Functional Characterization Of HSF4in Lens Development And Cataract Formation

Heat shock factors (HSFs) respond to environmental stresses through regulating the expression of heat shock proteins (HSPs) and play a crucial role in physiological function of cells. Heat shock factor4(HSF4) is expressed primarily in the ocular lens and has an essential role in the lens development. Mutations in the HSF4gene are responsible for the congenital and age-related cataract. However, the molecular mechanisms leading to this disease is still being undefined.Accumulation of DNA damage in lens is an important risk factor in the formation of age-related cataract, and its timely repair is crucial for maintaining the lens normal function. Our study firstly found that HSF4could promote to repair the UV induced DNA damage and DNA strand breaks. Yet, HSF4with cataract causative mutations failed. We confirm that DNA damage repair by HSF4is induced by the binding to the heat shock element in the Rad51promoter, a key catalytic kinase in the homologous recombination repair pathway. Wild type HSF4up-regulated Rad51mRNA and protein levels while HSF4mutations failed, and DNA damage were not repaired. Treatment with camptothecin abrogated the induction of Rad51expression, also disrupted the DNA damage repair promoted by HSF4. Moreover, when HSF4was knocked down in zebrafish, the protein levels of Rad51were obviously reduced, and DNA damage in zebrafish lens increased. Our study provids the first evidence that a congenital cataract responsible gene directly participates in DNA damage repair and may offer a novel understanding of congenital and age-related cataract formation.The degradation of nuclear DNA is crucial for the differentiation of lens fiber cells. DNase2β (DLAD) is mainly expressed in eye lens, and the DLAD--mice develop nuclear cataracts. In the present study, we found that HSF4induced the transcription and DNase activity of DLAD by directly binding to the HSE sequence among DLAD promoter. However, HSF4cataract causative mutations failed to bind to the DLAD promoter, and thus failed to induce the transcription and DNase activity of DLAD. We then conducted HSF4knockdown in zebrafish using the morpholino. Our results indicated that knockdown of HSF4in zebrafish led to incomplete de-nucleation of the lens, and reduced protein levels and DNase activity of DLAD. Our data imply that HSF4participates in lens differentiation through promoting the DLAD expression and DNase activity which is essential for the de-nucleation of lens fiber cells. Our results that HSF4cataract causative mutations failed to induce DLAD expression levels reveal a novel molecular mechanism about how HSF4mutations cause cataract formation. Meanwhile, we performed co-immunoprecipitation and pull down analysis and found that HSF4interacts with p53. This result could provide us a novel understanding of p53function in lens development and cataract formation, as well as the HSF4roles in p53related pathways.In a word, we firstly find that HSF4is involved in cataract formation through inducing the expression of Rad51which promotes the DNA damage repair. Secondly, HSF4enhanced the expression of DLAD and then promoted the de-nucleation of lens fibre cells. Finally, the interaction between HSF4and p53expands our view to understand the novel physiologic role of these two proteins.