Alternatively spliced insertions in the paired domain restrict the DNA sequence specificity of Pax6 and Pax8.
Transcription factors of the Pax family bind to their target genes via the paired domain which is known to be composed of two subdomains each recognizing distinct half-sites in adjacent major grooves of the DNA helix. We now demonstrate that the mammalian Pax8 gene gives rise, by alternative mRNA splicing, to a protein isoform containing an extra serine residue in the recognition alpha-helix 3 of the paired domain. This Pax8(S) protein does not interact with bipartite paired domain-binding sites, indicating that inactivation of the N-terminal DNA-binding motif severely restricts the sequence specificity of the paired domain. However, the Pax8(S) protein binds in vitro and in vivo to the 5aCON sequence which was previously identified as a high-affinity binding site for the Pax6(5a) splice variant carrying a 14-amino-acid insertion in the paired domain. The 5aCON sequence is shown to consist of four interdigitated 5' half-sites of the bipartite consensus sequence and is thus bound by four Pax8(S) molecules via the intact C-terminal DNA-binding motif of the paired domain. Together these data suggest that inactivation of the N-terminal region of the paired domain by alternative splicing is used in vivo to selectively target Pax transcription factors to gene regulatory regions containing highly specialized 5aCON-like sequences.
Kozmik
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Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria.
Czerny
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Busslinger
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D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins; D017930:Genes, Reporter; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D000071841:PAX6 Transcription Factor; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors; D011487:Protein Conformation; D012097:Repressor Proteins; D012694:Serine / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D000072636:PAX8 Transcription Factor; C083325:PAX8 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; C499496:Pax8 protein, mouse; D012097:Repressor Proteins; D015534:Trans-Activators; D012694:Serine