Autoregulation of Pax6 transcriptional activation by two distinct DNA-binding subdomains of the paired domain.
BACKGROUND
Pax6 is a transcription factor that plays a central role in eye development. Pax6 contains a DNA-binding domain called paired domain, which consists of a highly conserved N-terminal subdomain and a variable C-terminal subdomain. Recent findings have suggested that both subdomains possess distinct DNA-binding activities.


RESULTS
To understand the mechanism of DNA-binding and transcriptional activation by Pax6 via these subdomains, we employed Pax6 paired domain mutants previously isolated from patients with ocular disorders. Analysis of these mutants by gel shift assay revealed that the N-terminal and C-terminal subdomains can independently bind to their respective cognate sites, P6CON and 5aCON. Results from a luciferase assay, however, showed that the two functional subdomains negatively regulate their transactivation potentials each other. Wild-type Pax6 and its hyperactive variants show different patterns of DNA contact.


CONCLUSIONS
These results support a new model for the regulation of Pax6 transactivation: When one DNA-binding subdomain binds to its cognate site, the other subdomain also interacts with the flanking sequences nonspecifically, and this interaction constrains its structure to give a reduced level of transactivation.
Yamaguchi
Y
Y
Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Sawada
J
J
Yamada
M
M
Handa
H
H
Azuma
N
N
D000818:Animals; D002648:Child; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D005136:Eye Proteins; D005838:Genotype; D018398:Homeodomain Proteins; D006801:Humans; D008297:Male; D051379:Mice; D008958:Models, Molecular; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D014157:Transcription Factors