Pax-3 is a transcription factors important in normal embryonic development, as highlighted by its mutation in independent alleles of the Splotch mouse mutant and human Waardenburg syndrome, both characterized by pigmentary disturbances in the heterozygous state and limb muscle defects in the homozygotes. Pax-3 contains two structurally independent DNA-binding domains, a paired domain and a homeodomain. Through the analysis of a number of naturally occurring mutations, we have shown that both DNA-binding domains are functionally interdependent, as independent mutations in either domain can affect the DNA-binding activity of the other. In support of this finding, modeling the deleterious mutations into the three dimensional structure of the paired domain suggests that a series of consecutive phosphate contacts are essential for DNA-binding by both the paired domain and homeodomain of Pax-3. In addition, the paired domain can regulate the DNA-binding specificity and dimerization potential of the homeodomain and we have exploited the possibility to transfer this effect to a heterologous homeodomain to identify the protein segments involved in this functional interaction. Characterization of a series of chimeric proteins containing stepwise deletions within the paired domain provided two key findings: (i) the C-terminal subdomain of the paired domain does not play a major role in the regulation of the homeodomain DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for the modulation of homeodomain DNA-binding. In addition to intermolecular interactions, the functional association with other transcription factors can also modulate Pax-3 target gene selection and regulation. We found that Pax-3 can recruit PEA3, a member of the ETS family, to form a ternary complex with DNA sequences containing either a paired domain or homeodomain recognition site and a sub-optimal Ets site to which PEA3 normally does not bind. We have
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37617 |
| Date | January 2001 |
| Creators | Fortin, Anouk S. |
| Contributors | Gros, Philippe (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biochemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001802272, proquestno: NQ70019, Theses scanned by UMI/ProQuest. |
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