目的探讨CENP-A表达在人类胚胎绒毛染色体分离中的作用。方法应用荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测94例自然流产胚胎绒毛染色体数目,分别用q RT-PCR和Western blotting检测胚胎绒毛组织中CENP-A m RNA和...目的探讨CENP-A表达在人类胚胎绒毛染色体分离中的作用。方法应用荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测94例自然流产胚胎绒毛染色体数目,分别用q RT-PCR和Western blotting检测胚胎绒毛组织中CENP-A m RNA和蛋白质相对表达水平。结果 194例自然流产胚胎中检出异常结果病例数共48例,占总病例数的51.06%,其中阳性病例数30例,阳性率为31.91%。比较常见的异常类型有16三体、21三体、22三体、X单体和三倍体。2CENP-A m RNA在实验组和对照组表达水平差异无统计学意义(P>0.05)。3异常组胚胎绒毛组织中CENP-A蛋白相对表达水平明显高于正常组,差异具有统计学意义(P<0.05)。结论着丝粒蛋白CENP-A表达异常与染色体非整倍体引起的自然流产有关。展开更多
目的探讨CENP-A是否参与骨肉瘤细胞化疗耐药及与叉头框蛋白M1(forkhead box M1,FOXM1)的调控关系。方法采用Western blot法检测骨肉瘤亲本细胞(HOS、U2OS)、顺铂耐药细胞(HOS/R、U2OS/R)和稳定FOXM1过表达细胞(HOS/FOXM1、U2OS/FOXM1)中...目的探讨CENP-A是否参与骨肉瘤细胞化疗耐药及与叉头框蛋白M1(forkhead box M1,FOXM1)的调控关系。方法采用Western blot法检测骨肉瘤亲本细胞(HOS、U2OS)、顺铂耐药细胞(HOS/R、U2OS/R)和稳定FOXM1过表达细胞(HOS/FOXM1、U2OS/FOXM1)中CENP-A蛋白表达。转染siRNA后检测CENP-A蛋白表达;运用CCK-8细胞增殖实验观察siRNA敲低CENP-A后耐药骨肉瘤细胞对顺铂敏感性的影响;FOXM1抑制剂RCM1处理后检测CENP-A蛋白表达。Kaplan-Meier法分析GEPIA数据库中CENP-A表达与患者预后的关系,Spearman相关性分析CENP-A和FOXM1两者之间的关系。结果骨肉瘤顺铂耐药细胞中CENP-A蛋白表达比亲本细胞明显升高(0.52±0.03 vs 0.92±0.01,0.33±0.02 vs 1.12±0.01),FOXM1过表达细胞中CENP-A表达较空载体对照亦明显增高;在骨肉瘤顺铂耐药和FOXM1过表达细胞中转染siRNA-CENP-A后,CENP-A蛋白表达均明显降低(0.84±0.01 vs 0.60±0.02,0.98±0.01 vs 0.41±0.01)。转染siRNA敲低CENP-A后,骨肉瘤耐药细胞和FOXM1过表达细胞对顺铂的耐药性明显降低。10μmol/L RCM1 FOXM1抑制剂处理耐药细胞和FOXM1过表达细胞后,CENP-A蛋白表达均明显下降。生物信息学分析发现CENP-A高表达组患者总生存期显著低于低表达组(P<0.01)。Spearman相关性分析发现CENP-A和FOXM1表达呈正相关(r=0.79)。结论FOXM1可能通过调控CENP-A参与骨肉瘤化疗耐药,为骨肉瘤化疗耐药治疗策略提供新的理论基础。展开更多
Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chro...Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.展开更多
Centromere identity is defined by nucleosomes containing CENP-A,a histone H3 variant.The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner.We previously reported that the spatio...Centromere identity is defined by nucleosomes containing CENP-A,a histone H3 variant.The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner.We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68.However,a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading.Here,we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability.We also find that mice harboring the corresponding mutations are lethal.Together,these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.展开更多
Shugoshin-1(Sgo1)is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis.It has been reported that the localization of Sgo1 at the centromere is dependent on...Shugoshin-1(Sgo1)is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis.It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120.However,it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis.Here,we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis.Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A.Interestingly,the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere,resulting in aberrant chromosome segregation and premature chromatid separation.Taken together,these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1–CENP-A interaction guides accurate cell division.展开更多
Centromeres are chromosomal loci marked by histone variant Cen H3(centromeric histone H3)and essential for genomic stability and cell division.The budding yeast E3 ubiquitin ligase Psh1 selectively recognizes the yeas...Centromeres are chromosomal loci marked by histone variant Cen H3(centromeric histone H3)and essential for genomic stability and cell division.The budding yeast E3 ubiquitin ligase Psh1 selectively recognizes the yeast Cen H3(Cse4)for ubiquitination and controls the cellular level of Cse4 for proteolysis,but the underlying mechanism remains largely unknown.Here,we show that Psh1 uses a Cse4-binding domain(CBD,residues 1-211)to interact with Cse4-H4 instead of H3-H4,yielding a dissociation constant(Kd)of 27 nM.Psh1 recognizes Cse4-specific residues in the L1 loop and a2 helix to ensure Cse4 binding and ubiquitination.We map the Psh1-binding region of Cse4-H4 and identify a wide range of Cse4-specific residues required for the Psh1-mediated Cse4 recognition and ubiquitination.Further analyses reveal that histone chaperone Scm3 can impair Cse4 ubiquitination by abrogating Psh1-Cse4 binding.Together,our study reveals a novel Cse4-binding mode distinct from those of known Cen H3 chaperones and elucidates the mechanism by which Scm3 competes with Psh1 for Cse4 binding.展开更多
Wide species crosses often result in uniparental genome elimination and visible failures in centromere func- tion. Crosses involving lines with mutated forms of the CENH3 histone variant that organizes the centromere/...Wide species crosses often result in uniparental genome elimination and visible failures in centromere func- tion. Crosses involving lines with mutated forms of the CENH3 histone variant that organizes the centromere/ kinetochore interface have been shown to have similar effects, inducing haploids at high frequencies. Here, we propose a simple centromere size model that endeavors to explain both observations. It is based on the idea of a quantitative centromere architecture where each centromere in an individual is the same size, and the average size is dictated by a natural equilibrium between bound and unbound CENH3 （and its chaperones or binding proteins）. While centromere size is determined by the cellular milieu, centromere positions are heritable and defined by the interactions of a small set of proteins that bind to both DNA and CENH3. Lines with defective or mutated CENH3 have a lower loading capacity and support smaller centromeres. In cases where a line with small or defective centromeres is crossed to a line with larger or normal centromeres, the smaller/defective centromeres are selectively degraded or not maintained, resulting in chromosome loss from the small-centromere parent. The model is testable and generalizable, and helps to explain the coun- terintuitive observation that inducer lines do not induce haploids when crossed to themselves.展开更多
文摘目的探讨CENP-A表达在人类胚胎绒毛染色体分离中的作用。方法应用荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测94例自然流产胚胎绒毛染色体数目,分别用q RT-PCR和Western blotting检测胚胎绒毛组织中CENP-A m RNA和蛋白质相对表达水平。结果 194例自然流产胚胎中检出异常结果病例数共48例,占总病例数的51.06%,其中阳性病例数30例,阳性率为31.91%。比较常见的异常类型有16三体、21三体、22三体、X单体和三倍体。2CENP-A m RNA在实验组和对照组表达水平差异无统计学意义(P>0.05)。3异常组胚胎绒毛组织中CENP-A蛋白相对表达水平明显高于正常组,差异具有统计学意义(P<0.05)。结论着丝粒蛋白CENP-A表达异常与染色体非整倍体引起的自然流产有关。
文摘目的探讨CENP-A是否参与骨肉瘤细胞化疗耐药及与叉头框蛋白M1(forkhead box M1,FOXM1)的调控关系。方法采用Western blot法检测骨肉瘤亲本细胞(HOS、U2OS)、顺铂耐药细胞(HOS/R、U2OS/R)和稳定FOXM1过表达细胞(HOS/FOXM1、U2OS/FOXM1)中CENP-A蛋白表达。转染siRNA后检测CENP-A蛋白表达;运用CCK-8细胞增殖实验观察siRNA敲低CENP-A后耐药骨肉瘤细胞对顺铂敏感性的影响;FOXM1抑制剂RCM1处理后检测CENP-A蛋白表达。Kaplan-Meier法分析GEPIA数据库中CENP-A表达与患者预后的关系,Spearman相关性分析CENP-A和FOXM1两者之间的关系。结果骨肉瘤顺铂耐药细胞中CENP-A蛋白表达比亲本细胞明显升高(0.52±0.03 vs 0.92±0.01,0.33±0.02 vs 1.12±0.01),FOXM1过表达细胞中CENP-A表达较空载体对照亦明显增高;在骨肉瘤顺铂耐药和FOXM1过表达细胞中转染siRNA-CENP-A后,CENP-A蛋白表达均明显降低(0.84±0.01 vs 0.60±0.02,0.98±0.01 vs 0.41±0.01)。转染siRNA敲低CENP-A后,骨肉瘤耐药细胞和FOXM1过表达细胞对顺铂的耐药性明显降低。10μmol/L RCM1 FOXM1抑制剂处理耐药细胞和FOXM1过表达细胞后,CENP-A蛋白表达均明显下降。生物信息学分析发现CENP-A高表达组患者总生存期显著低于低表达组(P<0.01)。Spearman相关性分析发现CENP-A和FOXM1表达呈正相关(r=0.79)。结论FOXM1可能通过调控CENP-A参与骨肉瘤化疗耐药,为骨肉瘤化疗耐药治疗策略提供新的理论基础。
基金supported by the National Natural Science Foundation of China(No.30930065 and No.31271605)
文摘Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.
基金This work was supported by the Ministry of Science and Technology of China(2017YFA0504202 and 2019YFA0508903)the National Natural Science Foundation of China(31991161 and 32070604)+1 种基金the Beijing Municipal Science and Technology Committee(Z201100005320013)HHMI International Research Scholar grant(55008737).
文摘Centromere identity is defined by nucleosomes containing CENP-A,a histone H3 variant.The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner.We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68.However,a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading.Here,we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability.We also find that mice harboring the corresponding mutations are lethal.Together,these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.
基金supported by grants from the Ministry of Science and Technology of China(2022YFA1303100,2022YFA0806800,2022YFA1302700,and 2017YFA0503600)the National Natural Science Foundation of China(32090040,92254302,92153302,32170733,31621002,and 22177106)+1 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19040000 and XDB37010105)the Ministry of Education(IRT_17R102,20113402130010,and YD2070006001).
文摘Shugoshin-1(Sgo1)is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis.It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120.However,it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis.Here,we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis.Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A.Interestingly,the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere,resulting in aberrant chromosome segregation and premature chromatid separation.Taken together,these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1–CENP-A interaction guides accurate cell division.
基金supported by the grants from Natural Science Foundation of China(31521002,31970621,31871318,31671344,31801070)National Key Research and Development Program of China(2019YFA0508902)Strategic Priority Research Program(XDB37010100)。
文摘Centromeres are chromosomal loci marked by histone variant Cen H3(centromeric histone H3)and essential for genomic stability and cell division.The budding yeast E3 ubiquitin ligase Psh1 selectively recognizes the yeast Cen H3(Cse4)for ubiquitination and controls the cellular level of Cse4 for proteolysis,but the underlying mechanism remains largely unknown.Here,we show that Psh1 uses a Cse4-binding domain(CBD,residues 1-211)to interact with Cse4-H4 instead of H3-H4,yielding a dissociation constant(Kd)of 27 nM.Psh1 recognizes Cse4-specific residues in the L1 loop and a2 helix to ensure Cse4 binding and ubiquitination.We map the Psh1-binding region of Cse4-H4 and identify a wide range of Cse4-specific residues required for the Psh1-mediated Cse4 recognition and ubiquitination.Further analyses reveal that histone chaperone Scm3 can impair Cse4 ubiquitination by abrogating Psh1-Cse4 binding.Together,our study reveals a novel Cse4-binding mode distinct from those of known Cen H3 chaperones and elucidates the mechanism by which Scm3 competes with Psh1 for Cse4 binding.
文摘Wide species crosses often result in uniparental genome elimination and visible failures in centromere func- tion. Crosses involving lines with mutated forms of the CENH3 histone variant that organizes the centromere/ kinetochore interface have been shown to have similar effects, inducing haploids at high frequencies. Here, we propose a simple centromere size model that endeavors to explain both observations. It is based on the idea of a quantitative centromere architecture where each centromere in an individual is the same size, and the average size is dictated by a natural equilibrium between bound and unbound CENH3 （and its chaperones or binding proteins）. While centromere size is determined by the cellular milieu, centromere positions are heritable and defined by the interactions of a small set of proteins that bind to both DNA and CENH3. Lines with defective or mutated CENH3 have a lower loading capacity and support smaller centromeres. In cases where a line with small or defective centromeres is crossed to a line with larger or normal centromeres, the smaller/defective centromeres are selectively degraded or not maintained, resulting in chromosome loss from the small-centromere parent. The model is testable and generalizable, and helps to explain the coun- terintuitive observation that inducer lines do not induce haploids when crossed to themselves.
