Medical College of Wisconsin
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Functional analysis of HSF4 mutations found in patients with autosomal recessive congenital cataracts. Invest Ophthalmol Vis Sci 2013 Oct 11;54(10):6646-54

Date

09/21/2013

Pubmed ID

24045990

Pubmed Central ID

PMC3796937

DOI

10.1167/iovs.13-12283

Scopus ID

2-s2.0-84885441288 (requires institutional sign-in at Scopus site)   14 Citations

Abstract

PURPOSE: 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.

Author List

Merath K, Ronchetti A, Sidjanin DJ

Author

Danielle Sidjanin Maier PhD APP Inpatient 2 in the Medicine department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Blotting, Western
Cataract
Cells, Cultured
DNA
DNA Mutational Analysis
DNA-Binding Proteins
Electrophoretic Mobility Shift Assay
Genes, Recessive
Heat Shock Transcription Factors
Heat-Shock Proteins
Humans
Mutation
Transcription Factors
Transcriptional Activation