title: Domains of Brn-2 that mediate homodimerization and interaction with general and melanocytic transcription factors.
abstract: The class III POU gene brn-2, encoding the Brn-2/N-Oct-3 transcription factor, is widely expressed in the developing mammalian central nervous system. Brn-2 has also been found to regulate the melanocytic phenotype with N-Oct-3 DNA binding activity elevated in malignant melanoma, however, its mode of action is yet to be defined. The functional role of the Brn-2 transcription factor has been investigated through the analysis of protein-protein interactions it forms with a number of basal and melanocytic transcriptional regulatory proteins. In vivo interactions were tested by gene-cotransfection using the mammalian GAL4-Herpes Simplex viral protein 16 (VP16) two hybrid formation and direct protein binding by in vitro glutathione S-transferase (GST)-pull down assay. The Brn-2 protein was found to homodimerize in vivo with high affinity, using Brn-2 deletion constructs dimer complex formation was found to be dependent on the presence of both the homeodomain and linker regions of the POU-domain. However, the POU-homoedomain was dispensable for the formation of the dimerization interface in one of the partner molecules but not both, when the POU-linker region was removed the ability to interact was lost irrespective of the presence of the homeodomain. Dimerization of Brn-2/N-Oct-3 was also found to occur in DNA binding assays using melanoma cell line nuclear extracts and a recently reported dimer target sequence probe, which may have significant consequences for gene regulation in melanocytic tumours. Low affinity Brn-2 protein contacts have also been found with the basal transcription complex, including TATA binding protein (TBP) and the transcriptional coactivator p300, and with the Sox-10 and Pax-3 transcription factors that are known to play an important role in melanocyte cell formation.
authors.affiliation: Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: D J
authors.initials: DJ
authors.lastname: Smit
authors.forename: A G
authors.initials: AG
authors.lastname: Smith
authors.forename: P G
authors.initials: PG
authors.lastname: Parsons
authors.forename: G E
authors.initials: GE
authors.lastname: Muscat
authors.forename: R A
authors.initials: RA
authors.lastname: Sturm
mesh_terms: D001665:Binding Sites; D002457:Cell Extracts; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3; D000072041:PAX3 Transcription Factor; D050809:POU Domain Factors; D051761:Paired Box Transcription Factors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D055757:SOXE Transcription Factors; D017384:Sequence Deletion / Q000235:genetics; D035181:TATA-Box Binding Protein; D013698:Templates, Genetic; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D002457:Cell Extracts; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D050809:POU Domain Factors; C494173:POU5F1 protein, human; D051761:Paired Box Transcription Factors; C527923:SOX10 protein, human; D055757:SOXE Transcription Factors; C527924:Sox10 protein, mouse; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; C085076:transcription factor Brn-2; C068686:Pax3 protein, mouse; D004247:DNA