Upstream activating sequences of the rat aldolase C gene are shown here to confer brain-specific expression in transgenic mice. In addition to binding sites described previously for the brain-expressed POU proteins Brn-1 and Brn-2 (Skala, H., Porteu, A., Thomas, M., Szajnert, M. F., Okazawa, H., Kahn, A., and Phan-DinhTuy, F. (1998) J. Biol. Chem. 273, 31806 -31814), we have identified two novel DNA elements critical for an interaction with a brain-specific, high affinity DNA-binding protein. Characterization of this binding protein showed it to be sensitive to thiol oxidation and stable to heat at 100°C. This protein was purified on the basis of its thermostability and its selective adsorption to streptavidin magnetic particles via a biotinylated multimer of its target DNA binding site. Liquid chromatography coupled to tandem mass spectrometry analysis, binding competition with consensus oligonucleotides, and antibody supershift assays led to its identification as the homeodomain paired protein Pax-6. This result suggests that the brain-specific aldolase C gene could constitute a new target for the transcription factor Pax-6, which is implicated increasingly in neurogenesis.Regulation of gene transcription in active chromatin is dependent upon the interaction of discrete DNA motifs with regulatory proteins. These proteins either represent basal, ubiquitous elements of the transcriptional machinery or can be themselves expressed in a tissue-, development-, or stimulusspecific manner. Cell-type diversity in the different organs can be considered as the result of multiple DNA-protein and protein-protein interactions leading to the controlled expression of a very large number of genes. The central nervous system is one of the most complex structures and consequently the one for which such regulatory mechanisms are probably still the least elucidated. In light of this, identification of DNA binding sites for transcription factors involved in brain development will help to define their candidate target genes and to elucidate the complex gene regulatory network at play in the central nervous system. The aldolase C gene encodes a brain-specific glycolytic enzyme, the fructose-1,6 diphosphate aldolase (EC 4.1.2.13). In a previous paper (1), we reported that the transcriptional activation of the brain-specific promoter (115 bp) of the rat aldolase C gene in vivo requires a cooperation between distal and proximal upstream sequences. We identified in vitro in a 0.6-kb proximal activating fragment several POU/WH motifs (1) exhibiting overlapping binding sites for the brain-restricted class III Brn-1 and Brn-2 POU proteins (2) and for the protein HNF3, a member of the WH (winged helix) factor family, which has been shown to be involved in the early brain development (3, 4).In this paper, we first show that the distal and proximal regulatory sequences are also able to ensure the activation of the ubiquitous thymidine kinase (tk) 1 promoter specifically in the brain of transgenic mice. We also report that, in addition to the ...