The effects of disease mutations on the transcription factor Interferon Regulatory Factor 6

Abstract

Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip and cleft palate (CLP), lip pits, skin-folds, syndactyly and oral adhesions. VWS and PPS are caused by mutations in the transcription factor Interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved DNA-binding domain (DBD) and a less conserved protein-binding domain. Mutation analysis reveals a general genotype-phenotype correlation between the distribution of mutations and the manifestation of either VWS or PPS. However, the molecular mechanisms by which these mutations affect IRF6 function are still unknown. Therefore the aim of this project was to characterize the molecular function and regulation of IRF6 and subsequently determine the effects of VWS and PPS mutations on IRF6 function. DNA binding site-selection showed that the IRF6-DBD exhibited optimal binding site selectivity for the consensus sequence AACCGAAACC/T. This sequence was used to investigate the effects of VWS and PPS mutations in the DBD. Almost all mutations abrogated DNA-binding. It was subsequently demonstrated that a VP16AD-IRF6-DBD fusion protein was able to activate transcription from a promoter containing the derived binding site. However, wildtype IRF6 was shown to be autoinhibited and compartmentalized in the cytoplasm. To determine what signals are involved in the nuclear translocation of IRF6, internal signals that facilitate nucleocytoplasmic trafficking were mapped. The cytoplasmic localization of IRF6 suggested that the identified nuclear export signal (NES) 159IQDTFPFLNI168 is constitutively active and dominant over the nuclear localization signal 5PRRVRLK11. Disruption of the NES leads to nuclear retention; however nuclear localization alone is insufficient for functional activation of IRF6. Therefore, the role of phosphorylation in the regulation of IRF6 was assessed. Five novel phosphorylation sites were identified in IRF6 at residues S47, S131, S153, S424 and T425. Phosphorylation at S424/S425 was necessary for combinatorial phosphorylation at the serine/threonine rich region to induce nuclear retention, whereas, phosphorylation in the DBD at S47 abrogates DNA-binding. The understanding of IRF6 molecular function and mechanisms of regulation provides further insight into the pathways involved in the pathogenesis of VWS and PPS.

Date of Award	1 Aug 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Paul Shore (Supervisor) & Michael Dixon (Supervisor)