title: Functional analysis of HSF4 mutations found in patients with autosomal recessive congenital cataracts.
abstract: OBJECTIVE
  The goal of this study was to functionally evaluate three previously uncharacterized heat shock factor protein 4 (HSF4) mutations (c.595_599delGGGCC, c.1213C>T, c.1327+4A>G) encoding mutant HSF4 proteins (G199EfsX15, R405X, and M419GfsX29) with missing C-terminal ends. These HSF4 mutations were previously identified in families with congenital autosomal recessive cataracts.
  
  
  METHODS
  FLAG-tagged recombinant wild type (WT) and mutant HSF4 proteins were analyzed using the protein stability assay, cellular immunofluorescence, Western blotting, electrophoretic mobility shift assay (EMSA), and reporter activation.
  
  
  RESULTS
  HSF4 mutant proteins did not differ in the protein turnover rate when compared with WT HSF4. Immunofluorescence revealed that WT and mutant HSF4 proteins were properly trafficked to the nucleus. EMSA analysis revealed that the G199EfsX15 and M419GfsX29 proteins exhibited decreased heat shock element (HSE)-mediated DNA binding, whereas the R405X mutant exhibited increased HSE-mediated DNA binding when compared with WT HSF4. All three HSF4 mutant proteins exhibited abolished HSE-mediated luciferase reporter activation. Detailed evaluation of the C-terminal region identified three novel domains: two activation domains and one repression domain.
  
  
  CONCLUSIONS
  The three HSF4 autosomal recessive mutations evaluated here result in a loss of HSF4 function due to a loss of regulatory domains present at the C-terminal end. These findings collectively indicate that the transcriptional activation of HSF4 is mediated by interactions between activator and repressor domains within the C-terminal end.
authors.affiliation: Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin.
authors.forename: Kate
authors.initials: K
authors.lastname: Merath
authors.forename: Adam
authors.initials: A
authors.lastname: Ronchetti
authors.forename: Duska J
authors.initials: DJ
authors.lastname: Sidjanin
mesh_terms: D015153:Blotting, Western; D002386:Cataract / Q000151:congenital* / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005808:Genes, Recessive; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D006801:Humans; D009154:Mutation; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; C102948:HSF4 protein, human; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D014157:Transcription Factors; D004247:DNA
keywords: HSF4; cataracts; congenital; mutation
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