Truncation Mutations in the Transactivation Region of PAX6 Result in Dominant-Negative Mutants
1998; Elsevier BV; Volume: 273; Issue: 34 Linguagem: Inglês
10.1074/jbc.273.34.21531
ISSN1083-351X
AutoresSanjaya Singh, Hank Kejun Tang, Jing‐Yu Lee, Grady F. Saunders,
Tópico(s)Marine Ecology and Invasive Species
ResumoPAX6 is a transcription factor with two DNA-binding domains (paired box and homeobox) and a proline-serine-threonine (PST)-rich transactivation domain. PAX6 regulates eye development in animals ranging from jellyfish toDrosophila to humans. Heterozygous mutations in the humanPAX6 gene result in various phenotypes, including aniridia, Peter's anomaly, autosomal dominant keratitis, and familial foveal dysplasia. It is believed that the mutated allele ofPAX6 produces an inactive protein and aniridia is caused due to genetic haploinsufficiency. However, several truncation mutations have been found to occur in the C-terminal half ofPAX6 in patients with Aniridia resulting in mutant proteins that retain the DNA-binding domains but have lost most of the transactivation domain. It is not clear whether such mutants really behave as loss-of-function mutants as predicted by haploinsufficiency. Contrary to this theory, our data showed that these mutants are dominant-negative in transient transfection assays when they are coexpressed with wild-type PAX6. We found that the dominant-negative effects result from the enhanced DNA binding ability of these mutants. Kinetic studies of binding and dissociation revealed that various truncation mutants have 3–5-fold higher affinity to various DNA-binding sites when compared with the wild-type PAX6. These results provide a new insight into the role of mutant PAX6 in causing aniridia. PAX6 is a transcription factor with two DNA-binding domains (paired box and homeobox) and a proline-serine-threonine (PST)-rich transactivation domain. PAX6 regulates eye development in animals ranging from jellyfish toDrosophila to humans. Heterozygous mutations in the humanPAX6 gene result in various phenotypes, including aniridia, Peter's anomaly, autosomal dominant keratitis, and familial foveal dysplasia. It is believed that the mutated allele ofPAX6 produces an inactive protein and aniridia is caused due to genetic haploinsufficiency. However, several truncation mutations have been found to occur in the C-terminal half ofPAX6 in patients with Aniridia resulting in mutant proteins that retain the DNA-binding domains but have lost most of the transactivation domain. It is not clear whether such mutants really behave as loss-of-function mutants as predicted by haploinsufficiency. Contrary to this theory, our data showed that these mutants are dominant-negative in transient transfection assays when they are coexpressed with wild-type PAX6. We found that the dominant-negative effects result from the enhanced DNA binding ability of these mutants. Kinetic studies of binding and dissociation revealed that various truncation mutants have 3–5-fold higher affinity to various DNA-binding sites when compared with the wild-type PAX6. These results provide a new insight into the role of mutant PAX6 in causing aniridia. PAX6 is an evolutionarily conserved gene that regulates the development of the eye in animals ranging from jellyfish toDrosophila to humans (for review, see Ref. 1Callaerts P. Halder G. Gehring W.J. Annu. Rev. Neurosci. 1997; 20: 483-523Crossref PubMed Scopus (393) Google Scholar). The induction of ectopic compound eyes by overexpressing mouse and squidPax6 (2Halder G. Callaerts P. Gehring W.J. Science. 1995; 267: 1788-1792Crossref PubMed Scopus (1275) Google Scholar, 3Tomarev S.I. Callaerts P. Gehring W.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2421-2426Crossref PubMed Scopus (160) Google Scholar) clearly indicates that not only is the structure of PAX6 conserved but also its biochemical properties are conserved. Recent reports have shown that PAX6 is also involved in pancreas development (4Sander M. Neubuser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (468) Google Scholar, 5St-Onge L. Sosa-Pineda B. Chaowdhury K. Mansouri A. Gruss P. Nature. 1997; 387: 406-409Crossref PubMed Scopus (671) Google Scholar). Like other members of the PAX family, PAX6 functions as a transcriptional activator. Structural analysis of PAX6 has identified two DNA-binding domains (a paired domain at the N terminus and a paired like homeodomain in the middle), a glycine-rich hinge region that links the two DNA-binding domains, and a proline-serine-threonine-rich (PST) transactivation domain at the C terminus. Our recent studies designed to characterize the transactivation domain revealed that the four exons, which constitute the PST domain, synergistically stimulate transcriptional activation and that the transactivation potential is not localized but distributed throughout the PST domain (6Tang H.K. Singh S. Saunders G.F. J. Biol. Chem. 1998; 273: 7210-7221Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The transcription factors of the PAX family recognize their target genes via the DNA binding function of the paired domain (7Chalepakis G. Wijnholds J. Giese P. Schachner M. Gruss P. DNA Cell Biol. 1991; 13: 891-900Crossref Scopus (77) Google Scholar, 8Treisman J. Harris E. Desplan C. Genes Dev. 1991; 5: 594-604Crossref PubMed Scopus (289) Google Scholar). Several PAX6 paired-domain binding sequences have already been identified (reviewed in Ref. 1Callaerts P. Halder G. Gehring W.J. Annu. Rev. Neurosci. 1997; 20: 483-523Crossref PubMed Scopus (393) Google Scholar). However, studies of Czerny and Busslinger (9Czerny T. Busslinger M. Mol. Cell. Biol. 1995; 15: 2858-2871Crossref PubMed Scopus (263) Google Scholar) identifying the P3 site as the optional binding site for cooperative binding and transactivation by the PAX6 homeodomain, conservation of P3 in eye-specific promoters (10Wilson D. Gunther B. Desplan C. Kuriyan J. Cell. 1995; 82: 702-719Abstract Full Text PDF Scopus (305) Google Scholar), and the presence and requirement of the P3 site in the rhodopsin expression in Drosophila (11Fortini M.E. Rubin G.M. Genes Dev. 1990; 4: 444-463Crossref PubMed Scopus (170) Google Scholar) indicated that several genes could also be regulated through the homeodomain of PAX6. Mutations in PAX6 genes are responsible for several naturally occurring mutant phenotypes including aniridia in humans (12Glaser T. Walton D.S. Maas R.L. Nat. Genet. 1992; 2: 232-239Crossref PubMed Scopus (571) Google Scholar, 13Jordan T. Hansen I. Zaletayev D. Hodgson S. Prosser J. Seawright A. Hastie N.D. van Heyningen V. Nat. Genet. 1992; 1: 328-332Crossref PubMed Scopus (481) Google Scholar, 14Lyons L.A. Martha A. Mintz-Hittner H.A. Saunders G.F. Ferrell R.E. Genomics. 1992; 13: 925-930Crossref PubMed Scopus (34) Google Scholar, 15Hanson I.M. Seawright A. Hardman K. Hodgson S. Zeletayev D. 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Drechsler M. Royer-Pokora B. Collins F. Swaroop A. Strong L.C. Saunders G.F. Cell. 1991; 67: 1059-1074Abstract Full Text PDF PubMed Scopus (749) Google Scholar), and eyeless inDrosophila (22Quiring R. Walldorf U. Kloter U. Gehring W.J. Science. 1994; 265: 785-789Crossref PubMed Scopus (906) Google Scholar). The human and murine PAX6 proteins have an identical amino acid sequence (23Walthers C. Gruss P. Development. 1991; 113: 1435-1449PubMed Google Scholar, 54Ton C.C.T. Miwa H. Saunders G.F. Genomics. 1992; 13: 251-256Crossref PubMed Scopus (68) Google Scholar). Aniridia and small-eye are similar eye phenotypes (19Hill R.E. Favor J. Hogan B.L.M. Ton C.C.T. Saunders G.F. Hanson I.M. Posser J. Jordan T. Hastie N.D. van Heyningen V. Nature. 1991; 354: 522-525Crossref PubMed Scopus (1168) Google Scholar), with both being characterized by iris hypoplasia and cataracts. Mutations in PAX6 result in semidominant phenotypes,i.e. heterozygous mutations cause aniridia in humans and small-eye in rodents, while homozygous mutations lead to severe brain abnormalities, microencephaly, and early postnatal death with no eyes and no nose in rodents (20Matsuo T. Osumi-Yamashita N. Noji S. Ohuchi H. Koyama E. Myokai F. Matsuo N. Taniguchi S. Doi H. Iseki S. Ninomiya Y. Fujiwara M. Watanabe T. Eto K. Nat. Genet. 1993; 3: 299-304Crossref PubMed Scopus (260) Google Scholar, 24Schmahl W. Knoedlseder M. Favor J. Davidson D. Acta Neuropathol. 1993; 86: 126-135Crossref PubMed Scopus (180) Google Scholar) and humans (25Glaser T. Jepeal L. Edwards J.G. Young S.R. Favor J. Maas R.L. Nat. Genet. 1994; 7: 463-469Crossref PubMed Scopus (602) Google Scholar). In addition, mice with heterozygous mutations in PAX6 have lower levels of pancreatic hormones (4Sander M. Neubuser A. Kalamaras J. Ee H.C. Martin G.R. German M.S. Genes Dev. 1997; 11: 1662-1673Crossref PubMed Scopus (468) Google Scholar). A haploinsufficiency mechanism has been postulated for the heterozygous phenotype. According to this theory, the function of the protein product of the mutant allele is lost and the normal PAX6 protein produced by the wild-type allele does not reach the threshold level necessary for normal eye development (1Callaerts P. Halder G. Gehring W.J. Annu. Rev. Neurosci. 1997; 20: 483-523Crossref PubMed Scopus (393) Google Scholar). The mutation spectrum of PAX6 observed inaniridia and small-eye includes large deletions as well as intragenic mutations (1Callaerts P. Halder G. Gehring W.J. Annu. Rev. Neurosci. 1997; 20: 483-523Crossref PubMed Scopus (393) Google Scholar, 26Prosser J. van Heyningen V. Hum. Mutat. 1998; 11: 93-108Crossref PubMed Scopus (231) Google Scholar, 27Glaser T. Walton D.S. Cai J. Epstein J.A. Jepeal L. Maas R.L. Wiggs J.L. Molecular Genetics of Ocular Disease. Wiley-Liss, New York1995: 51-82Google Scholar, 41Saunders G.F. Chao L.Y. Human Mutat. 1998; : S207-S208Crossref PubMed Scopus (5) Google Scholar). Most intragenic mutations result in truncations of the PAX6 protein; only four of the detected mutants are due to missense mutations, one that affects the nuclear translocation signal (15Hanson I.M. Seawright A. Hardman K. Hodgson S. Zeletayev D. Fekete G. van Heyningen V. Hum. Mol. Genet. 1993; 2: 915-920Crossref PubMed Scopus (148) Google Scholar, 17Martha A. Ferrell R.E. Mintz-Hittner H. Lyons L.A. Saunders G.F. Am. J. Hum. Genet. 1994; 54: 801-811PubMed Google Scholar) and three that affect the paired DNA-binding domain (28Hanson I.M. Fletcher J.M. Jordan T. Brown A. Taylor D. Adams R.J. Punnett H.H. van Heyningen V. Nat. Genet. 1994; 6: 168-173Crossref PubMed Scopus (442) Google Scholar, 29Azuma N. Nishina S. Yanagisawa H. Okuyama T. Yamada M. Nat. Genet. 1996; 13: 141-142Crossref PubMed Scopus (170) Google Scholar, 30Tang H.K. Chao L. Saunders G.F. Hum. Mol. Genet. 1997; 6: 381-386Crossref PubMed Scopus (69) Google Scholar). The loss of function in the mutant PAX6 proteins that have lost the DNA binding can be clearly explained. What is not clear is the role of truncated PAX6 proteins that arise due to certain mutations that occur at the C-terminal region ofPAX6 and result in mutants that (i) lack all or part of the PST domain but retain both DNA-binding domains or (ii) lack all of the PST domain and part of the homeodomain but retain the paired-box DNA-binding domain (1Callaerts P. Halder G. Gehring W.J. Annu. Rev. Neurosci. 1997; 20: 483-523Crossref PubMed Scopus (393) Google Scholar, 26Prosser J. van Heyningen V. Hum. Mutat. 1998; 11: 93-108Crossref PubMed Scopus (231) Google Scholar, 27Glaser T. Walton D.S. Cai J. Epstein J.A. Jepeal L. Maas R.L. Wiggs J.L. Molecular Genetics of Ocular Disease. Wiley-Liss, New York1995: 51-82Google Scholar). In the study reported here, we demonstrate that these mutants are not simple loss-of-function mutants or unstable mutants. On the contrary, they have gained a stronger DNA-binding capacity, and since they lack most of the transactivation activity, thereby function as dominant-negative repressors. Surprisingly, we found that all those mutants with intact DNA-binding domains were able to repress the wild-type PAX6 activity by binding to the target site with a higher affinity than wild-type protein. NIH 3T3, a murine fibroblast cell line, was maintained in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum. HLEB3, a human lens epithelial cell line (31Andley U.P. Rhim J.S. Chylack L.T. Fleming T.P. Invest. Ophthalmol. Vis. Sci. 1994; 35: 3094-3102PubMed Google Scholar), was maintained in Eagle's minimal essential medium supplemented with 20% fetal calf serum. pRc-CMV-PAX6 expression plasmids were constructed by using a polymerase chain reaction cloning strategy. In brief, specific regions of PAX6 were amplified by polymerase chain reaction using the cDNA clone ph12 (21Ton C.C.T. Hirvonen H. Miwa H. Weil M.M. Monaghan P. van Heyningen V. Hastie N.D. Meijers-Heijboer H. Drechsler M. Royer-Pokora B. Collins F. Swaroop A. Strong L.C. Saunders G.F. Cell. 1991; 67: 1059-1074Abstract Full Text PDF PubMed Scopus (749) Google Scholar) as a template. Primers used to amplify specific regions of PAX6 are listed in Table I. All the C-terminal deletion constructs used a common 5′ primer containing a HindIII restriction site and a Kozak consensus sequence for translational initiation (PAX6(1–422)). The 3′ primers differed in sequence, but all contained an SpeI restriction site and a stop codon. Endonuclease digestion with SpeI and XbaI generated compatible cohesive ends. TheHindIII-SpeI-digested polymerase chain reaction products were ligated into the HindIII-XbaI restriction sites in the polylinker region of the expression vector pRc-CMV (Invitrogen). The construction of R26G and I87R mutants is described elsewhere (30Tang H.K. Chao L. Saunders G.F. Hum. Mol. Genet. 1997; 6: 381-386Crossref PubMed Scopus (69) Google Scholar).Table IOligos used for polymerase chain reaction cloning of pRc-CMV-PAX6 constructsConstructsPrimer sequencePAX6 (1–422)5′ primer....5′-GCCCAAGCTTCCAGCATGCAGAACAGTC-3′3′ primer....5′-GGACTAGTCTTACTGTAATCTTGGCCAGTA-3′PAX6 (1–395)5′ primer....same as used for PAX6 (1–422) above3′ primer....5′-GGACTAGTCTTACTGTAATCTTGGCCAGTA-3′PAX6 (1–344)5′ primer....same as used for PAX6 (1–422) above3′ primer....5′-GACTAGTCGGATCCTTATTGCATAGGCAGGTTATTTG-3′Pax6 (1–306)5′ primer....same as used for PAX6 (1–422) above3′ primer....5′-GACTAG-TCGGATCCTTAAACCGGTGTGGTGGGTTG-3′PAX6 (1–272)5′ primer....same as used for PAX6 (1–422) above3′ primer....5′-GACTAGTCATTTTTCTTCTCTTCTCCATTT-3′PAX6 (1–353)5′ primer....same as used for PAX6 (1–422) above3′ primer....5′-GGACTAGTCTCAGGAGGTCTGGCTGGGACTGG-3′PAX6 (1–317)5′ primer....same as used for PAX6 (1–422) above3′ primer....5′-GGACTAGTCTCAGCCCAACATGGAGCCAGATGT-3′PAX6 (1–267)5′ primer....same as used for PAX6 (1–422) above3′ primer....5′-GGACTAGTCTCATCTCCATTTGGCCCTTCGATT-3′PAX6 (1–240)5′ primer....same as used for PAX6 (1–422) above3′ primer....5′-GGACTAGTCTCAGGCAAACACATCTGGATAATG-3′ Open table in a new tab HLEB3 and NIH 3T3 cells were plated at a density of 4–6 × 105 cells/60-mm or 2 × 105 cells/35-mm tissue culture dish 24 h before transfection. Transfections for all cells were performed with plasmid DNA coated with the polycationic lipid LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's instructions. NIH 3T3 cells were transfected at 60–80% confluency using 15 μl (30 μg) of LipofectAMINE per 60-mm dish. HEB3 cells were transfected at 70–80% confluency using 6 μl (12 μg) of LipofectAMINE per 60-mm dish. For transfections with the pRc-CMV-PAX6 expression vectors, each 60-mm dish was transfected with 2 μg of CD19–2(A-ins)-luciferase (Czerny and Busslinger, 1995), 1 μg of pRc-CMV effector plasmid, and 0.4 μg of pSV2 β-galactosidase plasmid (Promega) as internal control. Cell extracts were prepared after 24–48 h of transfection. Luciferase activity was measured at room temperature using a luciferase assay kit (Tropix, Bedford, MA). Briefly, 50-μl aliquots of substrate A were placed into 75 × 12-mm luminometer sample tubes (Sarstedt). Then, 10 μl of individual cell extracts and 50 μl of substrate B were added sequentially into the tubes, which were then placed immediately into a luminometer for measurement. The luminometer was set to measure the luciferase signal for 10 s with a 2-s delay. β-galactosidase activity was detected using a Galacto-Light Plus chemiluminescent assay kit (Tropix) according to manufacturer's protocol. The measurement protocol was the same as for the luciferase assay. Luciferase activities were normalized relative to β-galactosidase activity. Crude nuclear extracts were prepared from transfected HEB3 or NIH 3T3 cells according to the method of Schreiber et al. (32Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3918) Google Scholar) as modified by Singh and Aggarwal (33Singh S. Aggarwal B.B. J. Biol. Chem. 1995; 270: 24995-25000Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar). The cells were harvested from each 60-mm culture dish and lysed in 0.2 ml of lysis buffer (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 2.0 mg/ml leupeptin, 2.0 mg/ml aprotinin, and 0.5 mg/ml benzamidine). The nuclear pellet was resuspended in 25 μl of extraction buffer (20 mm HEPES, pH 7.9, 0.4 mNaCl, 1 mm EDTA, 1 mm EGTA, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2.0 mg/ml leupeptin, 2.0 mg/ml aprotinin, and 0.5 mg/ml benzamidine). The protein concentration of each nuclear extract was measured by the Bradford assay and adjusted to equal protein concentration after dilution with the nuclear extraction buffer. Electrophoretic mobility shift assays were performed in a 20-μl binding reaction incubated at room temperature for 30 min in a binding buffer (25 mm HEPES, pH 7.9, 0.5 mm EDTA, 0.5 mm dithiothreitol, 1% Nonidet P-40, 5% glycerol, and 150 mm NaCl) (33Singh S. Aggarwal B.B. J. Biol. Chem. 1995; 270: 24995-25000Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar). Each binding reaction contained 0.25 to 1 μg of nuclear extracts, 1–3 μg of poly(dI-dC), and 16–100 fmol of probes labeled with 32P by T4 polynucleotide kinase. Protein-DNA complexes were separated from free probes on a 4.5 or 6% native polyacrylamide gel using a buffer containing 50 mmTris, 200 mm glycine, pH 8.5, and 1 mm EDTA. The gel was dried and then analyzed by a PhosphorImager (Molecular Dynamics) using Image Quant 3.3 software (Molecular Dynamics, Sunnyvale, CA). For supershift assays, the binding reaction mixture was incubated with the polyclonal antibody against PAX6 at room temperature for 30 min before loading onto the gel. The nuclear extracts prepared from transfected NIH 3T3 or HLEB3 cells were resolved by 10% SDS-PAGE, 1The abbreviations used are: PAGEpolyacrylamide gel electorphoresisEMSAelectrophoretic mobility shift assayC/EBPcAMP responese element-binding proteinlucluciferaseaaamino acid(s). electrotransfered to a nitrocellulose membrane, and then analyzed for PAX6expression by hybridization with polyclonal antibodies raised against PAX6(16–422) or against the paired domain or linker region of PAX6 (34Carriere C. Plaza S. Martin P. Quatannens B. Bailly M. Stehelin D. Saule S. Mol. Cell. Biol. 1993; 13: 7257-7266Crossref PubMed Scopus (112) Google Scholar). The protein band was detected by enhanced chemiluminescence using a horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (ECL, Amersham). The bands on the Western blots were quantitated using Personal Densitometer Scan v1.30 and Image Quanta v3.3 software (Molecular Dynamics). polyacrylamide gel electorphoresis electrophoretic mobility shift assay cAMP responese element-binding protein luciferase amino acid(s). Haploinsufficiency diseases such as aniridia and small-eye usually result from heterozygous mutations in which a mutant null allele coexists with a wild-type allele. In some cases, competition between the protein products of the mutant allele and the wild-type allele may result in phenotypic variability. PAX6 strongly activates transcription of the CD19-luc reporter, whereas most mutant PAX6 proteins with truncated PST domains have less than 30% of the wild-type activity (Figs. 2 A and 6). Since such mutant PAX6 proteins retain the paired domain, they may compete for target binding sites with the wild-type PAX6. To test this possibility in vivo, we co-transfected equal amounts of mutant and wild-type PAX6 expression plasmids (Fig. 1) with a CD19-luc reporter into NIH 3T3 cells, and then assessed the combined transactivation potential of mutant and wild-type PAX6 (Fig.2, A and B). Interestingly, all mutant PAX6 proteins with truncations in the PST domain repressed the wild-type PAX6 activity (Fig. 2, A andB). However, the missense mutants PAX6 (I87R) and PAX6 (R26G), which lose their DNA binding ability through the paired domain (30Tang H.K. Chao L. Saunders G.F. Hum. Mol. Genet. 1997; 6: 381-386Crossref PubMed Scopus (69) Google Scholar), did not repress the activity of wild-type PAX6. The repression by truncation mutants was much stronger than expected from a simple competition for DNA-binding sites of the wild-type and mutant PAX6 proteins and suggested that mutant PAX6 had dominant-negative effects.Figure 6Kinetic analysis of CD19–2(A-ins)-binding site to increasing amounts of PAX6 and truncation mutants. Fold differences in KD value between wild-type and truncated (mutant) PAX6 proteins. Since PAX6 binds DNA as monomer through its paired domain we calculated the fold difference inKD values using the following formula: Dis DNA, P is wild-type protein, and Pm is mutant protein: KDW/KDM = ([P][D]/[PD]) × ([PM D]/[PM][D]). At any point in the log phase of reaction where amount of probe and protein used were same: KDW/KDM = [PD]/PM D]. Increasing concentrations of wild-type and mutant proteins were individually incubated at the indicated concentrations with the32P-labeled probe as described under "Materials and Methods." The protein-DNA complexes were separated from free DNA by gel electrophoresis and the amount of free DNA and protein-DNA complexes were quantitated using a PhosphorImager and Image Quant program as described under "Materials and Methods." The data shown is mean ± S.E. of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Schematic diagram of wild-type and mutant PAX6 expression constructs. PAX6 cDNA and its mutant derivatives were inserted into the parental expression vector pRc-CMV. Schematic diagram of the full-length human PAX6(1–422) protein is shown at the top. The numbers abovethe diagram refer to amino acid number. The numbers inside the white bars indicate exon number. Thenumbers inside the parentheses in the name of each mutant construct correspond to the N-terminal and C-terminal amino acid residues. PD, paired domain; G, glycine-rich domain; HD, homeodomain; PST, proline/serine/threonine-rich domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To analyze the mechanism of the dominant-negative effects by mutant PAX6, we assessed the DNA binding ability of wild-type and mutant PAX6 in electrophoretic mobility shift assay (EMSA). We analyzed the DNA binding properties of the nuclear extracts prepared from NIH 3T3 cells co-transfected with mutant and wild-type PAX6 expression vectors. The wild-type PAX6 showed binding to the CD19–2(A-ins) probe (Fig.2 C, lane 2). In the presence of mutant PAX6, however, the wild-type PAX6 showed very weak DNA binding, whereas mutant PAX6 showed strong DNA binding (Fig. 2 C, lanes 3–6). No intermediate complex was detected between the wild-type and mutant PAX6-DNA complexes, suggesting that both mutant and wild-type PAX6 bound DNA as monomers. Assuming that both the mutant and wild-type PAX6 proteins accumulated in nuclei at comparable levels, the simplest model for the dominant repression effect of the mutants would be that deletion of the PST domain resulted in increased DNA binding ability of mutant PAX6. The same nuclear extracts used in the EMSA were assessed for expression of wild-type and mutant PAX6 proteins by Western blotting using an immune serum raised against Escherichia coli-produced recombinant PAX6 (16–422). As expected, the wild-type PAX6 protein was detected at constant levels in extracts transfected with wild-typePAX6 alone or co-transfected with wild-type and mutantPAX6 (data not shown), indicating that the reduction in DNA binding of wild-type PAX6 was due to the competition for DNA-binding sites by the mutant PAX6 rather than a reduction in protein level. Although the antibody used cannot quantitatively detect each mutant PAX6 protein because it was raised against the PAX6 with an intact PST domain, the mutant proteins had accumulated in sufficient amounts to be detected by the polyclonal antibody and EMSA. It is also possible that the mutant proteins were expressed in much higher quantity than the wild type, and therefore showed higher binding. However, later experiments shown in Figs. 5, A-C, 7, and 8 revealed that this was not the reason for lower PAX6 binding.Figure 7Dissociation analysis of complex of PAX6 or truncation mutants with CD19–2(A-ins)-binding site by increasing amounts of unlabeled binding sites. Using a fixed amount of either wild type or mutant (A) PAX6(1–317), (B) PAX6(1–353) protein and radiolabeled probe and increasing amounts of unlabeled oligonucleotides an EMSA was performed. Scatchard plots generated by plotting the ratio of bound labeled oligonucleotides to total labeled nucleotides versus amount of total oligonucleotides are shown. The trend lines were generated using "Microsoft Excel 97" of Microsoft to calculate the slope. The slope of trend line indicates the difference in affinities of wild-type and mutant proteins. The data shown is mean ± S.E. of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Dominant-negative effects of mutant PAX6 with truncated PST domains with the homeodomain-binding site. The histogram shows the repression of the transactivation activity of PAX6 by the truncation mutants. 0.5 μg of plasmid for wild-type or mutant proteins were used in each transfection. Total concentration of plasmid was maintained at 1.0 μg using empty expression vector. Mutant proteins were transfected alone or in the presence of PAX6. The relative luciferase activities of the reporter construct containing a homeodomain-binding site (P3) are shown as mean + S.E. of three separate transfection experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine the specificity of DNA binding of mutant and wild-type PAX6 in the nuclear extracts, we performed oligonucleotide competition and antibody supershift analyses in EMSA. Nuclear extracts were prepared from NIH 3T3 cells transfected with either a single plasmid of the wild-type PAX6, mutant PAX6(1–344), mutant PAX6(4V/G), or an equimolar mixture of wild-type PAX6 and mutant PAX6(1–344) plasmids. Since missense mutations have not been detected in aniridia patients, mutant PAX6(4V/G) was generated to find out the possible effect of missense mutations. This mutant did not show any significant difference in the transactivation potential nor on DNA binding (6Tang H.K. Singh S. Saunders G.F. J. Biol. Chem. 1998; 273: 7210-7221Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Protein expression of transfected mutant and wild-type PAX6was detected by the polyclonal antibody against the paired domain (data not shown). The protein-DNA complexes formed by the binding of each nuclear extract to the CD19–2(A-ins) probe (Fig.3 A, lanes 3, 7, 11, and15) were efficiently ablated by adding either a 100-fold excess of unlabeled CD19–2(A-ins) probe (Fig. 3 A, lanes 4, 8, 12, and 16) or polyclonal antibody against PAX6 (aa 16–422) (Fig. 3 A, lanes 5, 9, 13, and 17), but not by adding the nonspecific antibody (Fig. 3 A, lanes 6, 10, 14, and 18). Addition of the anti-PAX6 antibody also resulted in supershifting of the protein-DNA complexes (Fig. 3 A, lanes 5, 9, 13, and 17). Interestingly, the DNA-PAX6(1–344) complex was ablated less efficiently by the specific oligonucleotide and antibody than was the wild-type PAX6-DNA complex (Fig. 3 A, lanes 16 and 17), consistent with our finding that the DNA binding ability of the mutant PAX6(1–344) was stronger than that of the wild-type P
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