and MEIS2b is indicated by +; the presence of particular PDX1, PBX1b, and MEIS2b deletion mutants is indicated by their identifying numbers from Figs. 5, 6, and 7. Values are the mean, with standard error, of four or more transfections.

The PDX1·PBX1b·MEIS2b trimer activated the mini-enhancer nearly 4-fold in HeLa cells (Fig. 8B). Even though p48·HEB alone had no detectable effect on the mini-enhancer, it increased the activation by the trimer 2-fold. Addition of either PBX1b or MEIS2b to PDX1 in the presence of p48·HEB had little if any stimulatory effect, whereas adding PBX1b and MEIS2b together increased the activity of PDX1 with p48·HEB 4-fold. The increase in activity with the addition of p48·HEB was dependent on the presence of the A element, because p48·HEB had no effect on the activation of the B multimer by PDX1 or the PDX1·PBX1b·MEIS2b trimer in either HeLa or RIN-38 cells (data not shown). The A-element-dependent stimulation by p48·HEB represents cooperative activation with the trimer. Electrophoretic mobility shift experiments designed to detect a cooperative binding of IVT PDX1·PBX1b·MEIS2b trimer with IVT p48·HEB on adjacent B and A elements have been unsuccessful.5

To determine which domains of PDX1, PBX1b, and MEIS2b are responsible for the cooperative interaction of the heterotrimer with PTF1, key deletion mutants of the homeoproteins were tested for their ability to participate in activating the BA mini-enhancer (Fig. 8C). The activation domain of PDX1 was required, whereas the region of PDX1 C-terminal to the homeodomain is entirely dispensable for activity. All of the PBX1b and MEIS2b deletion constructs that formed heterotrimers on the B element could participate in the activation of the BA mini-enhancer. DNA binding by MEIS was not required for activity, because the deletion of the MEIS HD and C terminus (MEIS1-275) did not result in any loss of activity. These requirements mimic those of the trimer for transcriptional activation through the B-element multimer, which is independent of PTF1.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The trimeric complex of PDX1·PBX1b·MEIS2b binds a bipartite PBX-PDX1 site and is an order of magnitude more transcriptionally active in HeLa cells than PDX1 alone. The transcriptional activity of the complex is dependent on the activation domain of PDX1, as well as domains that mediate the interaction between PDX1 and PBX1b and between PBX1b and MEIS2b. Cooperative transcriptional activation with the pancreas-specific bHLH complex PTF1 requires the same structural domains and mimics the activity of the trimer within the context of the ELA1 enhancer in pancreatic acinar cells.

Organization of the DNA-bound Trimer-- Based on the behavior of the deletion mutants of the three proteins, we propose a model for the organization of the PDX1·PBX1b·MEIS2b complex bound to DNA (Fig. 9). The trimer binds DNA through the HDs of PDX1 and PBX1b. The HD of MEIS2b forms no sequence-specific DNA interactions and is dispensable for DNA binding; indeed, DNA binding of the trimer is enhanced by the absence of the MEIS HD. PBX1b is the centerpiece of the complex; PBX1b interacts with PDX1 and MEIS2b through separate polypeptide domains. PBX1b and PDX1 bind adjacent DNA half-sites and interact directly through helix 4 of the PBX1b homeodomain and the pentapeptide motif just N-terminal to the PDX1 homeodomain. PBX1b recruits MEIS2b into the complex through interactions involving the N-terminal halves of the two proteins. Our analyses did not reveal any direct interaction between PDX1 and MEIS2b.


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Fig. 9.   Model of the trimeric complex. PDX1 and PBX1b bind to adjacent half-sites on the B2 variant of the ELA1 enhancer B element. Protein·protein interactions occur between the PDX1 and PBX1b HDs, and between the pentapeptide (PP) of PDX1 and helix 4 (H4) of PBX1b. PBX1b and MEIS2b interact through their N-terminal domains, with the M2 domain of MEIS, and the PBC-A and PBC-B domains of PBX particularly important to the interactions. Binding of the MEIS2b HD to DNA is not required for trimer formation or transcriptional activity. PDX1 contains the only transcriptional activation domain (AD) in the complex.

Our results are consistent with previous biochemical and structural analyses of HOX·PBX and PBX·MEIS heterodimers. These analyses showed that when PBX and HOX proteins are bound at adjacent sites, the HOX partner reaches across the DNA duplex and inserts the tryptophan of the pentapeptide motif into a hydrophobic pocket formed by the PBX homeodomain and helix 4 (45, 46). The PBC-A domain (7, 44) and the M2 domain of MEIS (7) are of primary importance in PBX·MEIS complex formation; however, some interaction is maintained even in the absence of the PBC-A domain (7). Indeed, we have found that deletion of the PBC-B domain also disrupts PBX·MEIS interactions and eliminates formation of the DNA bound trimer (Fig. 6).

Recently, trimeric complexes containing HOX-like, PBX-like, and MEIS-like subunits have been reported with different modes of binding to DNA. Similar to the PDX1·PBX1b·MEIS2b complex, a HOXB1·PBX1·PREP1 complex also binds DNA through the HOX and PBX HDs, and DNA binding does not require the homeodomain of PREP1 (43). Other trimeric HOX·PBX-like·MEIS-like complexes require sequence-specific DNA binding of all three HDs (48-50), whereas at least one trimeric complex requires only an HOX binding site (51). Thus, in different contexts the trimeric complexes of HOX and HOX-like proteins with PBX-like and MEIS-like cofactors can require specific binding sites for only the HOX, for HOX and PBX, or for all three proteins.

Transcriptional Activity of the Trimer-- The polypeptide domains required for the increased transcriptional activity of the PDX1·PBX1b·MEIS2b trimer, in comparison to PDX1 alone, include those required for trimer binding to DNA (the PDX1 and PBX1b HDs) and for protein·protein interactions necessary for formation of a stable heterotrimer (the PDX1 pentapeptide, the PBX helix 4 and PBC domains, and MEIS M1 and M2 domains). The N-terminal activation domain of PDX1 is essential for transcriptional activity of the trimer, because a trimeric complex missing only this activation domain (PDX116-284) has no transcriptional activity (Fig. 5C).

The absence of transactivation potential in the trimer lacking the PDX1 activation domain indicates that PBX1b and MEIS2b do not have activation domains or that their activation domains do not function without the PDX1 activation domain. Co-expression of PBX1 and MEIS1 fails to activate a reporter gene driven by a multimer of PBX·MEIS binding sites (7). Other experiments also have failed to detect an activation domain in PBX1b (52, 53). The presence of PBX1b and MEIS2b does not appreciably increase the amount of PDX1 binding in gel shift assays, so it is unlikely that the effect of PBX1b and MEIS2b is to increase binding site occupancy by PDX1. Therefore, PBX1b and MEIS2b do not appear to contribute either activation domains to the trimeric complex or stabilization of PDX1 binding to DNA. Consequently, the basis of the increased transcriptional activity of the trimer, in comparison to PDX1 alone, is unclear.

Cooperative Transcriptional Activation with the p48·HEB Complex-- The ELA1 enhancer comprises the A and B elements, which bind the bHLH complex PTF1 and the PDX1·PBX1b·MEIS2b trimer, respectively, as well as a non-cell-specific element that binds unknown factors (10, 24, 26, 27). The pancreatic activity of the enhancer is dependent on synergy between the A and B elements in transgenic animals and transfected pancreatic acinar cell lines (25, 26). Although PDX1 bears the only activation domain within the PDX1·PBX1b·MEIS2b trimer, enhancer mutations that eliminate the PBX1 half-site and trimer binding without eliminating PDX1 binding are transcriptionally inactive (10). Thus, the requirement for PBX and MEIS, as well as PDX1, for the cooperative activation of an ELA1 mini-enhancer in HeLa (Fig. 8) is consistent with previous results with the complete ELA1 enhancer.

The ELA1 mini-enhancer, comprising three copies of adjacent PBX-PDX1 and PTF1 binding sites, directs high level expression in the pancreas and gut of transgenic mice (26). The pancreatic expression of this mini-enhancer is more than an order of magnitude greater than that seen with a multimer of the PBX-PDX1 binding site or a multimer of the PTF1 binding site. The activity of the mini-enhancer in HeLa cells is dependent on the activation domain of PDX1, within the context of the DNA-bound PDX1·PBX1b·MEIS2b complex (Fig. 8). The cooperative activation of the mini-enhancer in HeLa cells upon co-transfection with PDX1·PBX1b·MEIS2b and p48·HEB likely reflects the natural interaction of these two complexes in the acinar pancreas to activate the transcription of the transgenic mini-enhancer and the endogenous ELA1 gene as well.

Cooperation between homeoproteins and bHLH complexes have been shown to occur in two other instances. In the insulin gene enhancer, PDX1 contributes to synergistic activation in a manner different than its action in the ELA1 enhancer. PDX1 binds to the A3/4 region of the rat insulin I enhancer in the absence of PBX and MEIS proteins and activates transcription by cooperating with the bHLH protein E47, which binds to the adjacent E2 site (54). In this case, PDX1 contributes protein·protein interaction domains to recruit multiple proteins, including E47, BETA2/NeuroD, and high mobility group protein I(Y), to an activation complex on the E2A3/4 mini-enhancer. The transcriptional activity of this complex is believed to result from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery (54). In addition, a PBX·MEIS/PREP1 heterodimer and heterodimers of E2A and MyoD, myogenin, MRF-4, or MYF-5 exhibit cooperative DNA binding and transcriptional activation (55). This interaction requires a conserved peptide motif just N-terminal of the bHLH domain, which is not present in p48 (55); and p48 cannot substitute for the myogenic factors.5 Although the mechanism for the cooperation between the PDX1 and p48 complexes is not known, it is likely that the two complexes interact, either to stabilize each other's binding to DNA or to recruit coactivators to facilitate transcriptional activation.

    ACKNOWLEDGEMENTS

We thank Drs. Scott Rose, Christopher Wright, Richard Baer, and Pierre Chambon for providing cloned transcription factors, and Dr. Peter Wellauer for communicating results prior to publication. We thank Dr. Mike Peyton for many helpful discussions and communication of unpublished results.

    FOOTNOTES

* This research was supported by Grant DK-55266 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9148. Tel.: 214-648-1942; Fax: 214-648-1915; E-mail: swift@hamon.swmed.edu.

Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M100678200

2 S. D. Rose, R. J. MacDonald, M. J. Peyton, R. E. Hammer, and G. H. Swift, manuscript in preparation.

3 P. Wellauer, personal communication.

4 S. Rose and R. J. MacDonald, unpublished.

5 M. J. Peyton and R. J. MacDonald, unpublished data.

    ABBREVIATIONS

The abbreviations used are: bp, base pair(s); bHLH, basic helix-loop-helix; kb, kilobase(s); IVT, in vitro transcription and translation; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; AD, activation domain; HD, homeodomain; PP, pentapeptide motif.

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ABSTRACT
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DISCUSSION
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