Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

进化上保守的着丝粒组蛋白H3变体Cse4（人类中为CENP-A）及其伴侣Scm3（人类中为HJURP）在许多癌症中被证明是染色体分离所必需的。据报道，HJURP在肺癌和乳腺癌细胞中过表达和错定位，HJURP表达升高的患者生存率降低。HJURP过表达是否诱导肿瘤发生尚不清楚。我们发现HJURP和SCM3的不平衡化学计量导致人类和酵母细胞中染色体分离和着丝点完整性的缺陷，从而提供了HJURP过表达与癌症中有丝分裂缺陷之间的联系。现在，全基因组筛选将使我们能够识别抑制或增强与SCM3/HJURP过表达相关的表型的基因/途径，从而可能推断出癌症。在这些研究的继续，我们已经证明Pat1（与拓扑异构酶II相关的蛋白质）与Scm3相互作用。我们确定着丝粒染色质的结构完整性和忠实的染色体分离需要Pat1。在与Kerry Bloom的合作中，我们使用了一个pat1 null菌株来确定酵母着丝点上Cse4分子的数量。对PAT1的研究将帮助我们了解着丝粒染色质的拓扑结构如何调节染色体分离，这是一个目前尚未探索的研究领域。组蛋白的翻译后修饰（PTM）对包括染色体分离在内的许多染色质活动至关重要。组蛋白去乙酰化酶（HDAC）抑制剂被用于治疗某些癌症，然而，我们并不完全了解这些抑制剂的分子靶点。我们研究了着丝粒组蛋白PTM在出芽酵母中的性质和作用，目的是将PTM用于抗癌治疗。我们首次发现出芽酵母着丝粒含有低乙酰化的组蛋白H4，并且组蛋白H4在赖氨酸16 （H4K16）上乙酰化的增加导致染色体错误分离。我们还发现，H4K16乙酰转移酶Sas2和H4K16去乙酰化酶Sir2的平衡是染色体分离所必需的。值得注意的是，Sas2和Sir2都有人类同源物。我们现在将确定H4的乙酰化模式是否受细胞周期调节，改变的H4乙酰化是否影响着着丝粒染色质的结构以及组蛋白去乙酰化酶（HDAC）在染色体分离中的作用。我们建议将HDAC抑制剂与损害着丝点功能的药物联合使用，可能更有效地治疗癌症，对正常细胞的影响最小。为了研究Cse4的PTM，我们设计了一种创新的生化纯化Cse4的方法，这使得Cse4的PTM首次得到全面的分析。确定了Cse4中乙酰化、甲基化和磷酸化的保守位点。我们产生了磷酸化特异性抗体，发现磷酸化的Cse4与着丝粒存在关联，并确定Ipl1在体内和体外磷酸化Cse4以实现可靠的染色体分离。我们的研究表明，Cse4的磷酸化和甲基化调节酿酒酵母的染色体分离。在结肠直肠癌中观察到CENP-A的过表达和错误定位，并导致果蝇的非整倍体。我们发现酿酒葡萄球菌的spt4突变体表现出Cse4的错误定位和染色体分离缺陷，这些缺陷与人类的spt4互补。我们通过表明组蛋白剂量改变和Cse4错定位到非着丝点位点与染色体丢失相关，确定了Cse4错定位的原因和影响。我们的研究已经确定了Cse4的n端在其Ub介导的忠实染色体分离蛋白水解中的新作用。我们正在与Charlie Boone合作，确定介导Cse4蛋白水解的途径，以实现忠实的染色体分离。长期目标是确定特异性杀死过表达CENP-A的癌细胞的途径。我们的实验室最近报道了对基因组稳定性“单倍不足”（HI）基因的鉴定和表征。HI是一种基因的单个功能拷贝不足以维持正常活动并导致表型突变的情况。HI导致较高的肿瘤发生发生率，许多肿瘤表现为非整倍体。我们设计了一种新的筛选方法，利用代表几乎所有基因的半合子酵母缺失文库（6500）来鉴定和表征基因组稳定性“单倍不足”（HI）的基因。我们定义了BCY1和进化上保守的γ微管蛋白复合体作为染色体分离的HI的新作用。我们的研究确定了伽马微管复合体在纺锤体组织中的新作用。

Evolutionarily conserved Cse4, the centromeric histone H3 variant (CENP-A in humans) and its chaperone Scm3 (HJURP in humans) which are essential for chromosome segregation have been shown to be overexpressed in many cancers. Overexpression and mis-localization of HJURP has been reported in lung and breast cancer cells and patients with elevated HJURP expression show reduced survival rate. Whether HJURP overexpression induces tumorigenesis is not understood. We showed that imbalanced stoichiometry of HJURP and SCM3 lead to defects in chromosome segregation and kinetochore integrity in human and yeast cells thereby providing a link between HJURP overexpression and mitotic defects in cancers. Genome wide screens will now allow us to identify genes/pathways that suppress or enhance phenotypes associated with overexpression of SCM3/HJURP for possible extrapolation to cancers. In continuation of these studies we have shown that Pat1 (Protein associated with topoisomerase II) interacts with Scm3. We determined that structural integrity of centromeric chromatin and faithful chromosome segregation requires Pat1. In collaboration with Kerry Bloom we used a pat1 null strain to define the number of Cse4 molecules at the yeast kinetochore. Studies with PAT1 will help us understand how topological structure of centromeric chromatin regulates chromosome segregation an area of research that is largely unexplored at the present time. Post-translational modifications (PTM) of histones are critical for many chromatin activities including chromosome segregation. Histone deactylase (HDAC) inhibitors are used for treatment of certain cancers, however, we do not fully understand the molecular targets of these inhibitors. We investigated the nature and role of PTM of centromeric histones in budding yeast with the long-term objective of targeting PTM of histones for anti-cancer therapy. We showed for the first time that budding yeast centromeres contain hypoacetylated histone H4 and also that increased acetylation of histone H4 on lysine 16 (H4K16) leads to chromosome mis-segregation. We also discovered that a balance in H4K16 acetyltransferase, Sas2, and H4K16 deacetylase, Sir2, is required for chromosome segregation. Notably, both Sas2 and Sir2 have human homologs. We will now determine if the acetylation pattern of H4 is cell cycle regulated, if altered H4 acetylation affect the structure of centromeric chromatin and the role of histone deactylases (HDAC) in chromosome segregation. We propose that combining HDAC inhibitors with drugs that compromise kinetochore function may be more effective for cancer treatment with minimal effect on normal cells. To study PTM of Cse4 we devised an innovative approach for biochemical purification of Cse4 and this facilitated the first comprehensive analysis of PTMs of Cse4. Conserved sites for acetylation, methylation and phosphorylation in Cse4 were identified. We generated a phospho-specific antibody and showed the association of phosphorylated Cse4 with centromeres and determined that Ipl1 phosphorylates Cse4 in vivo and in vitro for faithful chromosome segregation Our studies have shown that phosphorylation and methylation of Cse4 regulate chromosome segregation in S. cerevisiae. Overexpression and mis-localization of CENP-A is observed in colorectal cancers and leads to aneuploidy in flies. We showed that S. cerevisiae spt4 mutants show mis-localization of Cse4 and chromosome segregation defects that are complemented by human SPT4. We established the cause and effect of Cse4 mis-localization by showing that altered histone dosage and mis-localization of Cse4 to non-centromeric loci correlates with chromosome loss. Our studies have defined a novel role for the N-terminus of Cse4 in its Ub mediated proteolysis for faithful chromosome segregation. We are collaborating with Charlie Boone to identify pathways that mediate the proteolysis of Cse4 for faithful chromosome segregation. The long-term objective is to identify pathways that will specifically lead to killing of cancer cells overexpressing CENP-A. Our laboratory recently reported on the identification and characterization of genes that are "haploinsufficient" (HI) for genome stability. HI is a condition where a single functional copy of a gene is insufficient to sustain normal activity and leads to a mutant phenotype. HI leads to higher incidences of tumorigenesis and many tumors display aneuploidy. We designed a novel screen to identify and characterize genes that are "haploinsufficient" (HI) for genome stability using the hemizygous yeast deletion library representing nearly all genes (6500). We defined novel roles for BCY1 and the evolutionarily conserved Gamma tubulin complex as HI for chromosome segregation. Our studies defined a novel role for the gamma tubulin complex in spindle organization.