Superactivation of Pax6-mediated Transactivation from Paired Domain-binding Sites by DNA-independent Recruitment of Different Homeodomain Proteins*

Ingvild MikkolaDagger§, Jack-Ansgar Bruun§, Turid Holm, and Terje Johansen||

From the Department of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway

Received for publication, September 28, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Pax6 genes encode evolutionary conserved transcription factors that act high up in the regulatory hierarchy controlling development of central organs such as the eyes and the central nervous system. These proteins contain two DNA-binding domains. The N-terminal paired domain is separated from a paired-type homeodomain by a linker region, and a transactivation domain is located C-terminal to the homeodomain. Vertebrate Pax6 genes express a paired-less isoform of Pax6 (Pax6Delta PD) from an internal start codon in the coding region between the paired domain and homeodomain. We now provide evidence for an interaction between the full-length isoform and Pax6Delta PD, which enhances the transactivation activity of Pax6 from paired domain-binding sites. The paired-like homeodomain protein Rax behaved similarly to Pax6Delta PD. Both Pax6Delta PD and Rax bound to the homeodomain of Pax6 in vitro in the absence of specific DNA binding. Coimmunoprecipitation experiments following cotransfection confirmed the existence of complexes between Pax6 and Pax6Delta PD, Pax6 and Rax, and Pax6Delta PD and Rax in vivo. Interestingly, the C-terminal subdomain of the paired domain and the homeodomain can interact with each other. The paired domain can also interact with itself. Surprisingly, GST pull-down assays revealed that the homeodomains of such diverse proteins as Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and HoxB1 could all bind to Pax6, and several of these enhanced Pax6-mediated transactivation upon coexpression. Since many homeodomain proteins are coexpressed with Pax6 in several tissues during development, our results indicate the existence of novel regulatory interactions that may be important for fine tuning of gene regulation.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Pax6 is a member of the evolutionary conserved Pax family of transcription factors, which plays pivotal roles during vertebrate development (1, 2). In addition to the paired domain (PD),1 encoded by the paired box, Pax6 contains another DNA binding domain, the paired-type homeodomain (HD). The C-terminal part of the protein is rich in proline, serine, and threonine (PST), and functions as a transcriptional activation domain (3-7). The mouse and human Pax6 proteins have an overall amino acid sequence identity of 100%, with zebrafish Pax6 being about 97% identical to the mammalian proteins (reviewed in Ref. 8). The PD and the HD are more than 90% conserved in the invertebrate proteins compared with the mammalian ones. In vertebrates, Pax6 is expressed in the developing nervous system, in the neuroretina and lens of the eye, in the nasal placode, and in the pancreas (reviewed in Ref. 8). Due to a gene duplication during insect evolution, Drosophila contains two Pax6 homologs, eyeless (ey) and twin of eyeless (toy). Toy regulates an eye-specific enhancer of ey and thus acts above Ey in activating the eye developmental program (9). Both these genes as well as mouse and zebrafish Pax6 induce ectopic eyes when overexpressed in the Drosophila embryo, indicating that the Pax6 proteins have not only a conserved structure but also a conserved function (9-11). Pax6 can also induce ectopic eyes in Xenopus embryos in a cell-autonomous manner (12). Mutations in Pax6 cause the small eye phenotype in mice and aniridia and Peters anomaly in humans (13-15). Studies of transgenic mice with homozygous targeted disruption of either the LIM homeodomain protein Lhx2 or the paired-type homeodomain proteins Rax/Rx and Hesx1 have revealed that, in addition to Pax6, also loss of function of these homeobox genes lead to anophthalmic (eyeless) embryos (16-19). The Rax protein contains a paired-like HD but no other domains with sequence homology to Pax6. Rax is, similar to Pax6, expressed in the developing forebrain and neuroretina (17, 18, 20) of mouse, Xenopus, and zebrafish.

There are several reported isoforms of the Pax6 protein. One of these, Pax6-5a, has a 14-amino acid insertion in the N-terminal subunit of the PD generating an altered DNA-binding specificity (21). This isoform is vertebrate-specific, since it is not found in invertebrates. Several additional isoforms of Pax6 have been reported in quail and bovine retina (22, 23). One of these is a 33/32-kDa isoform that lacks the PD due to the use of an alternative start codon for translation in the sequence between the PD and the HD (22). Interestingly, in C. elegans there are two transcripts of the Pax6 gene, one that encodes the full-length protein and one initiated at an internal promoter producing a transcript that does not encode the PD (24). Together with the fact that there exist natural "P3" HD binding sites in promoters of many retina genes (25), this indicates that the paired-less Pax6 isoform may play an important role in vivo during development and/or in the adult organism.

The paired-type HD in Pax proteins is distinguished from other HDs by having a serine in position 50 instead of a glutamine or a lysine. The paired-type HDs are able to bind cooperatively to palindromic DNA sequences of the type TAATN2-3ATTA, named P2 or P3 after the number of base pairs separating the two TAAT/ATTA palindromic core sequences (26). The HD of Pax6 is shown to bind preferentially to P3 sites (4). In contrast to the cooperative DNA binding/dimerization of other HDs (e.g. Hox), which relies on sequences extrinsic to the HD, the paired class HDs can rely entirely on the 60-amino acid-long HD to achieve cooperativity upon DNA binding (26).

Given the very conserved three-dimensional globular structure of HDs (25, 27-30), it seems reasonable that this structure is evolutionarily conserved not only for the purpose of DNA binding but also for preserving protein-protein contacts. Hence, the PD and HD of Drosophila Prd can bind cooperatively to DNA when present in the same or different peptides (31). Moreover, upon binding to DNA, the PD of Prd can interact with other paired-type HDs, and the same is true for the Prd HD regarding interactions with other PDs (31). Engrailed-1 (En-1) interacts with the PD of Pax6 and prevents DNA binding (32). En-1 is not dependent on binding DNA to exert this function. Pax5 is shown to function as a cell type-specific docking protein for an ETS family transcription factor at the B cell-specific mb-1 promoter (33). Recruitment of Net and Elk-1, but not SAP1a, by Pax5 defines a functional difference between closely related Ets proteins. The interactions between the Pax5 PD and the ETS domain have not been found in the absence of DNA. Nutt et al. (34) have recently shown that the Pax5 PD alone is sufficient for transcriptional activation from the mb-1 promoter. The PD of Pax5 also interacts with PU.1, and Pax5 represses the immunoglobulin kappa  3' light chain enhancer by targeting PU.1 function (35). The retinoblastoma protein, Rb, was shown to bind the HD of Pax3 (36). The HD of Pax6 and the partial HD of Pax5 can bind both Rb and TBP (37, 38). Finally, the paired-like HDs of Prd and Phox1 interact with the serum response factor (39).

Here we describe an interaction between the Pax6 protein and the paired-less Pax6 isoform, Pax6Delta PD, which enhanced the transactivation activity of Pax6 from PD-binding sites. Rax behaved similarly to Pax6Delta PD, indicating a more general function of paired-like HD interactions in modulation of transcriptional activation. Glutathione S-transferase (GST) pull-down experiments showed that in vitro these interactions were mediated by the paired-type HD of Pax6 by a DNA-independent mechanism. Coimmunoprecipitation experiments performed using extracts from transfected cells confirmed the existence of complexes between Pax6 and Pax6Delta PD, Pax6 and Rax, and Pax6Delta PD and Rax in vivo. Deletion of the HD indicated that other parts of Pax6 also could contribute to the interaction, and the C-terminal subdomain of the PD was identified as another interacting domain, whereas the C-terminal transactivation domain (TAD) by itself was unable to interact with Pax6 in GST pull-downs. Rax was also able to interact with the Pax6 PD. Interestingly, the PD also interacted with itself. We were surprised to find that HDs from various other HD proteins were also able to interact with Pax6 in GST pull-down assays, indicating a more general capability of protein-protein interactions involving the HD. Several of these HD proteins also gave superactivation of Pax6-mediated transactivation upon cotransfection.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Plasmid Constructs-- The pCI-Pax6 and pP6CON-LUC plasmids have been described previously (11). pCI-Pax6Delta PD was made by deleting a NheI fragment including the paired domain from pCI-Pax6. To construct pCI-Rax, pBSK/mouse Rax (kindly provided by C. L. Cepko) was cut with HindIII (made blunt) and XhoI and cloned into the NheI (made blunt) and XhoI sites of the pCI-neo expression plasmid (Promega). pcDNA3-HA (kindly provided by J. Moscat) contains an influenza virus hemaglutinin (HA) epitope tag inserted into the HindIII-EcoRI sites of pcDNA3 (Invitrogen). HA-Pax6 was constructed from a PCR product of full-length zebrafish Pax6.1 cDNA (primers 6.HA.5 and 6.HA.3; Table I) made blunt at the 5'-end and cut with XbaI in the 3'-end before cloning into the EcoRV and XbaI sites of pcDNA3-HA. The HA-Pax6Delta PD construct was made exactly the same way by use of the 6Delta PD.HA.5 and 6.HA.3 PCR primers (Table I). The PCR product of the coding sequence from mouse Rax cDNA was obtained using the HA-rax.5 and HA-rax.3 primers. The PCR product was cut with EcoRI and XhoI and inserted into the corresponding sites of pcDNA3-HA. The constructs of Pax6 lacking all of the homeodomain (HA-Pax6Delta HD) were made by use of specific primers containing XhoI, that would provide an open reading frame upon self-ligation (Table I). HA-Pax6 was used as a template, and primers delA and delD were used to make HA-Pax6Delta HD. In vitro mutagenesis was performed according to the instruction manual for the QuickChange site-directed mutagenesis kit (Stratagene), but XhoI was added during the digestion with DpnI, and the PCR fragments were self-ligated before they were transformed into bacteria. HA-Pax6Delta HD was used as a template and 6Delta PD.HA.5 and 6.HA.3 were used as primers when the HA-Pax6Delta PDDelta HD was constructed by PCR and cloned into the EcoRV and XbaI sites of the pcDNA3-HA vector as described above for the same primer set. The expression construct for the Pax6 HD was made using PCR with primers HA-6HD.5 and HA-6HD.3 followed by digestion with EcoRI and XbaI and insertion into the EcoRI-XbaI sites of pcDNA3-HA and pGEX-4T-3 (Amersham Pharmacia Biotech). To make vectors for expression of fusions of Pax6 or Pax6Delta PD to green fluorescent protein (GFP), a 1.7-kilobase pair SmaI-XbaI fragment (GFP-Pax6) or a 1.3-kilobase pair PmlI-XbaI fragment (GFP-Pax6Delta PD) from HA-Pax6 was inserted into SmaI-XbaI-cut pEGFP-C1 (CLONTECH). The homeodomains from mouse Rax (17), mouse Chx10 (40), mouse En-1 (41), mouse Six3 (42), mouse Lhx2 (43), human Prep1 (44), human Prox1 (45), and human HoxB1 (46) were amplified from cDNAs using the PCR primers described in Table I. The PCR products were then cut with EcoRI and XhoI and cloned into the corresponding sites of pGEX-4T-3. To express Chx10 and Lhx2 in mammalian cells, the coding regions were cloned into EcoRI-XhoI-cut pcDNA3-HA following PCR using primer pairs Chx10.HA5/Chx10.HA3 and Lhx2.HA5/Lhx2.HA3 (Table I) and digestion of the PCR products with EcoRI and XhoI. All PCRs were performed with the Pfu polymerase according to the instructions from the manufacturer (Stratagene), and all plasmid constructs were verified by sequencing.


                              
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Table I
Sequences of primers used for PCR

Transient Transfection Assays-- For transient transfection assays HeLa cells (ATCC CCL 2) were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum (Hyclone), nonessential amino acids (Life Technologies, Inc.), 2 mM L-glutamine, penicillin (100 units/ml) and streptomycin (100 µg/ml). About 8 × 104 HeLa cells/well in six-well tissue culture dishes were transfected by the calcium phosphate coprecipitation procedure using from 1 to 2.8 µg of DNA (see figure legends for details). To normalize for variations in transfection efficiency, 0.05 µg of pCMV-beta -galactosidase expressing beta -galactosidase from a cytomegalovirus (CMV) promoter was included in each transfection. Extracts were prepared 24 h following transfection using a Dual-Light luciferase and beta -galactosidase reporter gene assay system (Tropix) and analyzed in a Labsystems Luminoskan RT dual injection luminometer. Transfections were repeated with different DNA preparations.

GST Pull-down Assays-- GST fusion proteins were purified from Escherichia coli LE392 extracts using glutathione-agarose beads (Amersham Pharmacia Biotech). The proteins were not eluted but were left on the beads and stored at 4 °C in phosphate-buffered saline containing 1% Triton X-100 (PBT). The protocol for GST pull-down assays was adapted from Nead et al. (47). One µg of pCI-neo or pcDNA3-HA plasmids containing the genes of interest was in vitro transcribed and translated using the TNT system (Promega) according to the manufacturer's protocol in a total volume of 50 µl. For each pull-down experiment, 10 µl of in vitro translated 35S-labeled protein was diluted in 200 µl of ice-cold NET-N (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, pH 8.0, 0.5% Nonidet P-40 with one protease inhibitor mixture tablet (Roche Molecular Biochemicals) per 10 ml of buffer). GST-glutathione-agarose beads were added, and the tubes were placed on a rotating wheel at 4 °C for 15-30 min and then centrifuged at 1,600 rpm for 2 min in a microcentrifuge. Following this preincubation step, the supernatants were transferred to new tubes. About 2 µg of fusion protein immobilized on glutathione-agarose beads were added, and the interactions were allowed to proceed at 4 °C for 1 h on a rotating wheel. Both the GST beads used for preincubation and the GST fusion protein-containing beads were washed five times with NET-N, and as much as possible of the buffer was removed after the last wash before the beads were boiled in 25 µl of 2× SDS-polyacrylamide gel loading buffer. The samples were run on a 10% SDS-polyacrylamide gel, which was vacuum-dried before 35S-labeled proteins were detected by autoradiography and/or by the use of a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Preparation of Nuclear Extracts and Coimmunoprecipitation Experiments-- The protocol for harvesting cells was adapted from Nead et al. (47). The cells were trypsinized and washed twice with 5 ml of phosphate-buffered saline. They were counted in the last wash with phosphate-buffered saline and washed once with 0.5 ml of buffer A (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) containing the following protease and phosphatase inhibitors: 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, 20 mM beta -glycerophosphate, 25 mM NaF, 50 µM sodium vanadate, 0.7 µg/ml pepstatin A, and 200 µM phenylmethylsulfonyl fluoride. The cells were resuspended in 80 µl of buffer A containing 0.1% Nonidet P-40 per 107 cells, placed on ice for 10 min, vortexed briefly, and centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was discarded, and the pellet containing the nuclei was resuspended in 28 µl of buffer C (20 mM Hepes, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) containing the same protease and phosphatase inhibitors as buffer A. After 15 min on ice, the samples were vortexed briefly and centrifuged as described above. The supernatant was transferred to a new tube containing 140 µl of buffer D (20 mM Hepes, pH 7.9, 20% glycerol, 0.1 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) with protease and phosphatase inhibitors. The protein concentration was determined using the Bio-Rad protein assay reagent, and 60-100 µg of nuclear extracts were used for each immunoprecipitation. Protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) were washed twice in gel shift buffer (20 mM Hepes, pH 7.9, 60 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA) and saturated with bovine serum albumin in gel shift buffer for 0.5-1 h on a rotator at 4 °C. The beads were washed twice in gel shift buffer and resuspended to a 50% gel slurry in gel shift buffer containing the protease and phosphatase inhibitors mentioned above. Thereafter, 120-200 µg of nuclear extracts were preincubated with the beads for 15-20 min at 4 °C, before they were split into two portions and incubated at 4 °C with either 10 µl anti-HA hybridoma supernatant, containing the monoclonal 12CA5 antibody, per 100 µg of nuclear extracts or 4 µl of affinity-purified PC6 polyclonal antibody directed against the C-terminal of Pax6 (48, 49) in a total volume of 500 µl in gel shift buffer. After 2 h, 20 µl of the bovine serum albumin-saturated protein A-Sepharose beads were added, and the samples were left at 4 °C for 1 h. The beads were then washed three times in gel shift buffer containing 120 mM NaCl and boiled in 25 µl of 2× SDS-polyacrylamide gel loading buffer. The samples were run on a 10% SDS-polyacrylamide gel (Mighty Small, Hoefer) in a Tris-glycine buffer (25 mM Tris, 250 mM glycine, 0.1% SDS), and Western blots were performed as described below.

Western Blot Analyses-- Mighty Small (Hoefer) 10% SDS-polyacrylamide gels were blotted onto Hybond nitrocellulose membranes (Amersham Pharmacia Biotech), and the membranes were incubated with blocking buffer containing 5% nonfat dried milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 1% Tween 20) for 1 h at room temperature or overnight at 4 °C. The primary antibody was diluted 1:800 for the PC6 anti-Pax6 antibody or 1:50 for the 12CA5 anti-HA antibody in blocking buffer and incubated with the membrane as described above. The membrane was then washed in TBST 5-6 times during 30 min before the secondary antibody (diluted 1:5000 in blocking buffer) was applied. The secondary antibody was incubated with the membrane for 1 h at room temperature. Anti-rabbit horseradish peroxidase-conjugated antibody (Transduction Laboratories) was used for the Pax6 primary antibody, whereas anti-mouse horseradish peroxidase-conjugated antibody (Transduction Laboratories) was used for the HA primary antibody. The membrane was washed as described above and then incubated for 1 min in ECL solutions (Amersham Pharmacia Biotech) (1 part of each of the solutions mixed with 2 parts of water, 1:1:2). Signals were detected by autoradiography within the next 15-20 min.

Gel Mobility Shift Assay-- Nuclear extracts were prepared as described above and used at concentrations specified in the legend to Fig. 6. Binding of Pax6 to the 32P-labeled P6CON probe (50) was performed as described by Carrière et al. (3) with the exception that 4% Ficoll was used in the binding buffer instead of 10% glycerol. The binding reactions were incubated on ice for 20 min and then run on a 5% polyacrylamide gel in 0.5× TBE (45 mM Tris borate, 1 mM EDTA). The gel was dried, and bound probe was detected by autoradiography and/or by a PhosphorImager (Molecular Dynamics).


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The Paired-less Pax6 Isoform (Pax6Delta PD) and the Paired-type Homeodomain-containing Protein Rax Are Both Able to Enhance Transactivation by Pax6 from a Minimal Promoter Containing Consensus Binding Sites for the Pax6 Paired Domain-- In a careful study using antisera raised against different parts of quail Pax6, Carrière et al. (22) identified five Pax6 isoforms expressed in quail neuroretina. The 33- and 32-kDa isoforms lack the PD but contain the HD and the C-terminal TAD. This is due to the use of an internal start codon located in the linker region between the PD and HD. The presence of two proteins of 33 and 32 kDa was explained by alternative splicing of a TAD exon (22). For simplicity, we will refer to these paired-less isoforms as Pax6Delta PD and to the major full-length Pax6 isoform as Pax6. Consistent with previous findings for quail Pax6 (22), we observed that the Pax6Delta PD isoform is coexpressed with the full-length isoform in HeLa cells transiently transfected with an expression vector for the zebrafish Pax6.1 cDNA. This was also the case when the full-length Pax6 cDNA was in vitro transcribed and translated (data not shown). Since the Pax6 and Pax6Delta PD isoforms are coexpressed, we were interested in determining if they would affect each other's activities. Knowing that Pax6 proteins with and without the paired domain bind to different DNA sequences (4), it seems logical to assume that they may transactivate different target genes in vivo as well as sharing some target genes. It is also likely that the isoforms may compete for the same set of limiting coactivators or repressors and thereby indirectly affect each other's transactivation potential. To see if Pax6Delta PD could affect the transactivation exerted by Pax6 from a consensus Pax6 paired domain-binding site, HeLa cells were transfected with increasing amounts of expression vector for Pax6Delta PD and a fixed amount of Pax6 expression vector together with a luciferase reporter (pP6CON-LUC) containing six consensus Pax6 paired domain-binding sites upstream of the adenovirus E1b minimal promoter (11). Interestingly, coexpression of Pax6Delta PD caused an increase in Pax6-mediated transactivation from paired domain-binding sites. Thus, there seems not to be a competition for coactivator(s) or limiting factors in the transcriptional apparatus (e.g. squelching). While Pax6 by itself transactivated ~6-8-fold from the P6CON binding sites, cotransfection with 2 µg of expression plasmid for Pax6Delta PD resulted in a 3 times more efficient transcriptional activation with a ~20-fold increase above the background level defined by transfection with the empty vector control (Fig. 1A). The increased transcriptional activation was not caused by Pax6Delta PD activating the reporter by itself, since transfection with this expression construct alone gave no activation at all.



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Fig. 1.   Both Pax6Delta PD and Rax can enhance Pax6-mediated transactivation of a minimal promoter containing consensus Pax6 paired domain-binding sites. A, the paired-less Pax6 isoform, Pax6Delta PD, enhances the ability of full-length Pax6 to transactivate a promoter containing paired domain-binding sites. HeLa cells were cotransfected with 0.25 µg of pCI-Pax6 and increasing amounts (0.25-2.0 µg) of pCI-Pax6Delta PD or empty expression vector (pCI-neo), together with 0.5 µg of the luciferase reporter plasmid pP6CON-LUC. The pP6CON-LUC reporter contains six consensus binding sites for the Pax6 paired domain upstream of the adenovirus E1b minimal promoter. Control transfections with 0.25 µg of pCI-neo and 0.5 µg of pCI-Pax6Delta PD or vector control show that the empty expression vector and pCI-Pax6Delta PD alone are not able to transactivate the luciferase reporter gene. B, the paired-type HD protein Rax is also able to enhance Pax6 transactivation of a promoter with paired domain-binding sites. The experiments were conducted as in A except that an expression vector for Rax (pCI-Rax) was used instead of pCI-Pax6Delta PD. As seen for Pax6Delta PD in A, Rax alone is not able to transactivate the P6CON-LUC reporter. C, Pax6Delta PD can enhance transactivation mediated by a fusion protein containing the Pax6 HD and TAD (amino acids 175-437) fused to the DNA-binding domain of yeast GAL4. HeLa cells were transfected with 2 µg of reporter vector pG5E1BLUC, 2 µg of expression vectors for GAL4 fusions, and 2 µg of expression vector for Pax6Delta PD. The GAL4 DBD vector pSG424 (51) without insert and pcDNA3-HA served as vector controls. To normalize for variations in transfection efficiencies, 0.05 µg of pCMV-beta -galactosidase was included in all transfections, allowing measurements of beta -galactosidase activities. The data are expressed as the ratio between the measured luciferase and beta -galactosidase activities with the mean ± S.E. from transfection experiments performed in triplicate. The data shown in A, B, and C are representative of two other independent experiments yielding similar results. D, schematic drawing of the full-length Pax6 isoform, the Pax6Delta PD isoform, and the Rax protein with the PD, HD, and octapeptide (OP) indicated.

The above mentioned results could be explained if 1) the Pax6Delta PD protein titrates a negative cofactor that interacts with the HD and/or TAD; 2) Pax6Delta PD can interact directly with the Pax6 protein, thus recruiting an additional TAD to the promoter; or 3) a direct binding of Pax6Delta PD to Pax6 may induce a conformational change relieving intramolecular repression. If the enhanced activation is dependent on the HD, another paired-type HD protein could possibly have the same positive effect on transcriptional activation by Pax6. Rax (also known as Rx) is a candidate for such a positive effector. It has a paired-type HD but displays no other sequence homology to Pax6, and it is coexpressed with Pax6 and Pax6Delta PD in the neuroretina (17, 18, 20). Indeed, when cotransfected with Pax6 Rax strongly enhanced transcriptional activation from the P6CON binding sites (Fig. 1B). Cotransfection of 2 µg of Rax expression vector with 0.25 µg of Pax6 expression vector resulted in a 10-15-fold increase in transactivation compared with Pax6 alone and a 30-40-fold activation above the basal level. Thus, Rax is even more efficient than Pax6Delta PD in stimulating transactivation by Pax6. Since the HD is the only homologous sequence shared by Rax and Pax6Delta PD, these results indicate that the paired-type HD is necessary for the observed superactivation. Indeed, Pax6Delta PD was able to superactivate a construct containing amino acids 175-437, which includes part of the linker region, the HD and the TAD, of Pax6 fused to the DNA-binding domain of yeast GAL4 (Fig. 1C). In this experimental setting, transactivation of a luciferase reporter gene from a promoter containing five GAL4 binding sites upstream of the TATA box from the promoter of the adenovirus E1b gene is measured (51). A construct containing only the Pax6 TAD fused to the GAL4 DBD was not superactivated upon coexpression of Pax6Delta PD (Fig. 1C). Thus, when the PD of Pax6 is substituted with the GAL4 DBD, the Pax6Delta PD isoform causes superactivation, provided the HD is present in the GAL4 fusion protein. This also demonstrates that the superactivation mediated by Pax6Delta PD is not dependent on the presence of the PD as the DNA-binding domain. As previously reported (6), it is also evident from the data in Fig. 1C that the HD has an inhibiting effect on the TAD in GAL4-Pax6Delta PD.

To discriminate between 1) titration of a negatively acting factor and 2) recruitment of an additional TAD to the promoter via HD interacting with Pax6, we asked whether the HD alone was sufficient for superactivation. As seen from Fig. 2A, cotransfection of the Pax6 expression vector with a vector expressing only the HD of Pax6 did not cause any superactivation. Due to its small size and absence of a nuclear localization signal, the HD was evenly distributed between the nucleus and the cytoplasm of the transfected cells (data not shown). Using fusions to GFP, we have found both the full-length and the Delta PD isoforms of Pax6 to be strictly nuclear proteins in living cells (Fig. 2B). This is consistent with the location of a nuclear localization signal both in the PD and directly N-terminal to the HD (3). However, Carrière et al. (3) found the Delta PD isoform also in the cytoplasm. This could be due to leakage from the nucleus upon fixation of the cells. Alternatively, there may be cell-specific differences in subcellular localization. To be absolutely certain that enough of the Pax6 HD colocalized with Pax6 in the nucleus to observe a superactivation, we also coexpressed Pax6 with a GFP-HD fusion equipped with a strong nuclear localization signal. Cotransfections with different amounts of this construct did not cause any superactivation either (data not shown). Thus, although necessary for superactivation, the HD is clearly not sufficient, suggesting that it needs to be linked to a TAD.



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Fig. 2.   The Pax6 HD alone lacking the C-terminal transactivation domain is not able to enhance Pax6 transactivation from paired domain-binding sites. A, HeLa cells were transfected as described in the legend to Fig. 1 except that increasing amounts of an expression vector for Pax6 HD were used instead of pCI-Pax6Delta PD or pCI-Rax. The data shown represent the mean ± S.E. for two independent experiments performed in triplicate. B, both the full-length isoform and the paired-less isoform of Pax6 are exclusively nuclear proteins. HeLa cells were transfected with full-length Pax6 (GFP-Pax6) or Pax6Delta PD (GFP-Pax6Delta PD) fused to green fluorescent protein (GFP), and living cells were analyzed by fluorescence microscopy (left panels) 24 h post-transfection. The right panels represent phase-contrast micrographs of the same cells with "bleed-through" from the fluorescence filter showing that only the nuclei of the cells display green fluorescence.

The Pax6 HD Interacts with Pax6, Pax6Delta PD, and Rax in Vitro-- Pax6, Pax6Delta PD, and Rax all contain a paired-type HD (Fig. 1D). Furthermore, the results of the cotransfection experiments with Pax6 and the Pax6 HD suggested that the HD is not acting by sequestering any inhibitory factors. Thus, we performed GST pull-down experiments to determine whether the Pax6 HD fused to GST could interact directly with in vitro translated Pax6, Pax6Delta PD, and Rax proteins labeled with [35S]methionine. Fig. 3 shows that the HD of Pax6 is indeed capable of interacting with the full-length Pax6 isoform, Pax6Delta PD, and Rax. Paired-like HDs are the only HDs that can cooperatively dimerize on consensus palindromic TAATN2-3ATTA sites without the need for other parts of the protein (26). The P6CON-LUC reporter used in our transcriptional activation assays does not contain such a consensus palindromic site, but to be sure that the observed interactions in GST pull-down assays are not dependent on unspecific DNA binding, these assays were also performed in the presence of 100 µg/ml EtBr. EtBr is known to inhibit protein-DNA interactions (52). As can be seen from Fig. 3, the observed interactions are direct and not dependent on DNA binding.



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Fig. 3.   The HD of Pax6 interacts with full-length Pax6, Pax6Delta PD, and Rax in vitro in a DNA-independent manner. GST pull-down assays with the Pax6 HD fused to GST and immobilized on glutathione-agarose beads and full-length Pax6, Pax6Delta PD, and Rax proteins produced by in vitro transcription and translation in the presence of [35S]methionine. Twenty-µl portions of the in vitro translation reactions (of a total of 50 µl) were preincubated with GST immobilized on glutathione-agarose beads before incubation with the GST fusion protein containing the HD of Pax6 (GST-Pax6 HD). Both the GST beads used for preincubation and the GST-Pax6 HD beads were washed several times before they were boiled and run on a 10% SDS-polyacrylamide gel. The experiments were performed with (lanes 2, 5, and 8) and without (lanes 1, 4, and 7) 100 µg/ml EtBr to determine whether the interactions were dependent on DNA binding. Five µl of the in vitro translated proteins were run on the same gel to visualize the signal from 25% of the input (lanes 3, 6, and 9). The interactions with Pax6, Pax6Delta PD and Rax are specific, since no binding was observed to GST alone (not shown)

The C-terminal Subdomain of the Pax6 PD Can Also Interact with the HDs of Pax6Delta PD and Rax-- Since the results so far indicated that the HD was responsible for the interaction, the HD was deleted from the full-length Pax6 isoform. This construct, HA-Pax6Delta HD, was then in vitro transcribed and translated and used in a GST pull-down assay with the Pax6 HD fused to GST. Surprisingly, HA-Pax6Delta HD was still able to interact with the Pax6 HD (Fig. 4A). Consistently, transient transfections where HA-Pax6Delta HD was cotransfected with Pax6Delta PD and the pP6CON-LUC reporter showed that the isoform of Pax6 lacking the PD was able to superactivate Pax6Delta HD (Fig. 4B). GST fusions containing the HD, PD, and TAD of Pax6 were therefore used in a GST pull-down assay to see if domains other than the HD would make contact with full-length Pax6. The results clearly showed that the PD but not the TAD was able to do this (Fig. 4C). Within the PD, the C-terminal subdomain (PD-C) displayed a significant interaction, while the N-terminal subdomain did not (Fig. 4D). This was also the case for Pax6Delta HD and Pax6Delta PD. Thus, interactions both between the PD (Pax6Delta HD) and between the HD (Pax6Delta PD) with the C-terminal subdomain of the PD were observed, suggesting that in full-length Pax6 both domains could in principle contribute to this interaction. However, when both the PD and the HD were missing from the Pax6 protein (HA-Pax6Delta PDDelta HD), this construct was neither capable of superactivating Pax6 when cotransfected nor able to interact with the Pax6 PD or HD in GST pull-down assays (Fig. 4, E and F, respectively). We also found that the isolated Pax6 HD and PD fused to GST both interacted strongly with in vitro translated Rax (Fig. 4G). Taken together, these results show that the paired-type HD of Pax6 and Rax can interact with each other/themselves and with the C-terminal subdomain of the PD. In addition, the PD can also interact with itself.



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Fig. 4.   Both the C-terminal subdomain of the Pax6 paired domain and the homeodomain are involved in protein-protein interactions. A, the Pax6 HD can bind to a Pax6 protein with the HD deleted. GST pull-down assay, performed as described in the legend to Fig. 3, showing that in vitro translated Pax6Delta HD binds to a GST-Pax6HD fusion protein immobilized on glutathione-agarose beads. B, Pax6Delta PD can enhance transactivation by a Pax6 protein lacking the HD. HeLa cells were transfected with 0.25 µg of pcDNA3-HA or HA-Pax6 or HA-Pax6Delta HD and 0.5 µg of pCI-Pax6Delta PD or empty expression vector (pCI-neo), together with 0.5 µg of the luciferase reporter plasmid pP6CON-LUC and 0.05 µg of pCMV-beta -galactosidase. C, the full-length isoform of Pax6 binds to both the PD and the HD but not to the TAD in vitro. D, the C-terminal subdomain (PD-C), but not the N-terminal subdomain (PD-N), of the paired domain can interact with full-length Pax6 in a GST pull-down assay (upper panel). Such an interaction can be mediated by both the PD (HA-Pax6Delta HD; middle panel) and the HD (Ha-Pax6Delta PD; lower panel) of Pax6. E, a deletion mutant of Pax6 lacking both the PD and the HD (Pax6Delta PDDelta HD) cannot enhance Pax6-mediated transactivation of the pP6CON-LUC reporter. HeLa cells were transfected with 0.25 µg of pCI-neo or pCI-Pax6 and 0.75 µg of HA-Pax6Delta PD, HA-Pax6Delta PDDelta HD, or empty expression vector (pcDNA3-HA), together with 0.5 µg of the luciferase reporter plasmid pP6CON-LUC and 0.05 µg of pCMV-beta -galactosidase. F, the deletion mutant of Pax6 lacking both the PD and the HD (Pax6Delta PDDelta HD) cannot bind to either the HD or the PD. G, Rax binds to both the HD and the PD in vitro. A GST pull-down assay is shown, in which the HD, PD, and PD-linker-HD from Pax6 fused to GST on glutathione-agarose beads are shown to bind in vitro translated Rax. H, schematic drawing of the full-length and Pax6Delta PD isoforms and the deletion mutants Pax6Delta HD and Pax6Delta PDDelta HD. The latter retains only the linker region and the TAD. The GST pull-down assays (A, C, D, F, and G) were performed as described in the legend to Fig. 3. Similar amounts of GST fusion proteins immobilized on beads were used in all experiments. The reporter gene assays (B and E) were performed essentially as described in the legend to Fig. 1, and the data shown are expressed as the ratio between the measured luciferase and beta -galactosidase activities with the mean ± S.E. from transfection experiments performed in triplicate. The results are representative of two other independent experiments yielding similar results.

Pax6Delta PD and Rax Form Protein Complexes with Full-length Pax6 in Vivo-- To see if the interaction observed in vitro in GST pull-down assays could be observed in vivo, HeLa cells were transfected with Pax6 alone or Pax6 in combination with either HA epitope-tagged Pax6Delta PD or Rax. A polyclonal affinity-purified antibody against the C terminus of Pax6 effectively coimmunoprecipitated HA-Rax, as seen on a Western blot stained with the anti-HA antibody (Fig. 5A, lane 3). The anti-Pax6 antibody does not by itself precipitate the HA-Rax protein (data not shown). The same nuclear extracts were used for immunoprecipitations with the HA antibody, with the purpose of detecting coimmunoprecipitated Pax6 proteins on Western blots using the anti-Pax6 antibody. However, a strong band of about 50 kDa was detected in all lanes of these blots, corresponding to the Ig heavy chain of the HA antibody, making it impossible to detect the 48-kDa Pax6 protein (data not shown). To avoid this problem, we transfected the paired-less isoform of Pax6, Pax6Delta PD, with HA-Pax6 or HA-Rax. As seen in Fig. 5B, the anti-Pax6 antibody was equally efficient this time in coimmunoprecipitating the HA epitope-tagged Rax protein (lane 3). This proves that the paired domain of Pax6 is not required for the interaction with Rax, since Rax is coimmunoprecipitated both with Pax6 and Pax6Delta PD. When the HA antibody was used for the immunoprecipitation, however, no detectable amounts of Pax6Delta PD was coimmunoprecipitated. This can possibly be explained by smaller amounts of the Pax6Delta PD protein in this extract compared with the others (compare lanes 11 and 12, Fig. 5B). It is also likely that the binding of the HA antibody to HA-Rax and the binding of Pax6Delta PD to HA-Rax are mutually exclusive. HA-Pax6 and Pax6Delta PD coimmunoprecipitated (Fig. 5B, lane 5), proving that Pax6 and Pax6Delta PD can interact in vivo. Based on the in vitro interactions described above, it seems reasonable to assume that the two proteins interact directly with each other also in vivo.



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Fig. 5.   Pax6Delta PD and Rax coimmunoprecipitate with the full-length Pax6 isoform from nuclear extracts of transfected HeLa cells. A, full-length Pax6 expressed from a pCI-neo vector was cotransfected with expression vectors for either HA-tagged Pax6Delta PD or HA-Rax. The PC6 polyclonal affinity-purified antibody directed against the C terminus of the Pax6 protein (alpha -Pax6) was used to immunoprecipitate (IP) Pax6 from 100 µg of HeLa cell nuclear extracts, and the monoclonal 12CA5 anti-HA antibody (alpha -HA) was used to detect coimmunoprecipitated HA-Rax (lane 3) by Western blotting (WB). The HA- Pax6Delta PD protein is also detected by the anti-HA antibody (lane 2), since this protein is also precipitated by alpha -Pax6. Lanes 4-6 show a Western blot of 5 µg of the nuclear extract used for immunoprecipitation stained with the alpha -Pax6 antibody. The upper band corresponds to full-length Pax6 (48 kDa); the lower band is Pax6Delta PD (34 kDa) coexpressed from an internal start codon of the Pax6 cDNA. The intermediate band observed in lane 5 is the cotransfected HA epitope-tagged Pax6Delta PD (36 kDa). B, to be able to detect coimmunoprecipitated Pax6 with the alpha -Pax6 antibody, we cotransfected HA-Pax6 and HA-Rax with the truncated version of Pax6, Pax6Delta PD. HeLa cell nuclear extracts (60 µg) were either immunoprecipitated with alpha -Pax6 antibody (lanes 1-3) or alpha -HA antibody (lanes 4-6), and then the alpha -HA antibody or alpha -Pax6 antibody was used to detect coimmunoprecipitated HA-tagged Pax6 (lane 2), HA-Rax (lane 3), or Pax6Delta PD proteins (lanes 4-6), respectively. Lane 5 shows that Pax6Delta PD is coimmunoprecipitated with HA-Pax6. No detectable amounts of Pax6Delta PD is coimmunoprecipitated with HA-Rax (lane 6) in this experiment. However, HA-Rax is clearly coimmunoprecipitated with Pax6Delta PD (lane 3). Lanes 7-9 and lanes 10-12 show Western blots of 3 µg of nuclear extracts stained with alpha -HA or alpha -Pax6 antibodies, respectively. The upper band in lane 11 is HA epitope-tagged Pax6.

Neither Pax6Delta PD nor Rax Affects the Binding of Pax6 to a Paired Domain-binding Site-- The nuclear extracts used for coimmunoprecipitations were used in gel mobility shift assays to see if the presence of Pax6Delta PD or Rax caused a shift in the migration of the Pax6 complex binding to the P6CON probe. No shifted complexes were detected, and the DNA binding was not affected at all upon cotransfection with Pax6Delta PD or Rax (data not shown). To be able to adjust the amounts of Pax6, Pax6Delta PD, and Rax individually, they were separately transfected into HeLa cells, and nuclear extracts were made. Gel mobility shift assays were performed with 1 µg of nuclear extract containing Pax6 combined with 1 or 4 µg of nuclear extracts containing Pax6Delta PD or Rax. As can be seen from Fig. 6, the extracts containing Pax6Delta PD or Rax alone (lanes 11-14) display no binding to the P6CON probe. Importantly, the binding of the Pax6 protein to the P6CON probe is not affected in any way by the addition of extracts containing either Pax6Delta PD (lanes 5 and 6) or Rax (lanes 9 and 10).



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Fig. 6.   Pax6Delta PD and Rax cannot bind to the P6CON binding site and do not affect the binding of Pax6 to this site. Nuclear extracts (N.E.) from HeLa cells transfected with the indicated expression constructs were used in gel mobility shift assays as described under "Materials and Methods." One µg of nuclear extract containing Pax6 bound the P6CON consensus Pax6 paired domain-binding site as seen in lane 2. This binding was neither affected by the addition of 1 or 4 µg of nuclear extract from HeLa cells transfected with Pax6Delta PD (lanes 5 and 6) or HA-tagged Rax (lanes 9 and 10) nor by the addition of nuclear extract from cells transfected with the empty expression vectors for these constructs (pCI-neo (lanes 3 and 4) and pcDNA3-HA (lanes 7 and 8), respectively). As can be seen from lanes 11-14, Pax6Delta PD and Rax are not able to bind the P6CON probe.

The Ability to Interact with Pax6 in Vitro and to Enhance Transactivation Is Not Limited to Paired-type HD Proteins-- Pax6 is coexpressed with various other HD-containing proteins during development and in the adult. To see if Pax6 can interact with other HDs as well as with the paired-type HDs of Rax and Pax6Delta PD, a selection of various HDs were fused to GST and tested for their ability to bind to in vitro translated Pax6 in a pull-down assay. These isolated HDs were from Pax6 itself as a control, Rax, Six3, Lhx2, Chx10, En-1, Prep1, Prox1, and HoxB1, and the pull-down experiments were performed both in the presence and absence of EtBr. Surprisingly, all of the different HDs were able to interact with Pax6, and this interaction was not dependent on DNA (Fig. 7A). We also tested if the full-length proteins of three very different HD proteins (Pbx-1, HoxB1, and Chx10) could interact with the PD or the HD of Pax6. As seen from Fig. 7B, these proteins could interact with both these domains. All of the tested HDs come from transcription factors coexpressed with Pax6 during development, and their HD sequences range from 65 to only 13% sequence identity with the Pax6 HD (Fig. 7C). To test whether coexpression of these HD proteins with Pax6 could affect Pax6-mediated transactivation from paired domain-binding sites, we carried out cotransfection experiments in HeLa cells with pP6CON-LUC as the reporter. This assay functions as a mammalian cell one-hybrid assay for protein-protein interactions in vivo if the protein interacting with Pax6 contains a transactivating domain. Completely consistent with this notion, Pbx1, HoxB1, Lhx2, and Chx10 were able to superactivate Pax6-mediated transactivation, while Prep1 and Prox1 did not give any significant effects. Pbx1, HoxB1, and Lhx2 contain experimentally proven TADs (53, 54). In addition, we find that both the paired-type HD proteins Rax and Chx10 act as transactivators using a reporter containing Pax6 HD-binding sites (data not shown). Prep1 definitely lacks a conventional TAD (54), while for Prox1 this is not known. Thus, these results are consistent with in vivo interactions between Pax6 and these HD proteins.



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Fig. 7.   Homeodomains from divergent homeodomain proteins interact with Pax6 in vitro, and several of these induce superactivation of Pax6-mediated transactivation. A, the HDs of Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and HoxB1 all interact with in vitro translated Pax6 in a DNA-independent manner. The GST pull-down assays were performed as described in the legend to Fig. 3. The experiments were performed with (middle panel) and without (upper panel) 100 µg/ml EtBr to determine whether the interactions were dependent on DNA binding. The asterisk indicates the Pax6Delta PD isoform, which also is translated from the full-length cDNA. The lower panel shows a Coomassie-stained SDS-polyacrylamide gel of the same amounts of GST-HD fusion proteins immobilized on beads as used in the pull-down experiments. B, full-length versions of the HD proteins Pbx1, HoxB1, and Chx10 can interact with both the PD and the HD of Pax6 in vitro. The GST-pull downs were done as described in the legend to Fig. 3. C, phylogram of the HD sequences used in the GST pull-down assay in A. The sequence identity between the Pax6 HD and the individual HDs are given in parentheses. The phylogram was made using the Growtree program of the GCG software package with Jukes-Cantor distances and the UPGMA method. D, Pbx1, HoxB1, Lhx2, and Chx10 enhance Pax6-mediated transactivation of a minimal promoter containing consensus Pax6 paired domain-binding sites. HeLa cells were cotransfected with 0.25 µg of pCI-Pax6 or empty expression vector (pCI-neo) and 0.75 µg of expression vector for the different homeodomain proteins together with 0.5 µg of pP6CON-LUC and 50 ng of pCMV-beta -galactosidase. Pbx1, Prep1, and HoxB1 were expressed from the pSG5 expression vector, while Prox1, Lhx2, Chx10, and Pax6Delta PD were expressed from pcDNA3-HA. The data shown are representative of at least three other independent experiments with each experiment performed in triplicate. The numbers above the gray columns indicate -fold activation relative to the background (white columns) level observed for transfections with expression vectors for the different homeodomain proteins alone together with empty expression vector for Pax6 (vector control).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown that the Pax6Delta PD isoform and Rax both enhance the transcriptional activation mediated by the full-length Pax6 isoform using a reporter containing P6CON consensus paired domain-binding sites upstream of a minimal promoter. The Delta PD isoform and Rax are unable to bind the P6CON binding site and do not by themselves cause transactivation from the P6CON reporter; nor do they seem to work by enhancing the DNA binding of the Pax6 PD to the reporter. In vitro the PD and HD of Pax6 are able to bind full-length Pax6, Pax6Delta PD, and Rax proteins in GST pull-down assays, and in vivo Pax6 is coimmunoprecipitated with Pax6Delta PD or Rax from HeLa cell nuclear extracts. The in vitro interactions also occur in the presence of EtBr, suggesting that they are DNA-independent. Our results show that the HDs interact with each other and with the PD and that the PD can also interact with itself. The C-terminal subdomain of the PD was found to be important as a protein-protein interaction surface.

The most surprising finding was, however, that rather divergent HDs were able to interact with Pax6 in vitro. Several of these HD proteins containing TADs could also superactivate Pax6-mediated transactivation, strongly indicating their ability to interact also in vivo. Our results illustrate the potential influence that different coexpressed HD transcription factors may have on Pax6-mediated gene regulation by interacting directly with DNA-bound Pax6 without binding to a specific DNA sequence themselves.

During the last few years, evidence indicating important roles for homeodomains in protein-protein interactions has accumulated. A number of proteins interacting with different DNA-bound HD proteins have been identified (55-62). In many cases, the HD itself is necessary and sufficient for the interactions. Hence, the cardiogenic HD protein Nkx-2.5 interacts both with the serum response factor and with the zinc finger transcription factor GATA-4 via its HD (58, 63). The RAG1 HD directly interacts with both HMG boxes of HMG1 and -2 (64). Smad1 interacts with the HD of HoxC8, leading to relief of HoxC8-mediated repression of osteopontin gene transcription (65). There are also examples of HD proteins interacting with each other independent of DNA binding. Oct-1 and Pit-1 associate in solution through an interaction mediated, in part, by the POU homeodomain of Pit-1 (66). HoxB8 and Pbx1 interact in the absence of DNA (67). The interaction was dependent on the HD and a highly conserved hexapeptide for the Hox protein and was dependent on the HD and a region immediately C-terminal of the HD for Pbx1. Prep1 and Pbx1 dimerize efficiently in solution independent of DNA (68). Similar to the results that we report here for Pax6, the DBD of the glucocorticoid receptor was found to bind to all HDs from the seven different HD proteins tested in a GST pull-down assay (69). Expression of the glucocorticoid receptor DBD in one-cell stage zebrafish embryos severely affected their development, while expression of a point mutant that could no longer bind HDs developed normally (69). As with our findings with Pax6, the extent of glucocorticoid receptor DBD-HD binding in vivo remains to be established. If, and to what degree, interactions occur in different tissues where Pax6 and other in vitro interacting HD proteins are coexpressed must be evaluated carefully for each potential interaction.

We show for the first time that the HD and the PD of Pax6 can interact off DNA. Several previous reports show that the PD and HD can interact with each other both intermolecularly (31) and intramolecularly (70) while bound to DNA. The PD/HD cooperativity reported by Jun and Desplan is not specific for domains within the same protein, since the Prd PD can cooperatively interact with the Engrailed HD to bind to DNA. The Pax6 PD and Pax8 PD can likewise cooperatively bind together with the Prd HD to DNA. DNA binding by the Pax3 PD and HD is reported to be functionally interdependent, since certain mutations in the PD affect the DNA binding by the HD, and vice versa (71-73). Recently, it was reported that the R26G mutation in the N-terminal subdomain of the PD of Pax6, which abolishes DNA binding by the PD, lead to increased DNA binding by the HD. Furthermore, the I87R mutation in the C-terminal subdomain of the PD resulted in loss of the ability to bind DNA via either the PD or the HD (74). The first effect suggests interactions or at least conformational changes affecting both domains. The latter effect may be related to our finding that it is the C-terminal subdomain of the PD that interacts with the HD. Perhaps such an interaction could be involved in stabilizing the DNA binding conformation of both domains. Of other reported intermolecular interactions involving paired-type HD proteins, Phox1 interacts with the serum response factor, and this interaction seems to apply for other paired-type HDs as well. The products of the Drosophila paired and orthodenticle genes can bind serum response factor, but the more distantly related homeobox genes fushi tarazu and Deformed can not (39, 75). Rb was recently reported to interact directly with factors containing paired-like HDs, including Pax3 (36). The residual HD homologous sequence of Pax5 interacts with Rb and TBP (38), and the HD of Pax6 does the same (37). The repressor protein hDaxx and the WD40 repeat protein HIRA have both been shown to interact with the HD of Pax3 and Pax7 (76, 77). The HD protein Cdx-2/3 was recently found to interact with both the PD and the HD of Pax6 (78). This is completely consistent with our results for Rax, Chx10, Pbx1, and HoxB1. As mentioned in the introduction, En-1 interacts with the PD of Pax6 (32) and Pax5, via its PD, acts as a cell type-specific docking protein for an ETS family transcription factor at the B cell-specific mb-1 promoter (33). In fact, the Pax5 PD alone is sufficient for transcriptional activation from this promoter (34). By interacting with another ETS family protein, PU.1, Pax5 mediates repression of the immunoglobulin kappa  3' light chain enhancer (35). The Pax5 PD has also been found to interact with the runt DBD of AML1 (79). Several observations of Pax gene behavior can be attributed to the PD and/or HD being involved in protein-protein interactions. First, the Pax3 HD alone fused to a GAL4 binding site can work as a repressor of transcription (80). Second, Pax3 can repress activity from the N-CAM promoter through a mechanism independent of DNA binding (81). Third, Pax6 has recently been found to be a repressor of the beta B1-crystallin promoter (82). The repression activity was dependent on the PD and the HD but not the TAD. Pax6-5a or the PD alone could not repress transcription. Fourth, Pax5 has been reported to act as both an activator and a repressor of genes in B cells dependent on the promoter context (83). Together, these results indicate that Pax protein interactions with other proteins on the promoter is important in determining the function of the Pax proteins as activators or repressors. This has also been reported for some Hox proteins (see Ref. 84 and references therein).

What is the biological relevance of Pax6 interaction with Pax6Delta PD, Rax, other paired-type HD proteins, and even very divergent HD proteins? All of the proteins we have tested in this work are coexpressed with Pax6 in some tissue(s) during development. Except for Hox1, Pbx1, and Prep1, the other six HD proteins are all expressed in the developing eye and crucial for normal development of the eyes. For example, Pax6 and Rax are expressed in overlapping areas of the neuroretina. They are both expressed uniformly in the early retina and then restricted to the inner nuclear layer (8, 17). The distinct and overlapping expression patterns of Pax6 and Rax and/or other HD proteins could possibly cause a fine tuning of the transcriptional regulation of target genes. It is known that the eyes are especially dosage-sensitive to the expression of Pax6 protein (13-15). If Pax6 function is dependent upon specific interactions with other proteins through its PD or HD, this could contribute to the observed dosage sensitivity. In this scenario, both loss-of-function and gain-of-function of Pax6 gene expression would disturb the delicate balance between factors participating in these interactions. In conclusion, our studies suggest that both the HD and the PD of Pax6 should be viewed not only as DNA-binding domains but also as protein-protein interaction domains capable of both intramolecular and intermolecular interactions.


    ACKNOWLEDGEMENTS

We are grateful to J. Botas, C. L. Cepko, P. Gruss, A. Joyner, R. R. McInnes, S. Tomarev, and V. Zappavigna for the generous gift of cDNA clones for murine Lhx2, Rax, Six3, En-1, Chx10, human Prox1, Prep1, and HoxB1, respectively.


    FOOTNOTES

* This work was supported by grants from the Norwegian Cancer Society, the Norwegian Research Council, the Aakre Foundation, and the Blix Foundation (to T. J.).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 Fellow of the Norwegian Research Council.

§ These authors contributed equally to this work.

Fellow of the Norwegian Cancer Society.

|| To whom correspondence should be addressed: Dept. of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway. Tel.: 47-776-44720; Fax: 47-776-45350; E-mail: terjej@fagmed.uit.no.

Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008882200


    ABBREVIATIONS

The abbreviations used are: PD, paired domain; GST, glutathione S-transferase; DBD, DNA-binding domain; GFP, green fluorescent protein; HD, homeodomain; PCR, polymerase chain reaction; TAD, transactivation domain; HA, hemagglutinin; CMV, cytomegalovirus.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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