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Modulation of PAX6 homeodomain function by the paired domain
S Singh1, C M Stellrecht, H K Tang
1Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
The Journal of Biological Chemistry
|April 5, 2000
Summary
Mutations in the PAX6 gene affect its DNA binding and transactivation functions. Structural changes in PAX6
Area of Science:
- Developmental biology
- Molecular genetics
- Human genetics
Background:
- PAX6 is crucial for the development of the eye, central nervous system, and nose.
- PAX6 possesses two DNA binding domains within its paired domain, linked by a glycine-rich region, and a transactivation domain.
- Previous research suggests distinct target genes for PAX6's DNA binding domains, but their independent function remains unclear.
Purpose of the Study:
- To investigate the impact of structural alterations in the paired domain on PAX6 function, particularly its homeodomain activity.
- To analyze the functional consequences of specific PAX6 mutations (R26G and I87R) found in patients with varying clinical phenotypes.
Main Methods:
- Site-directed mutagenesis to create R26G and I87R PAX6 mutants.
- DNA binding assays to assess the binding affinity of wild-type and mutant PAX6 proteins to their target sites.
- Transactivation assays to measure the transcriptional activity mediated by wild-type and mutant PAX6 proteins.
Main Results:
- The I87R mutation abolished both DNA binding and transactivation functions of PAX6.
- The R26G mutation impaired DNA binding via the paired domain but enhanced DNA binding and transactivation through the homeodomain.
- The 5a isoform of PAX6 exhibited increased DNA binding compared to wild-type PAX6.
Conclusions:
- The two subdomains of the PAX6 paired domain differentially influence the homeodomain's function.
- These findings provide a molecular explanation for the distinct clinical phenotypes observed in patients with R26G and I87R PAX6 mutations.