Vol. 4, 1041-1050, December 1993 Cell Growth & Differentiation 1041 Quail PAX-6 (PAX-QNR) Encodes a Transcription Factor Able to Bind and Trans-activate Its Own Promoter’ Serge Plaza, Christine Dozier, and Simon Saule2 Laboratoire de Diff#{233}renciation Cellulaire et Mol#{233}culaire, Centre National de Ia Recherche Scientifique E.P. J0056, Institut Pasteur, 1 Rue Calmette, 5901 9 Lille Cedex, France Abstract Proper growth and development of multicellular organisms requires precise regulation of developmental genes. One aspect of this regulation is at the level of transcription from the gene promoters. As an initial approach to understanding the regulation of the Pax-6 gene, which plays an important role in eye development and perhaps in other developmental processes, we characterized a promoter region of the quail Pax-6 (Pax-QNR) gene. Sequence analysis of the 5’ flanking region revealed a TATA-like box and a CAAT box as well as several putative cis-regulatory elements. A 1 .5-kilobase pair fragment, containing 1 386 base pairs of 5’ flanking sequence, the first exon, and a portion of the first intron, was able to efficiently promote expression of the bacterial CAT gene in quail neuroretina cells. Cotransfection of the Pax-QNR promoter with a vector expressing the 46 kilodalton Pax-QNR protein resulted in an increase in Pax-QNR promoter activity. By electrophoretic migration shift assay and immunoselection experiments, we showed that the Pax-QNR protein can interact directly with the Pax-QNR promoter. By footprinting experiments, we identified the binding sites for the Pax-QNR protein within the promoter region. These results show that Pax-QNR encodes a transcriptional activator and that it potentially trans-activates its own promoter. Introduction A large number of vertebrate developmental control genes have been identified by their homology to Drosophila genes that regulate pattern formation and contain conserved DNA binding domains, like the homeobox on the paired box (1-3). Through homology to the paired box sequences of Drosoph- i/a, nine pained box-containing genes, the Pax genes, have been isolated in vertebnates(4, 5). Each member of the family shows spatially and temporally restricted expression patterns during embryonic development (6). The pained box is pre- sent alone (in the munine genes Pax-1, Pax-2, Pax-S, and Pax-8) on together with the homeobox domain (Pax-3, Pax-4, Pax-6, and Pax-7) (6). The ability of the pained do- main and homeodomain to bind specific DNA sequences suggests that the proteins containing such structures may be involved in transcriptional control. Recently, this hypothesis has been verified by demonstrating thatthe munine Pax-i (7), Pax-2 (8), Pax-5 (9), and Pax-8 proteins (1 0) are transcnip- tional activators. The importance of these genes in development is dem- onstrated by the developmental alterations in the mouse as- sociated with mutations in the Pax genes (6). Mutations in the Pax-6 gene have been associated with the mouse mutant small eye (1 1 ). Moreover, the corresponding human gene (AN) has been found to be deleted or mutated in some cases of the human congenital disorder aninidia (1 2, i 3). The Pax-6 gene is expressed in the developing central nervous system, the optic cup, the lens, and the overlying epithelium prior to morphological differentiation, and later in the neu- ronal layer ofthe retina (1 4, 1 5). All of this strongly suggests that Pax-6 may be involved in the regulation of some of the inductive events that occur during the formation of the eye. Moreover, the restricted expression of this gene suggests transcriptional regulation of expression, with sequence- specific DNA-binding proteins interacting with tissue- specific enhancer and promoter elements. However, to date, nothing is known about the regulation and the role of this gene. We have recently characterized the quail Pax-6 gene, named Pax-QNR3 (1 5), and elucidated its complete exon- intron organization (1 6). We have identified two mRNAs, named MC29-QNR2 and Bi , differing by their 5’ UTR and resulting from alternative splicing ofthe Pax-QNRgene. We now report the structural and functional analysis of the pro- moter region initiating the MC29-QNR2 mRNA, as a step toward elucidating the mechanisms involved in the control of its expression. We also show that the 46 Kda Pax-QNR protein is a transcriptional activator, able to trans-activate its own promoter. These results identify the first target for Pax-6 gene product. Results Cloning and Sequencing of the Pax-QNR Promoter. We have previously determined the organization and structure of the quail Pax-QNR gene and showed that the 5’ UTR of the MC29-QNR2 cDNA is organized in 4 exons (exons 0, 1, 2, and 3, the latter containing the initiation AUG) (1 6). The restriction map of the genomic region containing the up- stream untranslated sequences of the MC29-QNR2 cDNA (exon 0 of the Pax-QNR gene) is shown in Fig. 1 A. We de- tenmined the nucleotidesequenceofa l.5-kbp Xbal-Asp718 genomic fragment (Fig. 1 B), which hybridized to sequences corresponding to the 5’ terminus ofthe MC29-QNR2 cDNA. Received 8/31/93; revised 9/27/93; accepted 10/1/93. 1 This work was supported by grants from the Centre National de Ia Recherche Scientifique, the Institut Pasteur de Lille, the Association de Recherche contre le Cancer, and the Association Fran#{231}aise Retinitis Pigmentosa. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: QNR, quail neuroretina cells; QEC, quail em- bryo cells; 5’ UTR, 5’ untranslated region; Kda, kilodalton(s); cDNA, comple- mentary DNA; bp, base pair(s); kbp, kilobase pair(s); CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; EMSA, electrophoretic migration shift assay; CRE, cyclic AMP response element; a-gal, -galactosidase.