【作者】 张军；

【导师】 沈倍奋；

【作者基本信息】 河南大学 ， 高分子化学与物理， 2010， 博士

【摘要】 背景热休克转录因子4(heat shock transcription factor 4,Hsf4)被认为是一种新的与白内障发生相关基因,在Hsf4 DNA结合域或其它调节域的错义突变与人先天性或家族常染色体显性或隐性遗传性白内障密切相关,这些基因突变与动物先天性白内障发生也密切相关,小鼠模型中Hsf4基因缺失可妨碍晶状体上皮细胞增殖和纤维细胞终未分化,从而导致出生后晶状体核白内障。Hsf4有Hsf4a、Hsf4b两种亚型,Hsf4b是调节鼠晶状体发育中唯一亚型,已报道Hsf4b基因缺失不仅可以下调许多热休克蛋白(例如:Hsp25γ-晶体蛋白,β-晶体蛋白和骨骼肌蛋白)的表达,而且可以上调鼠晶状体组织中许多基因的表达(例如:FGF家族成员),这些研究表明Hsf4b通过对其下游靶基因转录激活和转录抑制双重作用而调控晶状体发育,但是,调节Hsf4b转录活性的信号通路还不清楚。目的探讨Hsf4调控晶状体发育的分子机制。通过探究Hsf4在上述信号转导通路中的作用,揭示Hsf4基因突变所致先天性白内障的致病机理,为早期诊断和治疗Hsf4突变相关的先天性白内障提供理论依据。方法用人心脏cDNA文库为模板,应用囊括Hsf4b全长的引物进行PCR并在Hsf4bcDNA的N-端加入Flag标签。将PCR产物经Kpn I和EcoR I酶切后,与该二酶线性化pcDNA3.0质粒一起连接获得重组质粒pcDNA-Flag-Hsf4b,将克隆好的pcDNA-flag-Hsf4b转染HEK293T细胞,并用抗Flag抗体进行免疫印迹分析,免疫沉淀实验和体内Pull down实验证明Hsf4b可与MAP激酶P38结合,激酶实验结果显示P38可体外磷酸化Hsf4b。用Hsf4-/-小鼠的晶状体上皮细胞转染SV40,T-抗原,G418筛选建立永生化细胞系MLEC/Hsf4-/-。应用不同的含有CMV启动子的质粒(如腺病毒载体),在mLEC细胞和其他细胞系中表达Hsf4b,测定Hsf4b对CMV启动子的抑制作用。DNA-蛋白沉淀实验和DNA-蛋白结合实验体外测定Hsf4b与CMV启动子的结合。免疫印迹检测Hsf4b/S299磷酸化抑制Hsf4b的转录活性,免疫沉淀和细胞内GST pull down实验证明Hsf4蛋白可以结合转录抑制调节因子Daxx,免疫荧光染色实验发现Hsf4b被摹集到核内Daxx的POD核小体内。结果成功构建热休克转录因子4b (Hsf4b)的真核表达载体并在真核细胞HEK293T中表达,研究发现Hsf4b可与MAP激酶P38结合,Hsf4b的C-端转录调控区参与和P38的结合, P38可体外磷酸化Hsf4b。Hsf4b具有转录抑制功能,Hsf4b可与CMV启动子中176bp处TTC(HSE序列)的序列直接结合,抑制CMV启动子活性,把此序列中TTC突变为GCC可抑制Hsf4b对CMV活性的负性调节作用。Hsf4b转录抑制功能通过与转录抑制调节因子Daxx结合实现,Hsf4b被摹集到核内Daxx的POD核小体内,Hsf4b与Daxx的结合受Hsf4b/S299磷酸化调控。结论实验首次证明Hsf4b与P38结合而被磷酸化,并且发现Hsf4b具有转录抑制功能,Hsf4b/S299磷酸化可调控Hsf4b与Daxx结合从而调控Hsf4b转录抑制活性。为进一步探讨Hsf4b在晶状体发育过程中的作用提供了新的信号通路。更多还原

【Abstract】 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.更多还原