Functional analysis of the Hsf4lop11 allele responsible for cataracts in lop11 mice

PURPOSE Lens opacity 11 (lop11) is a spontaneous autosomal recessive mouse mutation resulting in cataracts. Insertion of an early transposable element (ETn) in intron 9 of heat shock factor 4 (Hsf4) was previously identified as responsible for lop11 cataracts. Although molecular analysis showed that the ETn insertion resulted in an aberrant Hsf4 transcript encoding a truncated mutant HSF4(lop11) protein, the function of the mutant HSF4(lop11) protein was not investigated. The goal of this study was to functionally evaluate the mutant HSF4(lop11) protein and to establish the onset and progression of cataracts in lop11 lenses. METHODS HSF4 is expressed as two alternatively transcribed isoforms Hsf4a and Hsf4b. Given that only Hsf4b is expressed in the lens we pursued evaluation of the mutant Hsf4b isoform only. Recombinant wild type HSF4b and mutant HSF4b(lop11) proteins were analyzed using elecrophoretic mobility shift, reporter transactivation, western blotting and protein half-life assays in HEK293 cells. Prenatal and postnatal wild type and lop11 lenses were evaluated using a combination of clinical, histological, and immunohistological analyses. RESULTS HSF4b(lop11) stability and nuclear translocation of did not differ from wild type HSF4b. However, HSF4b(lop11) exhibited abolished HSE-mediated DNA binding and transactivation. Further investigation identified that HSF4b(lop11) fails to form trimers. Histological analysis of lop11 lenses indicated the persistence of nuclei in lens fiber cells as early as postnatal day 0.5 (P0.5). No differences were observed between wild type and lop11 in lens epithelial cell proliferation and spatio-temporal differentiation to fiber cells. However, by P10-12, lop11 lenses develop severely vacuolated cataracts commonly accompanied by rupture of the lens capsule and release of the lenticular material in the vitreous cavity. Clinically, lop11 vacuolated cataracts were visible upon eyelid opening between P12-14. CONCLUSIONS The ETn insertion in lop11 mice results in abolished HSF4b function. Loss of 132 amino acids from the COOH-terminus in HSF4b(lop11) results in the failure of trimer formation and subsequent failure of HSE-mediated DNA binding and transactivation. These findings highlight the importance of the COOH-terminal region for normal function. The persistence of nuclei in postnatal lop11 lens fiber cells was identified as the initial lens abnormality, thus confirming a previously identified role of HSF4b in denucleation of lens fiber cells. By P14 lop11 lenses develop severe fiber cell vacuoles although how the loss of HSF4b function results in this process remains unknown. Collectively, these findings further our understanding of the mechanism of HSF4 loss of function as well as the resulting implications on lop11 cataractogenesis.