Human Molecular Genetics Advance Access published online on May 9, 2009
Human Molecular Genetics, doi:10.1093/hmg/ddp221
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Pathogenic mechanisms of tooth agenesis linked to paired domain mutations in human PAX9
1 Department of Biomedical Sciences, Texas A&M University Health Science Center Baylor College of Dentistry, Dallas, TX 75246, USA 2 Maxillofacial Orthognathics, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549 Japan 3 New York University Medical Center, Institute of Reconstructive Plastic Surgery, 560 First Avenue, New York, NY 10016
* To whom correspondence should be addressed: Hitesh Kapadia, New York University Medical Center, Institute of Reconstructive Plastic Surgery, 560 First Avenue, New York, NY 10016. Phone: 212 263 5891; FAX: 212 263 6002; E-mail: hkapadia{at}bcd.tamhsc.edu
Received February 13, 2009; Revised April 13, 2009; Accepted May 6, 2009
Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis that preferentially affects posterior dentition. Of the eighteen mutations identified to date, eight are phenotypically well-characterized missense mutations within the DNA-binding paired domain. We determined the structural and functional consequences of these paired domain missense mutations and correlated our findings with the associated dental phenotype variations. In vitro testing included subcellular localization, protein-protein interactions between MSX1 and mutant PAX9 proteins, binding of PAX9 mutants to a DNA consensus site and transcriptional activation from the Pax9 effector promoters Bmp4 and Msx1 with and without MSX1 as co-activator. All mutant PAX9 proteins were localized in the nucleus of transfected cells and physically interacted with MSX1 protein. Three of the mutants retained the ability to bind the consensus paired domain recognition sequence; the others were unable or only partly able to interact with this DNA fragment and also showed a similarly impaired capability for activation of transcription from the Msx1 and Bmp4 promoters. For seven of the eight mutants the degree of loss of DNA-binding and promoter activation correlated quite well with the severity of the tooth agenesis pattern seen in vivo. One of the mutants however showed neither reduction in DNA-binding nor decrease in transactivation; instead, a loss of responsiveness to synergism with MSX1 in target promoter activation and a dominant negative effect when expressed together with wildtype PAX9 could be observed. Our structure-based studies, which modeled DNA binding and subdomain stability, were able to predict functional consequences quite reliably.