Novel target sequences for Pax-6 in the brain-specific activating regions of the rat aldolase C gene.
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-Dinh-Tuy, 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 degrees 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.
Skala-Rubinson
Henriette
H
Département de Génétique, Développement et Pathologie Moléculaire, Institut Cochin, INSERM, CNRS, Université René Descartes, 24, rue du faubourg Saint Jacques, 75014 Paris, France.
Vinh
Joëlle
J
Labas
Valérie
V
Kahn
Axel
A
Phan
Dinh Tuy Francoise
DT
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D002853:Chromatography, Liquid; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D013058:Mass Spectrometry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010084:Oxidation-Reduction; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D005136:Eye Proteins; D018398:Homeodomain Proteins; D009841:Oligonucleotides; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; C000605733:Pax6 protein, rat; D012097:Repressor Proteins; D004247:DNA; D003850:Deoxyribonuclease I; D005634:Fructose-Bisphosphate Aldolase