Dissection of the Transactivation Function of the Transcription Factor Encoded by the Eye Developmental Gene PAX6
1998; Elsevier BV; Volume: 273; Issue: 13 Linguagem: Inglês
10.1074/jbc.273.13.7210
ISSN1083-351X
AutoresHank Kejun Tang, Sanjaya Singh, Grady F. Saunders,
Tópico(s)Animal Genetics and Reproduction
ResumoPAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 isproline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. We dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. Our data consistently showed that in both systems all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act synergistically to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6, although the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. Our data suggest that these mutants may antagonize the wild-type PAX6 activity by competing for target DNA-binding sites. We conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree and that its full transactivation activity relies on the synergistic action of the four exon fragments. PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 isproline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. We dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. Our data consistently showed that in both systems all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act synergistically to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6, although the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. Our data suggest that these mutants may antagonize the wild-type PAX6 activity by competing for target DNA-binding sites. We conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree and that its full transactivation activity relies on the synergistic action of the four exon fragments. Aniridia is a congenital eye disorder characterized by complete or partial absence of the iris (1Shaw M.W. Fall H.F. Neel J.V. Am. J. Hum. Genet. 1960; 12: 389-415PubMed Google Scholar, 2Nelson L.B. Spaeth G.L. Nowinski T.S. Margo C.E. Jackson L. Surv. Ophthalmol. 1984; 28: 621-642Abstract Full Text PDF PubMed Scopus (352) Google Scholar). The gene responsible for aniridia in humans, PAX6, was isolated by positional cloning (3Ton 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 (750) Google Scholar) and documented by mutations found in both familial and sporadic aniridia (4Glaser T. Walton D.S. Maas R.L. Nat. Genet. 1992; 2: 232-239Crossref PubMed Scopus (573) Google Scholar, 5Jordan 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 (483) Google Scholar, 6Lyons L.A. Martha A. Mintz-Hittner H.A. Saunders G.F. Ferrell R.E. Genomics. 1992; 13: 925-930Crossref PubMed Scopus (35) Google Scholar, 7Hanson I.M. Seawright A. Hardman K. Hodgson S. Zeletayev D. 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Baumgertner S. Frigerio G. Noll M. Cell. 1986; 47: 1033-1040Abstract Full Text PDF PubMed Scopus (368) Google Scholar), is conserved in the genome of diverse organisms ranging fromDrosophila to humans (12Noll M. Curr. Opin. Genet. Dev. 1993; 3: 595-605Crossref PubMed Scopus (295) Google Scholar). The PAX6 genes are conserved in both vertebrates and invertebrates, including humans (3Ton 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 (750) Google Scholar,4Glaser T. Walton D.S. Maas R.L. Nat. Genet. 1992; 2: 232-239Crossref PubMed Scopus (573) Google Scholar), mice (13Walther C. Gruss P. Development. 1991; 113: 1435-1449Crossref PubMed Google Scholar, 14Ton C.C.T. Miwa H. Saunders G.F. Genomics. 1992; 13: 251-256Crossref PubMed Scopus (68) Google Scholar), quail (15Martin P. Carriere C. Dozier C. Quatannens B. Mirabel M.A. Vandenbunder B. Stehelin D. Saule S. Oncogene. 1992; 7: 1721-1728PubMed Google Scholar), chicks (16Li H.-S. Yang J.-M. Jacobson R.-D. Pasko D. Sundin O. Dev. Biol. 1994; 162: 181-194Crossref PubMed Scopus (253) Google Scholar), Xenopus (17Hirsch N. Harris W.A. J. Neurobiol. 1997; 32: 45-61Crossref PubMed Scopus (192) Google Scholar), zebrafish (18Krauss S. Johansen T. Korzh V. Moens U. Ericson J.U. Fjose A. EMBO J. 1991; 10: 3609-3619Crossref PubMed Scopus (187) Google Scholar, 19Puschel A.W. Gruss P. Westerfield M. Development. 1992; 114: 643-651Crossref PubMed Google Scholar), Drosophila (20Quiring R. Walldorf U. Kloter U. Gehring W.J. Science. 1994; 265: 785-789Crossref PubMed Scopus (909) Google Scholar), sea urchins (21Czerny T. Busslinger M. Mol. Cell. Biol. 1995; 15: 2858-2871Crossref PubMed Scopus (263) Google Scholar), squid (22Tomarev S. Callaerts P. Kos L. Zinovieva R. Halder G. Gehring W. Piatigorsky J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2421-2426Crossref PubMed Scopus (161) Google Scholar), and ribbonworms Lineus sanguineus (23Loosli F. Kmita-Cunisee M. Gehring W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2658-2663Crossref PubMed Scopus (66) Google Scholar) and Caenorhabditis elegans (24Chisholm A.D. Horvitz H.R. Nature. 1995; 377: 52-55Crossref PubMed Scopus (149) Google Scholar, 25Zhang Y. Emmons S.W. Nature. 1995; 377: 55-59Crossref PubMed Scopus (132) Google Scholar). Mutations in Pax6 are responsible for several naturally occurring mutant phenotypes including aniridia in humans, small-eye in rodents (14Ton C.C.T. Miwa H. Saunders G.F. Genomics. 1992; 13: 251-256Crossref PubMed Scopus (68) Google Scholar, 26Hill 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 (1171) Google Scholar,27Matsuo 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), and eyeless in Drosophila (20Quiring R. Walldorf U. Kloter U. Gehring W.J. Science. 1994; 265: 785-789Crossref PubMed Scopus (909) Google Scholar). The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a transactivation domain when fused to a heterologous DNA-binding domain, GAL4 (aa 1–97) (21Czerny T. Busslinger M. Mol. Cell. Biol. 1995; 15: 2858-2871Crossref PubMed Scopus (263) Google Scholar, 28Tang K. Saunders G.F. J. Cell. Biochem. Suppl. 1994; 8A: 203Google Scholar, 29Glaser T. Jepeal L. Edwards J.G. Young S.R. Favor J. Maas R.L. Nat. Genet. 1994; 7: 463-469Crossref PubMed Scopus (605) Google Scholar). PST sequences are also found in the C terminus of other PAX proteins and have been shown to constitute transactivation domains (30Kozmik Z. Kurzbauer R. Dorfler P. Busslinger M. Mol. Cell. Biol. 1993; 13: 6024-6035Crossref PubMed Scopus (143) Google Scholar, 31Chalepakis G. Jones F.S. Edelman G.M. Gruss P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12745-12749Crossref PubMed Scopus (123) Google Scholar, 32Dorfler P. Busslinger M. EMBO J. 1996; 15: 1971-1982Crossref PubMed Scopus (136) Google Scholar). The C-terminal regions of the Pax5, Pax2, and Pax8 proteins also contain a repression domain that is adjacent to the transactivation domain (32Dorfler P. Busslinger M. EMBO J. 1996; 15: 1971-1982Crossref PubMed Scopus (136) Google Scholar). The PST region of the PAX6 protein has not been analyzed in detail. PAX6 can be inactivated by various mutations scattered throughout the gene. The majority (91%) of these are truncation mutations (reviewed in Ref. 33Hanson I. van Heyningen V. Trends Genet. 1995; 11: 268-272Abstract Full Text PDF PubMed Scopus (166) Google Scholar), while others are missense mutations that affect the nuclear translocation signal (7Hanson I.M. Seawright A. Hardman K. Hodgson S. Zeletayev D. Fekete G. van Heyningen V. Hum. Mol. Genet. 1993; 2: 915-920Crossref PubMed Scopus (149) Google Scholar, 34Glaser 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) and the paired DNA-binding domain (35Hanson 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 (444) Google Scholar, 36Azuma N. Nishina S. Yanagisawa H. Okuyama T. Yamada M. Nat. Genet. 1996; 13: 141-142Crossref PubMed Scopus (174) Google Scholar, 37Tang H.K. Chao L. Saunders G.F. Hum. Mol. Genet. 1997; 6: 381-386Crossref PubMed Scopus (69) Google Scholar). Twelve mutations have been detected in the PST region of PAX6. 2Summarized on the World Wide Web at http://craigellachie. hgu.mrc.ac.uk/Softdata/PAX6/. Two of them are simple nonsense mutations that result in mutant PAX6 with residual PST domains, and the other 10 are splicing mutations that result in truncation of the PST domain and fusion to a nonsense peptide. Residual PST domains can constitute substantial transactivation function when fused to the GAL4 DNA-binding domain (29Glaser T. Jepeal L. Edwards J.G. Young S.R. Favor J. Maas R.L. Nat. Genet. 1994; 7: 463-469Crossref PubMed Scopus (605) Google Scholar), which has led to the prediction that PAX6 mutants with truncated PST domains may retain partial transactivation function. However, the functions of these residual PST domains are unclear in the context of native PAX6. In this paper, we report a detailed dissection of the PST region of PAX6 in two independent systems, a heterologous system (GAL4) and a native system (PAX6). We found that the transactivation function of the PST domain is dispersed into the four constituent exons (exons 10–13). The four exon fragments act synergistically to stimulate transcription, but none of them can function individually as an independent transactivation domain. Although residual PST domains can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, they surprisingly do not produce as much activity in the context of native PAX6, although the mutant PAX6 proteins are stable and retain their DNA binding function intact. Our data suggest that these mutants may antagonize the wild-type PAX6 activity by competing for target DNA-binding sites. HeLa, a human cervical carcinoma cell line (CCL2, ATCC), was maintained in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. NIH 3T3, a murine fibroblast cell line, was maintained in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum. HEB3, a human lens epithelial cell line (38Andley 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. GAL4-PAX6 fusion expression vectors were generated by polymerase chain reaction (PCR) cloning. In brief, specific regions of PAX6 were amplified by PCR using the cDNA plasmid clone ph12 (3Ton 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 (750) Google Scholar) as a template. The primers used are listed in Table I. All 5′-end primers contained an XbaI restriction site. All 3′-end primers contained a BamHI restriction site. The PCR products were digested with endonucleases XbaI and BamHI, inserted into the XbaI–BamHI restriction sites of the expression vector PCGGAL (39Wong M.-W. Pisegna M. Lu M.-F. Leibham D. Perry M. Dev. Biol. 1994; 166: 683-695Crossref PubMed Scopus (39) Google Scholar), and then fused in-frame to the N-terminal GAL4 DNA-binding domain (G4; aa 1–97).Table IOligonucleotides used for PCR cloning of the GAL4-PAX6 constructs Open table in a new tab pRc-CMV-PAX6 expression plasmids were constructed by a similar PCR cloning strategy. Specific regions of PAX6 were amplified by PCR using the cDNA clone ph12 (3Ton 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 (750) Google Scholar) as a template. Primers used to amplify specific regions of PAX6 are listed in Table II. 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. the HindIII–SpeI-digested PCR products were ligated into the HindIII–XbaI restriction sites in the polylinker region of the expression vector pRc-CMV (Invitrogen).Table IIOligonucleotides used for PCR cloning of Rc-CMV-PAX6 constructs Open table in a new tab The PAX6-SacII construct used to make internal deletions by site-directed mutagenesis was created by recombinant PCR (40Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13540) Google Scholar). The 5′-primer contained a HindIII restriction site and a Kozak consensus sequence. The 3′-primer contained a stop codon followed by an XbaI restriction site. The two internal mutant primers contained a SacII restriction site designed to fit in-frame between aa 277 and 278 of PAX6. All of the internal deletion constructs were constructed using a common 3′-primer that contained an XbaI restriction site and a stop codon but different 5′-primers that all contained a SacII restriction site. The various SacII–XbaI endonuclease-digested PCR products were then inserted between the SacII and XbaI restriction sites of the PAX6-SacII construct to create various internal deletion mutants. The PAX6-(4V/G) construct was similarly created by recombinant PCR using primers designed to mutate Val401, Val403, Val405, and Val407 into glycines simultaneously. HeLa and NIH 3T3 cells were plated at a density of 4–6 × 105 cells/60-mm tissue culture dish 24–36 h prior to transfection. Transfections for all cells were carried out with plasmid DNA coated with the polycationic lipid lipofectamine (Life Technologies, Inc.) according to the manufacturer's instructions. HeLa cells were transfected at 50–70% confluence using 6 μl (12 μg) of lipofectamine/60-mm dish. NIH 3T3 cells were transfected at 60–80% confluence using 15 μl (30 μg) of lipofectamine/60-mm dish. HEB3 cells were transfected at 70–80% confluence using 6 μl (12 μg) of lipofectamine/60-mm dish. For the GAL4 fusion experiments, each dish was transfected with 0.8 μg of the reporter plasmid 4 × GAL4-fos-CAT, 0.025–0.4 μg of GAL4 effector plasmid, 0.8 μg of the internal control plasmid pSV2 β-galactosidase (Promega), and pBluescript carrier plasmid (Strategene, La Jolla, CA) to bring the total amount of plasmids to 3.2 μg. For transfections with the pRc-CMV-PAX6 expression vectors, each dish was transfected with 2 μg of reporter plasmid P6CON-CAT (41Epstein J. Cai J. Glaser T. Jepeal L. Maas R. J. Biol. Chem. 1994; 269: 8355-8361Abstract Full Text PDF PubMed Google Scholar) or CD19–2-(A-ins)-luciferase (21Czerny T. Busslinger M. Mol. Cell. Biol. 1995; 15: 2858-2871Crossref PubMed Scopus (263) Google Scholar), 1 μg of pRc-CMV effector plasmid, and 0.4 μg of pSV2 β-galactosidase (Promega) as internal control. Cell extracts were prepared after 24–72 h of transfection. The cells on each 60-mm culture dish were rinsed twice with ice cold 1× Dulbecco's phosphate-buffered saline, detached using a rubber policeman into 160 μl of ice-cold 0.25 mm Tris, pH 7.8, and then transferred into a microcentrifuge tube. The cells were then lysed by three cycles of freezing in dry ice and ethanol and thawing at 37 °C. The microcentrifuge tube was then centrifuged in a microcentrifuge at full speed for 5 min at 4 °C, after which the supernatant was transferred to a fresh microcentrifuge tube and used immediately or stored at −70 °C for future use. The cell extracts were first assayed for β-galactosidase activity and then normalized by dilution with 0.25 mm Tris, pH 7.8. CAT assays were performed according to standard procedures (55Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and extracts were diluted as necessary to keep the assay in the linear range. The percentage of acetylation of [14C]chloramphenicol was quantitated directly from thin layer chromatography plates using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Cell extracts were prepared after 24–48 h of transfection. The cells on each 60-mm tissue culture dish were rinsed twice with 3 ml of 1× phosphate-buffered saline and lysed at room temperature in 250 μl of lysis solution (100 mm potassium phosphate, pH 7.8, 0.2% Triton X-100, 1 mm dithiothreitol added fresh prior to use). The cell lysates were detached from the culture plate using a rubber policeman or cell scraper, transferred to a microcentrifuge tube, and centrifuged for 2 min to pellet any debris. Supernatant was transferred to a fresh microcentrifuge tube. Cell extracts were then used immediately for luciferase and β-galactosidase assays or frozen at −70 °C for future use. Luciferase activity was measured at room temperature using a luciferase assay kit (Tropix, Bedford, MA; catalog number BC100L). All reagents and cell extracts were warmed to room temperature before use. Briefly, 50 μl of substrate A was aliquoted into 75 × 12-mm luminometer sample tubes (Sarstedt; catalog number 55.476). Then 10 μl of individual cell extracts and 50 μl of substrate B were added sequentially into the tube, which was 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; catalog number BL100G). All reagents and cell extracts were warmed to room temperature before use. Briefly, 200 μl of reaction buffer containing Galacton-Plus substrate was dispensed into 75 × 12-mm luminometer sample tubes. Then, 5 μl of individual cell extracts was added to the tube, which was incubated at room temperature for 1 h. Then, 300 μl of Accelerator reagent was added to each tube, which was immediately placed in a luminometer for measurement. 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 HeLa or NIH 3T3 cells according to the method of Schreiber et al. (42Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3921) Google Scholar), as modified by Singh and Aggarwal (43Singh S. Aggarwal B.B. J. Biol. Chem. 1995; 270: 24995-25000Abstract Full Text Full Text PDF PubMed Scopus (1303) 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 ice-cold 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 Bradford assay. Electrophoretic mobility shift assays (EMSAs) 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) (43Singh S. Aggarwal B.B. J. Biol. Chem. 1995; 270: 24995-25000Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). Each binding reaction contained 0.5 μg of nuclear extracts, 1.5 μg of poly(dI-dC), and 16 fmol of probes labeled with 32P. Protein-DNA complexes were resolved on a 4.5% native polyacrylamide gel using a buffer containing 50 mm Tris, 200 mm glycine, pH 8.5, and 1 mm EDTA (43Singh S. Aggarwal B.B. J. Biol. Chem. 1995; 270: 24995-25000Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). The gel was analyzed by a PhosphorImager using ImageQuant software (Molecular Dynamics). For supershift assays, the binding reaction mixture was incubated with the polyclonal antibody against PAX6-(16–422) for 30 min at room temperature before loading onto the gel. The nuclear extracts prepared from transfected HeLa, NIH 3T3, or HEB3 cells were resolved by 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then analyzed for PAX6 expression by hybridization with polyclonal antibodies raised against the paired domain (PD) of PAX6 (aa 1–127) (44Carriere 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 kit, Pharmacia Amersham Biotech). The bands on the Western blots were quantitated using Personal Densitometer Scan version 1.30 and ImageQuant Version 3.3 software (Molecular Dynamics). The identification and characterization of functional protein domains are critical to understanding the molecular role of PAX6. We (28Tang K. Saunders G.F. J. Cell. Biochem. Suppl. 1994; 8A: 203Google Scholar) and others (21Czerny T. Busslinger M. Mol. Cell. Biol. 1995; 15: 2858-2871Crossref PubMed Scopus (263) Google Scholar, 29Glaser T. Jepeal L. Edwards J.G. Young S.R. Favor J. Maas R.L. Nat. Genet. 1994; 7: 463-469Crossref PubMed Scopus (605) Google Scholar) have demonstrated previously that the PST region of PAX6 can confer a potent transactivation function when fused to the DNA-binding domain of the yeast transcription activator GAL4-(1–97). To examine the functional relationship of the PST region with other domains, we fused full-length PAX6 to the DNA-binding domain of GAL4 and tested its ability to activate transcription from a promoter bearing GAL4 binding sites. The reporter plasmid, 4 × GAL4-fos-CAT, contains four GAL4 binding sites inserted upstream of the c-fos TATA box linked to the CAT gene. Cotransfection of the effector plasmid and the reporter plasmid into HeLa cells revealed that the full-length PAX6 fused with GAL4 did not show transcriptional activation (Fig. 1 A), whereas deletion of the PD resulted in an efficient transcriptional activation of the CAT gene. This suggests that the presence of the PD could either interfere with the DNA binding of GAL4 or conformationally interfere with the interaction between the PST domain and the general transcriptional machinery. Further deletion of the linker region (G) caused a reduction in transactivation. This reduction was likely due to the negative effects of the homeodomain (HD), which became stronger after removal of the G region or when the HD was placed in proximity to GAL4. Although the function of the G region is unknown, it is not directly involved in transactivation, since this region had no activity when tested either alone or in association with the PD or HD. Finally, deletion of the entire PD-G-HD region showed that the C-terminal 152-aa PST region produced the highest activation (Fig. 1 A). In contrast, removal of the PST region resulted in the complete loss of transactivation in all C-terminal deletion mutants. These results demonstrate that the transactivation function of PAX6 resides in the C-terminal PST region, which can function independently of the other regions of PAX6 when fused with an appropriate DNA-binding domain. They also show that PD and the HD have negative effects on the transactivation function of the GAL4-PAX6 fusion protein. Similar negative effects of PD and HD have been observed with the GAL4-Pax3 fusion protein (31Chalepakis G. Jones F.S. Edelman G.M. Gruss P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12745-12749Crossref PubMed Scopus (123) Google Scholar). To dissect the transactivation domain, we divided the PST region at the exon boundaries into four fragments: E10 (composed of 35 aa), E11 (39 aa), E12 (50 aa), and E13 (28 aa). These four exons were fused individually to GAL4-(1–97) and tested for their ability to transactivate the reporter gene 4 × GAL4-fos-CAT in HeLa cells (Fig. 2 B). None of the four exon fragments caused significant activation, indicating disruption of the transactivation domain. To verify the protein expression of the transfected GAL4 fusion constructs, we performed Western blot analysis of nuclear extracts prepared from HeLa cells transfected under the same experimental conditions as used for CAT assays. The GAL4 fusion proteins of the expected sizes were detected in the nuclear extracts by an immune serum raised against the GAL4 DNA-binding domain (GAL4-DBD) (data not shown). We next tested combinations of adjacent exons: E10 and E11 of 74 amino acids, E11 and E12 of 89 amino acids, and E12 and E13 of 78 amino acids. All three combinations resulted in synergistic activation at similar levels, albeit with more activity shifted to the C-terminal exons. Further combination of three exons, E10, E11, and E12, produced higher activity than combination of two exons. Protein expression of transfected constructs was verified by Western blot using the polyclonal antibody against the GAL4-DBD (data not shown). Some of the GAL4 fusion constructs, e.g. G4-E12+13 and G4-E12, gave variable expression in different experiments. These experiments were repeated several times, and the transactivation data shown represent the activity of similar levels of protein. If we assume that combination of E10 and E11 reconstituted a relatively weak transactivation domain and that combination of E12 and E13 reconstituted a relatively strong transactivation domain, then combination of the two disrupted activation domains, E11 and E12, would not have reconstituted a third domain. Thus, the full activity of transactivation was dispersed among the four exon fragments. These data demonstrate that the integrity of the PST region is crucial for a high level of activation. A minimal deletion in the C-terminal exon (E13) can result in a 50% reduction in transactivation, which may be sufficient to cause aniridia. The residual PST domains retain substantial transactivation function and thereby may cause a relatively mild phenotype (29Glaser T. Jepeal L. Edwards J.G. Young S.R. Favor J. Maas R.L. Nat. Genet. 1994; 7: 463-469Crossref PubMed Scopus (605) Google Scholar), although there was no prior evidence that the truncated PAX6 proteins would be stable in the context of the native PAX6 protein. To test the hypothesis that the PST domain activated transcription by protein-protein interactions with coactivators, we tested whether a PST pep
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