Background and objective Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer.PR-Set7(also known as Set8)is a cell cycle regulated enzyme that ca...Background and objective Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer.PR-Set7(also known as Set8)is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20(H4K20me1)to promote chromosome condensation and prevent DNA damage.Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage.This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage.Methods We developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A.We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo.With this model,staining of PR-Set7 in the preneoplastic and tumor lesions in Adeno Cre-induced mouse lungs was performed.Meanwhile we identified higher protein level changes ofγ-tubulin and pericentrin by IHC.Results The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A.We also identified higher levels of the proteins pericentrin andγ-tubulin in Cul4A mouse lungs induced by Adeno Cre.Conclusion PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.展开更多
目的探讨组蛋白甲基转移酶PR-Set7在亚砷酸钠致人永生化皮肤角质形成细胞(Ha Ca T细胞)DNA损伤中的作用机制。方法将处于对数生长期的Ha Ca T细胞暴露于含终浓度分别为0(对照)、1.25、2.5、5、10μmol/L亚砷酸钠的DMEM高糖培养基染毒24 ...目的探讨组蛋白甲基转移酶PR-Set7在亚砷酸钠致人永生化皮肤角质形成细胞(Ha Ca T细胞)DNA损伤中的作用机制。方法将处于对数生长期的Ha Ca T细胞暴露于含终浓度分别为0(对照)、1.25、2.5、5、10μmol/L亚砷酸钠的DMEM高糖培养基染毒24 h。采用MTT法测定Ha Ca T细胞的存活情况。另设对照(24 h)组、亚砷酸钠(10μmol/L,24 h)染毒组、PR-Set7小干扰RNA干扰组(Si PR-Set7,50 nmol/L,48 h)和联合暴露(50 nmol/L Si PR-Set7干扰48 h后,10μmol/L亚砷酸钠暴露24 h)组。分别采用q RT-PCR法检测PR-Set7 m RNA的表达水平,采用Western blot法检测H4K20me1和PR-Set7蛋白的表达水平,采用单细胞凝胶电泳法检测DNA损伤水平。结果与对照组相比,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞的存活率均较低,差异有统计学意义(P<0.05)。与对照组比较,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均升高,而H4K20me1蛋白的表达水平降低;2.5、5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞尾部DNA%及尾矩升高,差异有统计学意义(P<0.05)。随着亚砷酸钠染毒浓度的升高,Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均呈上升趋势,而H4K20me1蛋白和m RNA的表达水平呈下降趋势,DNA损伤水平(尾部DNA%及尾矩)呈上升趋势。与对照组比较,Si PR-Set7干扰组Ha Ca T细胞PR-Set7蛋白的表达大幅下降;亚砷酸钠染毒组、Si PRSet7干扰组和联合暴露组Ha Ca T细胞H4K20me1蛋白的表达水平均降低,而尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。与亚砷酸钠染毒组比较,联合暴露组Ha Ca T细胞PR-Set7蛋白和m RNA以及H4K20me1蛋白的表达均降低,而Ha Ca T细胞尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。结论在对Ha Ca T细胞进行PR-Set7干扰后,亚砷酸钠可能通过抑制组蛋白甲基转移酶PR-Set7蛋白及m RNA的表达水平,从而降低H4K20me1的修饰水平,影响Ha Ca T细胞DNA损伤修复能力,使DNA损伤加重。展开更多
Background and Aims:Multiple regulatory mechanisms play an important role in arsenic-induced liver injury.To investigate whether histone H3 lysine 4(H3K4)methyltransferase(SET7/9)and histone H3K4 demethyltransferase(L...Background and Aims:Multiple regulatory mechanisms play an important role in arsenic-induced liver injury.To investigate whether histone H3 lysine 4(H3K4)methyltransferase(SET7/9)and histone H3K4 demethyltransferase(LSD1/KDM1A)can regulate endoplasmic reticulum stress(ERS)-related apoptosis by modulating the changes of H3K4 methylations in liver cells treated with arsenic.Methods:Apoptosis,proliferation and cell cycles were quantified by flow cytometry and real-time cell analyzer.The expression of ERS-and epigenetic-related proteins was detected by Western blot analysis.The antisense SET7/9 expression vector and the overexpressed LSD1 plasmid were used for transient transfection of LO_(2) cells.The effects of NaAsO_(2) on the methylation of H3 in the promoter regions of 78 kDa glucose-regulated protein,activating transcription factor 4 and C/EBP-homologous protein were evaluated by chromatin immunoprecipitation assay.Results:The protein expression of LSD1(1.25±0.08 vs.1.77±0.08,p=0.02)was markedly decreased by treatment with 100μM NaAsO_(2),whereas the SET7/9(0.68±0.05 vs.1.10±0.13,p=0.002)expression level was notably increased,which resulted in increased H3K4me1/2(0.93±0.64,1.19±0.22 vs.0.71±0.13,0.84±0.13,p=0.03 and p=0.003).After silencing SET7/9 and overexpressing LSD1 by transfection,apoptosis rate(in percentage:3.26±0.34 vs.7.04±0.42,4.80±0.32 vs.7.52±0.38,p=0.004 and p=0.02)was significantly decreased and proliferation rate was notably increased,which is reversed after inhibiting LSD1(in percentage:9.31±0.40 vs.7.52±0.38,p=0.03).Furthermore,the methylation levels of H3 in the promoter regions of GRP78(20.80±2.40 vs.11.75±2.47,20.46±2.23 vs.14.37±0.91,p=0.03 and p=0.01)and CHOP(48.67±4.04 vs.16.67±7.02,59.33±4.51 vs.20.67±3.06,p=0.004 and p=0.001)were significantly increased in LO_(2) cells exposed to 100μM NaAsO_(2) for 24 h.Conclusions:Histone methyltransferase SET7/9 and histone demethyltransferase LSD1 jointly regulate the changes of H3K4me1/me2 levels in arsenic-induced apoptosis.NaAsO_(2) induces apoptosis in LO_(2) cells by activating the ERS-mediated apoptotic signaling pathway,at least partially by enhancing the methylation of H3 on the promoter regions of ERS-associated genes,including GRP78 and CHOP.展开更多
Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms under...Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.展开更多
基金support from the Kazan, Mc Clain, Abrams, Fernandez, Lyons, Greenwood, Harley & Oberman Foundation, Incthe Estate of Robert Griffiths+3 种基金the Jeffrey and Karen Peterson Family FoundationPaul and Michelle Zygielbaumthe Estate of Norman Mancinithe Barbara Isackson Lung Cancer Research Fund
文摘Background and objective Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer.PR-Set7(also known as Set8)is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20(H4K20me1)to promote chromosome condensation and prevent DNA damage.Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage.This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage.Methods We developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A.We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo.With this model,staining of PR-Set7 in the preneoplastic and tumor lesions in Adeno Cre-induced mouse lungs was performed.Meanwhile we identified higher protein level changes ofγ-tubulin and pericentrin by IHC.Results The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A.We also identified higher levels of the proteins pericentrin andγ-tubulin in Cul4A mouse lungs induced by Adeno Cre.Conclusion PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.
文摘目的探讨组蛋白甲基转移酶PR-Set7在亚砷酸钠致人永生化皮肤角质形成细胞(Ha Ca T细胞)DNA损伤中的作用机制。方法将处于对数生长期的Ha Ca T细胞暴露于含终浓度分别为0(对照)、1.25、2.5、5、10μmol/L亚砷酸钠的DMEM高糖培养基染毒24 h。采用MTT法测定Ha Ca T细胞的存活情况。另设对照(24 h)组、亚砷酸钠(10μmol/L,24 h)染毒组、PR-Set7小干扰RNA干扰组(Si PR-Set7,50 nmol/L,48 h)和联合暴露(50 nmol/L Si PR-Set7干扰48 h后,10μmol/L亚砷酸钠暴露24 h)组。分别采用q RT-PCR法检测PR-Set7 m RNA的表达水平,采用Western blot法检测H4K20me1和PR-Set7蛋白的表达水平,采用单细胞凝胶电泳法检测DNA损伤水平。结果与对照组相比,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞的存活率均较低,差异有统计学意义(P<0.05)。与对照组比较,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均升高,而H4K20me1蛋白的表达水平降低;2.5、5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞尾部DNA%及尾矩升高,差异有统计学意义(P<0.05)。随着亚砷酸钠染毒浓度的升高,Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均呈上升趋势,而H4K20me1蛋白和m RNA的表达水平呈下降趋势,DNA损伤水平(尾部DNA%及尾矩)呈上升趋势。与对照组比较,Si PR-Set7干扰组Ha Ca T细胞PR-Set7蛋白的表达大幅下降;亚砷酸钠染毒组、Si PRSet7干扰组和联合暴露组Ha Ca T细胞H4K20me1蛋白的表达水平均降低,而尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。与亚砷酸钠染毒组比较,联合暴露组Ha Ca T细胞PR-Set7蛋白和m RNA以及H4K20me1蛋白的表达均降低,而Ha Ca T细胞尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。结论在对Ha Ca T细胞进行PR-Set7干扰后,亚砷酸钠可能通过抑制组蛋白甲基转移酶PR-Set7蛋白及m RNA的表达水平,从而降低H4K20me1的修饰水平,影响Ha Ca T细胞DNA损伤修复能力,使DNA损伤加重。
基金The present study was supported by the National Natural Science Foundation of China(Grant No.81100284)Guizhou Science and Technology Cooperation Platform Personnel[2018](Grant No.5779-10,5779-19)Science and Technology Foundation of Guizhou Province(Grant No.ZK[2021]-364)。
文摘Background and Aims:Multiple regulatory mechanisms play an important role in arsenic-induced liver injury.To investigate whether histone H3 lysine 4(H3K4)methyltransferase(SET7/9)and histone H3K4 demethyltransferase(LSD1/KDM1A)can regulate endoplasmic reticulum stress(ERS)-related apoptosis by modulating the changes of H3K4 methylations in liver cells treated with arsenic.Methods:Apoptosis,proliferation and cell cycles were quantified by flow cytometry and real-time cell analyzer.The expression of ERS-and epigenetic-related proteins was detected by Western blot analysis.The antisense SET7/9 expression vector and the overexpressed LSD1 plasmid were used for transient transfection of LO_(2) cells.The effects of NaAsO_(2) on the methylation of H3 in the promoter regions of 78 kDa glucose-regulated protein,activating transcription factor 4 and C/EBP-homologous protein were evaluated by chromatin immunoprecipitation assay.Results:The protein expression of LSD1(1.25±0.08 vs.1.77±0.08,p=0.02)was markedly decreased by treatment with 100μM NaAsO_(2),whereas the SET7/9(0.68±0.05 vs.1.10±0.13,p=0.002)expression level was notably increased,which resulted in increased H3K4me1/2(0.93±0.64,1.19±0.22 vs.0.71±0.13,0.84±0.13,p=0.03 and p=0.003).After silencing SET7/9 and overexpressing LSD1 by transfection,apoptosis rate(in percentage:3.26±0.34 vs.7.04±0.42,4.80±0.32 vs.7.52±0.38,p=0.004 and p=0.02)was significantly decreased and proliferation rate was notably increased,which is reversed after inhibiting LSD1(in percentage:9.31±0.40 vs.7.52±0.38,p=0.03).Furthermore,the methylation levels of H3 in the promoter regions of GRP78(20.80±2.40 vs.11.75±2.47,20.46±2.23 vs.14.37±0.91,p=0.03 and p=0.01)and CHOP(48.67±4.04 vs.16.67±7.02,59.33±4.51 vs.20.67±3.06,p=0.004 and p=0.001)were significantly increased in LO_(2) cells exposed to 100μM NaAsO_(2) for 24 h.Conclusions:Histone methyltransferase SET7/9 and histone demethyltransferase LSD1 jointly regulate the changes of H3K4me1/me2 levels in arsenic-induced apoptosis.NaAsO_(2) induces apoptosis in LO_(2) cells by activating the ERS-mediated apoptotic signaling pathway,at least partially by enhancing the methylation of H3 on the promoter regions of ERS-associated genes,including GRP78 and CHOP.
基金This work was supported in part by the National Natural Science Foundation of China(31430054,31320103904,31621002,31671405,91854203,91853115,21922706,31671407,31871359,31601097,and 21672201)the National Key Research and Development Program of China(2017YFA0503600 and 2016YFA0100500)+2 种基金Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19000000)Chinese Academy of Sciences Center for Excellence in Molecular Cell Science(2015HSC-UE010)MOE Innovative Team(IRT_17R102),and the US National Institutes of Health(CA164133and DK26929).
文摘Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.
