Dissection of the transactivation function of the transcription factor encoded by the eye developmental gene PAX6.
PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. We dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. Our data consistently showed that in both systems all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act synergistically to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6, although the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. Our data suggest that these mutants may antagonize the wild-type PAX6 activity by competing for target DNA-binding sites. We conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree and that its full transactivation activity relies on the synergistic action of the four exon fragments.
Tang
H K
HK
Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Singh
S
S
Saunders
G F
GF
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D005091:Exons; D005123:Eye / Q000254:growth & development*; D005136:Eye Proteins / Q000502:physiology*; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005136:Eye Proteins; C052130:GAL4 protein, S cerevisiae; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA