Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

染色体传递和细胞周期调节的分子决定因素

• 批准号: 10926077
• 负责人: Munira Basrai
• 金额: $ 234.36万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10926077
• 关键词: Acetylation; Address; Affect; Aneuploidy; Area; Basic Science; Biochemical; Biological Assay; Cell Cycle; Cell Cycle Regulation; Cell Line; Cell model; Cells; Cellular biology; Centromere; Chromatin; Chromosomal Instability; Chromosomal Stability; Chromosome Segregation; Chromosomes; Clinic; Clinical Trials; Complement; DAXX gene; DNA; DNA Sequence; Defect; Diagnosis; Diploidy; Dose; Drosophila polo protein; Ensure; Euchromatin; Excision; F-Box Proteins; Future; Gene Dosage; Genes; Genetic; Genetic Screening; Genome; Hela Cells; Heterogeneity; Histone Acetylation; Histone Deacetylase Inhibitor; Histone H3; Histone H4; Histones; Homologous Gene; Human; In Vitro; Incidence; Kinetochores; Length; Link; Lysine; Malignant Neoplasms; Mediating; Methylation; Microtubules; Mitosis; Mitotic; Molecular; Molecular Chaperones; Mus; Nucleosomes; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Post Translational Modification Analysis; Post-Translational Protein Processing; Process; Prognosis; Proliferating; Proteins; Proteolysis; Regulation; Reporting; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Sister Chromatid; Site; Solid Neoplasm; Sumoylation Pathway; Topoisomerase II; Translating; Ubiquitin-mediated Proteolysis Pathway; Ubiquitination; Variant; Xenograft procedure; Yeast Model System; Yeasts; anticancer research; cancer cell; cancer therapy; centromere protein A; chromosome loss; chromosome missegregation; cohesin; cohesion; constitutive expression; daughter cell; dosage; established cell line; fly; genome wide screen; genome-wide; human model; in vivo; inducible gene expression; insight; interdisciplinary approach; micronucleus; mouse model; multidisciplinary; mutant; novel; overexpression; prevent; segregation; stoichiometry; targeted treatment; tool; transmission process; tumorigenesis

We use multi-organismal (yeast, mouse and human cells) and multi-disciplinary (genetic, cell biology, biochemical and genome-wide) approaches to study faithful chromosome segregation, a fundamental process of every living cell. Genetic screens served as a starting point and in-depth mechanistic studies have provided evidence for new roles for kinetochore genes and the identification of new kinetochore genes. We have identified and defined roles for post-translational modifications (acetylation, methylation, phosphorylation, sumoylation and ubiquitination) of Cse4 in chromosome segregation. Our research is focused on understanding the role of Cse4-associated proteins in chromosome segregation and defining pathways that prevent mislocalization of Cse4 to non-centromeric regions. In the first project we defined roles for Scm3, Pat1, Cdc5 and Sgo1 for the assembly of centromeric chromatin and characterized role of post-translational modifications of centromeric histones in faithful chromosome segregation. We determined that imbalanced stoichiometry of a Cse4 chaperone, Scm3 (HJURP in humans) leads to chromosome mis-segregation in both human and yeast cells thereby providing a link between HJURP overexpression and mitotic defects in cancers (Mishra et al., 2011). Scm3 interacts with Pat1 (Protein associated with topoisomerase II) and Pat1 regulates the topology of centromeric chromatin (Mishra et al., 2013). We used a pat1 deletion strain to define the number of Cse4 molecules at the yeast kinetochore (Hasse, Mishra 2013, Mishra et al., 2015) and provided evidence for a structural role for Pat1 in the structural integrity of centromeric chromatin and localization of Cse4 for faithful chromosome segregation. In addition to kinetochore proteins, association of cohesins with centromeres and along the length of the chromosomes ensures faithful segregation of sister chromatids during mitosis. We reported that evolutionarily conserved polo kinase, Cdc5 associates with centromeric chromatin to facilitate the removal of centromeric cohesins (Mishra et al., 2016) and Cdc5-mediated phosphorylation of Cse4 regulates faithful chromosome segregation (Mishra et al., 2019). Furthermore, evolutionarily conserved Sgo1 which protects centromeric cohesion interacts with Cse4 and this is required for faithful chromosome segregation (Mishra et al., 2018). We recently reported that evolutionarily conserved Hpr1 prevents the accumulation of R-loops at centromeric chromatin affects the assembly of kinetochore and leads to chromosomal instability (Mishra et al., 2021). We have done a comprehensive analysis of Post-translational modifications (PTMs) of Cse4 and identified conserved sites for acetylation, methylation, and phosphorylation (Boeckmann et al., 2013). We determined that evolutionarily conserved Aurora B/Ipl1 kinase phosphorylates Cse4 in vivo and in vitro for faithful chromosome segregation (Boeckmann et al., 2013) and that cell cycle regulated methylation of Cse4 prevents CIN (Mishra et al., 2023). Using budding yeast with a single nucleosome we provided the first evidence that yeast centromeres contain hypoacetylated histone H4 and that increased acetylation of histone H4 on lysine 16 (H4K16) leads to chromosome mis-segregation (Choy et al., 2011). Even though HDAC inhibitors (HDACi) are used in clinical trials we do not fully understand their mode of action. A genome-wide screen with an HDACi was used to identify pathways that are vulnerable to altered histone acetylation. Our results showed that chromosome segregation mutants are more sensitive to HDACi (Choy et al., 2015). Future studies will allow us to understand the molecular role of PTMs of Cse4 in chromosome segregation and determine if these PTMs are conserved in human CENP-A. In the second project we have focused on the identification of pathways that prevent mislocalization of Cse4 and CIN. We showed previously that S. cerevisiae spt4 mutants show mislocalization of Cse4 and chromosome segregation defects that are complemented by human SPT4 (Basrai et al, 1996 and Crotti and Basrai 2004). We established the cause and effect of Cse4 mislocalization by showing that altered histone dosage and mislocalization of Cse4 to non-centromeric chromatin correlate with chromosome loss (Au et al., 2008). We identified a novel role for the N terminus of Cse4 in ubiquitin (Ub)-mediated proteolysis for faithful chromosome segregation (Au et al., 2013) and showed that Cse4 is sumoylated and ubiquitination of sumoylated Cse4 by Slx5 regulates its proteolysis to prevent mislocalization to euchromatin (Ohkuni et al., 2016, 2018, 2020). Genome-wide approaches have been used to identify regulators that prevent mislocalization of Cse4 to euchromatin and these studies revealed a role for histone chaperones (Ciftci-Yilmaz et al., 2018). F-box proteins Cdc4 and Met30 in Cse4 proteolysis (Au et al., 2020), Dbf4 dependent kinase (DDK) (Eisenstatt et al., 2020) and Cdc48 (Ohkuni et al., 2022). Furthermore, reduced dosage of histone H4 prevents mislocalization of Cse4 (Eisenstatt et al., 2021). In the third project, we have focused on causes and consequences of mislocalization of CENP-A in human cells and xenograft mouse model. Mislocalization of CENP-A has been observed in many cancers and this correlates with poor prognosis. Hence, it is critical to understand how CENP-A overexpression contributes to tumorigenesis and whether CENP-A expression can be exploited for prognosis, diagnosis and targeted treatment of CENP-A overexpressing cancers. We established cell lines and optimized cell biology-based assays to address a long-standing question of whether mislocalization of overexpressed CENP-A contributes to CIN. We determined that constitutive or inducible expression of CENP-A in HeLa and stable diploid RPE1 cells results in mislocalization of CENP-A to non-centromeric regions. Comprehensive analysis for mitotic effects showed a dose-dependent effect of CENP-A overexpression on chromosome segregation defects and higher incidence of micronuclei. Altered localization of kinetochore proteins contributes to a weakening of the native kinetochore in CENP-A overexpressing cells. Depletion of the histone chaperone DAXX prevents CENP-A mislocalization and rescues the CIN phenotype in CENP-A overexpressing cells. These results show that mislocalization of CENP-A is one of the major contributors for CIN in CENP-A overexpressing cells. Our studies provide the first evidence for how mislocalization of CENP-A to non-centromeric chromatin contributes to CIN in human cells and provide mechanistic insights into how CENP-A overexpression may contribute to aneuploidy in CENP-A overexpressing cancers (Shrestha et al., 2017). We recently reported that mislocalization of overexpressed CENP-A in pseudodiploid DLD1 cell line and xenograft mouse model contribute to CIN, aneuploidy with karyotypic heterogeneity (Shrestha et al., 2021). We are pursuing studies with human homologs of the yeast genes identified in genome wide screens and using genome-wide approaches to identify and characterize pathways that prevent mislocalization of CENP-A and CIN. We recently reported that histone H3 chaperone, CHAF1B prevents mislocalization of CENP-A and CIN in human cells (Shrestha et al., 2023). n summary, our studies using multi-organismal and multi-disciplinary approaches have provided mechanistic insights for how defects in kinetochore function contribute to aneuploidy in human cancers. We are optimistic that our studies will help translate basic science research to the clinic and aid in the diagnosis, prognosis and treatment of cancers that show overexpression of CENP-A.

我们使用多生物（酵母，小鼠和人类细胞）以及多学科（遗传，细胞生物学，生化和全基因组）方法来研究忠实的染色体隔离，这是每个活细胞的基本过程。遗传筛选是起点，深入的机械研究为动物学基因的新作用提供了证据和新的动型基因的鉴定。我们已经确定了CSE4染色体分离中CSE4的翻译后修饰（乙酰化，甲基化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化，磷酸化和泛素化）的作用。我们的研究集中在理解CSE4相关蛋白在染色体分离中的作用，并定义途径，以防止CSE4错误地定位到非中心粒子区域。在第一个项目中，我们定义了scm3，pat1，cdc5和sgo1的作用，用于集中丝粒染色质的组装，并在忠实的染色体隔离中的centromeric组蛋白的翻译后修饰作用。我们确定CSE4伴侣伴侣SCM3（人类的HJURP）的化学计量学不平衡会导致人和酵母细胞中的染色体错误分离，从而在Cancers中提供Hjurp过表达和有丝分裂缺陷之间的联系（Mishra等人，Mishra等人，2011年）。 SCM3与PAT1相互作用（与拓扑异构酶II相关的蛋白质），PAT1调节着丝粒染色质的拓扑（Mishra等，2013）。我们使用PAT1缺失菌株来定义酵母动力学上的CSE4分子的数量（Hasse，Mishra，2013； Mishra等，2015），并为PAT1在CENTROMERIC染色质的结构完整性和CSE4定位的结构完整性中提供了证据，并为忠实的染色体染色体植物的定位提供了证据。除了动力学蛋白外，粘连蛋白与丝粒的缔合以及沿染色体的长度确保了有丝分裂过程中姐妹染色单体的忠实隔离。我们报道说，进化保守的polo激酶，CDC5与丝粒染色质相关，以促进去除丝粒粘着素（Mishra等，2016）和CDC5介导的CSE4磷酸化CSE4调节忠实的染色体染色体分离（Mishra等人（Mishra等人，2019年）。此外，保护着丝粒凝聚力的进化保守的SGO1与CSE4相互作用，这对于忠实的染色体隔离是必需的（Mishra等，2018）。我们最近报道，进化保守的HPR1阻止了R-loops在丝粒染色质上的积累会影响动力学的组装并导致染色体不稳定性（Mishra等，2021）。我们已经对CSE4的翻译后修饰（PTM）进行了全面分析，并确定了乙酰化，甲基化和磷酸化的保守位点（Boeckmann等，2013）。我们确定，进化保守的Aurora B/IPL1激酶在体内和体外磷酸化CSE4的忠实染色体分离（Boeckmann等，2013），该细胞周期调节CSE4的甲基化CIT（Mishra等，2023）。使用单个核小体的出现酵母，我们提供了第一个证据，表明酵母中心粒含有低乙酰化组蛋白H4，并且在赖氨酸16（H4K16）上增加组蛋白H4的乙酰化会导致染色体错误播种（Choy等，2011）。即使HDAC抑制剂（HDACI）用于临床试验中，我们也不完全了解其作用方式。带有HDACI的全基因组筛选用于识别容易改变组蛋白乙酰化的途径。我们的结果表明，染色体分离突变体对HDACI更敏感（Choy等，2015）。未来的研究将使我们能够理解CSE4 PTM在染色体分离中的分子作用，并确定这些PTM是否在人CENP-A中保守。在第二个项目中，我们专注于识别防止CSE4和CIN错误定位的途径。我们先前表明，酿酒酵母SPT4突变体表明CSE4和染色体隔离缺陷的错误定位是由人类SPT4补充的（Basrai等，1996以及Crotti和Basrai 2004）。我们通过表明CSE4错误定位的原因和作用，表明组蛋白剂量改变了CSE4与非中心染色质与染色体损失相关（Au等，2008）。 We identified a novel role for the N terminus of Cse4 in ubiquitin (Ub)-mediated proteolysis for faithful chromosome segregation (Au et al., 2013) and showed that Cse4 is sumoylated and ubiquitination of sumoylated Cse4 by Slx5 regulates its proteolysis to prevent mislocalization to euchromatin (Ohkuni et al., 2016, 2018，2020）。全基因组方法已被用来识别防止CSE4错误定位到白染色质的调节剂，这些研究揭示了组蛋白伴侣的作用（Ciftci Yilmaz等，2018）。 CSE4蛋白水解中的F-box蛋白Cdc4和Met30（Au等，2020），DBF4依赖性激酶（DDK）（Eisenstatt等，2020）和Cdc48（Ohkuni等人，2022年）。此外，组蛋白H4的剂量降低可防止CSE4的错误定位（Eisenstatt等，2021）。在第三个项目中，我们专注于人类细胞和异种移植小鼠模型中CENP-A错误定位的原因和后果。在许多癌症中已经观察到CENP-A的错误定位，这与预后不良有关。因此，重要的是要了解CENP-A过表达如何有助于肿瘤发生，以及CENP-A表达是否可以用于预后，诊断和靶向CENP-A过表达癌症的靶向治疗。我们建立了细胞系和优化的基于细胞生物学的测定方法，以解决一个长期的问题，即过表达CENP-A是否有助于CIN。我们确定CENP-A在HELA和稳定的二倍体RPE1细胞中的组成型或诱导表达导致CENP-A将CENP-A错误定位到非中心粒子区域。全面的有丝分裂作用分析表明，CENP-A过表达对染色体隔离缺陷的剂量依赖性作用和较高的微核发生率。动力学蛋白的定位改变改变了CENP-A过表达细胞中天然动力学的弱化。组蛋白伴侣DAXX的耗竭可防止CENP-A错误定位并挽救CENP-A过表达细胞中的CIN表型。这些结果表明，CENP-A的错误定位是CENP-A过表达细胞中CIN的主要因素之一。我们的研究提供了第一个证据，证明CENP-A对非中心染色质的错误定位如何有助于人类细胞中的CIN，并为CENP-A过表达如何有助于CENP-A过表达的CENP-A过表达癌症提供机械洞察力（Shrestha等，2017）。我们最近报道说，假二倍体DLD1细胞系中过表达的CENP-A和异种移植小鼠模型中过表达的CENP-A模型有助于CIN，cIN，具有核型异质性的非整倍性（Shrestha等，2021）。我们正在研究基因组宽筛查中鉴定出的酵母基因的人类同源物，并采用全基因组方法来识别和表征防止CENP-A和CIN错误定位的途径。我们最近报道，组蛋白H3伴侣CHAF1B可防止人类细胞中CENP-A和CIN的错误定位（Shrestha等，2023）。总而言之，我们使用多生物和多学科方法的研究为动物学功能缺陷如何有助于人类癌症的非整倍性提供了机械见解。我们乐观的是，我们的研究将有助于将基础科学研究转化为诊所，并有助于诊断，预后和治疗表现出CENP-A过表达的癌症。

项目成果

N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric Chromatin.

• DOI: 10.1534/g3.117.300419
• 发表时间: 2018-03-28
• 期刊: G3 (Bethesda, Md.)
• 作者: Ohkuni K;Levy-Myers R;Warren J;Au WC;Takahashi Y;Baker RE;Basrai MA
• 通讯作者: Basrai MA

Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae.

• DOI: 10.1091/mbc.e12-12-0893
• 发表时间: 2013-06
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Boeckmann L;Takahashi Y;Au WC;Mishra PK;Choy JS;Dawson AR;Szeto MY;Waybright TJ;Heger C;McAndrew C;Goldsmith PK;Veenstra TD;Baker RE;Basrai MA
• 通讯作者: Basrai MA

SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin.

SUMO 靶向泛素连接酶 (STUbL) 可降低与亨廷顿突变、易聚集片段相关的毒性和异常转录活性。

• DOI: 10.3389/fgene.2018.00379
• 发表时间: 2018
• 期刊: Frontiers in genetics
• 影响因子: 3.7
• 作者: Ohkuni,Kentaro;Pasupala,Nagesh;Peek,Jennifer;Holloway,GraceLauren;Sclar,GloriaD;Levy-Myers,Reuben;Baker,RichardE;Basrai,MuniraA;Kerscher,Oliver
• 通讯作者: Kerscher,Oliver

Yeast hEST1A/B (SMG5/6)-like proteins contribute to environment-sensing adaptive gene expression responses.

• DOI: 10.1534/g3.113.006924
• 发表时间: 2013-10-03
• 期刊: G3 (Bethesda, Md.)
• 作者: Lai X;Beilharz T;Au WC;Hammet A;Preiss T;Basrai MA;Heierhorst J
• 通讯作者: Heierhorst J

Dosage suppression genetic interaction networks enhance functional wiring diagrams of the cell.

• DOI: 10.1038/nbt.1855
• 发表时间: 2011-05-15
• 期刊: Nature biotechnology
• 影响因子: 46.9
• 作者: Magtanong L;Ho CH;Barker SL;Jiao W;Baryshnikova A;Bahr S;Smith AM;Heisler LE;Choy JS;Kuzmin E;Andrusiak K;Kobylianski A;Li Z;Costanzo M;Basrai MA;Giaever G;Nislow C;Andrews B;Boone C
• 通讯作者: Boone C