Investigation Of The Molecular Mechanism Of Congenital Cataract Induced By Hsf4 Mutant

BackgroundThe heat shock factor 4 has been identified as a novel cataract related gene. Multiple missense-mutations in the Hsf4 DNA binding domain or in other regulatory regions have been linked to human congenital or family inheriting chromosome autosome or recessive cataracts. These mutations are also associated with the occurrence of animal congenital cataracts. In mouse model, deletion of the Hsf4 gene induces nucleated cataract of postnatal lens by interfering with both lens epithelial cell proliferation and fiber cell terminal differentiation. However, the transcription activity of Hsf4 during lens development is still not completely defined.Hsf4 has two isoforms, Hsf4a and Hsf4b. Hsf4b is the unique isoform that regulates the mouse ocular lens development. In lens tissues, Hsf4b was reported to negatively regulate the expression of some FGF subfamily members but positively regulate the expression of Hsp25, some members of the gamma-crystallin family, and vemintin. The studies imply that signaling factors that balance Hsf4b transcription activation and inhibition are important in maintaining lens development. However, the signal pathways that can modulate Hsf4b to act as a transcription activator or inhibitor are still not clear.ObjectiveTo discuss the molecular mechanism how Hsf4b regulates the mouse ocular lens development. the investigation of the biological function of hsf4 and its associating signal pathways during lens development will be significant for early diagnosis of hsf4-realted congential cataract. MethodsThe total RNA of human heart tissues were prepared. Hsf4b cDNAs were then synthesized with RT-PCR,The PCR products were digested with Kpn I and EcoR I and sublconed into pcDNA3.0, pcDNA-Flag-Hsf4b was transfected into HEK293T cells,The expression of Hsf4b was testified with western blotting. The interaction between Hsf4b and P38 was assayed by Immunoprecipitation .In vivo pull down GST demonstrated that Hsf4b(196-493) could interact withP38, P38 phosphorylation of Hsf4b were testified with Kinase assay.We established the mouse lens epithelial cell line (mLEC/hsf4-/-) by immortalizing the expanded Hsf4 deficient mouse lens epithelial cells with SV40 large T-antigen. Hsf4b can directly interact with the CMV promoter by Immunoblotting, Immunoprecipitation and GST pull down in vivo results indicate phosphorylation of Hsf4b S299 determines Hsf4 transcription suppression activity. both Hsf4b and Daxx are expressed in the nucleus. Over-expressed Daxx formed granules and was co-localized with Hsf4b in the nucleus and some nuclear granules by Immunofluorescent staining.ResultsTo construct eukaryotic plasmid expressing Hsf4b, Hsf4b was over-expressed in HEK293T cells. further studies demonstrated that Hsf4b could interact with and phosphorylated by MAP kinase P38.Hsf4b C-terminal participates the association with P38. Hsf4b has transcription-inhibitory function. Hsf4b inhibits CMV promoter activity by directly binding to nGAAn (HSE motif) in the CMV promoter at176 bp. Mutation of GAA in this motif into GCC demolishes Hsf4b's negative regulation of CMV activity. The phosphorylation of Hsf4b/S299 participates in the negative regulation of the CMV promoter. Moreover, we found that Hsf4b inhibits CMV promoter activity by associating with transcriptional inhibitor Daxx. Hsf4b is co-localized with Daxx in the nuclear POD body, and phosphorylation of Hsf4b/S299 regulates their association. ConclusionHsf4b could interact with and phosphorylated by MAP kinase P38. the transcription-inhibitory function of Hsf4b is regulated by the phosphorylation of Hsf4b/S299 and its phosphorylation-dependent association with Daxx. Our results will provide more evidence for understanding the signal regulation of Hsf4b transcription activity during lens development.