| BackgroundCataracts are a leading cause of blindness.UV irradiation,abnormal metabolism,gene mutation and aging can lead to lens metabolism disorders and lens opacity of eyes,which are the main causes of cataracts.The lens of the mammalian eye is a clear structure composed of two types of cells,epithelial and fibrous.The lens in the mammalian eye is a transparent structure consisting of two types of cells,epithelial cells and fibrous cells.Epithelial cells,which are mainly arranged in the anterior hemisphere of the lens.They are composed of monolayer cells and have the ability of lifelong proliferation and differentiation.The rest is composed of terminally differentiated fibrous cells,which mainly maintain the internal stability and transparency of the lens.In the mammalian lens,epithelial cells stagnate through the cell cycle,elongate and differentiate into lens fiber cells.Lens epithelium maintains the stable state of lens fiber,protects internal fiber cells from stimulation and oxidative damage,provides nutrients for fiber cells,and plays a crucial role in the mutual transport of liquid and ions within the lens,affecting the growth,differentiation and stability of the lens.Senile cataract is the most common type of cataract,mainly manifested as opacity of lens.The aging of lens epithelial cells will inhibit cell proliferation and block cell cycle,leading to the occurrence of cataract.Heat shock factor(HSF)is the main transcription factor mediating heat shock response,which mainly regulates the expression of heat shock protein(Hsps).Heat shock proteins act as molecular chaperones to protect cells from damage such as protein toxicity.In heat shock or under certain stress conditions,heat shock factors bind to heat shock elements(HSE)on the heat shock protein(HSPs)gene promoter to activate the transcription of heat shock protein genes.In mammals,heat shock factor is composed of at least three members,namely Hsf1,Hsf2 and Hsf4.Hsf4,a member of the HSF family,is mainly expressed in the lens and is one of the common genes associated with congenital cataract.Hsf4 contains conserved DNA binding domains and trimerization domains.Compared with other heat shock transcription factors,there is no structural domain that inhibits trimerization,so Hsf4 has always been in the form of trimers,which regulate the expression of heat shock proteins as transcriptional activators.Mutations of Hsf4,such as R74 H and L115 P,lead to dominant and recessive congenital cataracts with normal staining,while mutations of Q62 R,R117H and other sites can lead to age-related cataracts.The mechanism of Hsf4 dysfunction leading to senile cataract formation remains unclear.The causes of cell senescence regulation are complex and diverse,involving a variety of molecular pathways.The most ordinary molecular pathways include ATM-p53-p21,p16-Rb,etc.In our previous study,we found that the knockout of Hsf4 in mouse lens epithelial cells resulted in increased expression of age-related cyclin-dependent kinase inhibitor(CDKN1A)p21.However,how the Hsf4 in lens epithelial cells regulates p21 expression and participates in the regulation of cellular senescence is not clear.ObjectiveBy revealing the molecular mechanism of Hsf4 regulating p21 expression,the molecular function and mechanism of Hsf4 involved in lens cell aging was elaborated.It provides a new direction for understanding the molecular mechanism of senile cataract and the development of targeted drugs.Methods1.Wild type zebrafish of different age groups and adult zebrafish with Hsf4 knockout were anesthetized with 0.003% Tricaine anesthetic,and the eyes of zebrafish were observed under the slit lamp.2.The aging phenotype of lens epithelial cells in zebrafish was detected by β-galactosidase staining of adult zebrafish lens with wild-type and Hsf4 knockout.RNA from the lens of adult zebrafish with wild-type and Hsf4 knockout was taken and real-time quantitative PCR was used to detect the expression of senescence related secretory factors.3.Constructed Hsf4-knockout mouse lens epithelial cells(m LEC)were used to detect the expression levels of senescence related genes p21,p53 and p-p53 in the condition of UV stimulation.4.Luciferase reporter plasmids with different truncated bodies of p21 promoter were constructed,and the binding of Hsf4 and p21 promoter was detected by using double Luciferase reporter technology.Direct binding of Hsf4 to p21 promoter was detected by chromatin immunoprecipitation(CHIP)assay.5.RNA of lens of wild-type and knockout Hsf4 zebrafish of different ages was taken and the expression of p21 was detected by real-time quantitative PCR.6.P21 interfering RNA was transfected into 3a and Hsf4-knockout mouse lens cells,and the aging phenotype was detected by β-galactosidase staining.7.Epigenetic modification regulation detection of p21 promoter by Hsf4: western blot detect of trimethylation of histone H3 lysine in m LEC/Hsf4b-/-and HA-hsf4 b cells.Chromatin immunoprecipitation(CHIP)was used to detect the binding of H3K27M3 to p21 promoter in m LEC/Hsf4-/-and HA-Hsf4 cells.p21 promoter DNA labeled with biotin was used for DNA pull down technology to detect the binding of methyl transferase EZH2,G9 a and p21 promoter.8.The detection method for the interaction between Hsf4 and methyltransferase EZH2 and G9a: in m LEC/Hsf4-/-and HA-Hsf4 b cells,immunoprecipitation(IP)technology was used to detect the interaction between EZH2,G9 a and Hsf4 protein by ha-tag antibody immunoprecipitation of Hsf4 and western blot assay.GST-Hsf4(1-230),GST-Hsf4(196-493)and GST-Hsf4(320-493)with GST tag were used to test the interaction between each truncated body of Hsf4 and EZH2 and G9 a by GST Pull down experiment.Co-localization of Hsf4 with EZH2 and G9 a was detected by an immunofluorescence assay in m LEC/Hsf4b-/-and HA-Hsf4 b cells.Results1.It was observed under slit lamp that with the growth of age,severe cataract symptoms would appear in the eyes of zebrafish with Hsf4 knockout.2.Compared with wild-type zebrafish,the lens epithelial cells of Hsf4 zebrafish with knockout showed a cellular aging phenotype;the expression of z IL15,z IF,and z-CXCL12 a in Hsf4 zebrafish lens was increased,while the expression of z IL1 b,z IL7 alf,z IL12 a,z IL13,and zp16 in Hsf4 zebrafish lens remained unchanged.The results showed that the lens of Hsf4 knockout zebrafish had senescence related types.3.Compared with wild lens epithelial cells,Hsf4 was knocked out in mouse lens epithelial cells,resulting in increased p21 expression independent of p53.4.The results of dual luciferase reporting technique showed that Hsf4 binds HSE of p21 promoter and negatively regulates p21 expression.Mean while,chromatin immunoprecipitation(CHIP)showed that Hsf4 could bind directly to the p21 promoter.5.Compared with wild zebrafish,the expression of p21 in the lens of Hsf4 knockout zebrafish was significantly increased.6.The knockout of Hsf4 in mouse lens epithelial cells interferes with p21,which weakens the aging phenotype of cells.The cell aging caused by Hsf4 knockout depends on the increase of p21 expression.7.Compared with m LEC/Hsf4-/-mouse lens epithelial cells,Hsf4 promoted the trimethylation level of H3K27 on p21 promoter in the lens epithelial cell lines of overexpressed Hsf4 mice.8.Compared with m LEC/Hsf4-/-mouse lens epithelial cells,Hsf4 protein interacts with methyltransferase EZH2 and G9 a protein in the overexpressed mouse lens epithelial cells.Conclusions1.In animal models,the lens of Hsf4 zebrafish was knocked out for the cataract phenotype,and the expression of aging related secretory factors and aging related gene p21 was increased.2.In lens epithelial cells,Hsf4 trimethylates H3K27 on p21 promoter by recruiting methyltransferase EZH2 and G9 a,resulting in decreased expression of p21,which may be the cause of aging of lens epithelial cells caused by Hsf4 knockout. |
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