Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

进化上保守的Cse4及其伴侣蛋白Scm3（人类中的HJURP）对染色体分离至关重要，已被证明在许多癌症中过表达。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。Pat1的研究为着丝粒染色质的拓扑结构如何调节染色体分离提供了重要的见解，这是一个目前尚未探索的研究领域。忠实的染色体分离也受到着丝粒组蛋白和着丝粒蛋白的翻译后修饰（PTM）的调节。我们研究了着丝粒组蛋白H4和Cse4的PTM在出芽酵母中的性质和作用，目的是将组蛋白的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）和组蛋白去乙酰化酶（HDAC） 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。我们正在研究H4乙酰化模式是否受细胞周期调节，H4乙酰化改变是否影响着着丝粒染色质的结构，以及HDAC在染色体分离中的作用。HDAC抑制剂用于治疗某些癌症，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PTM在Cse4中的作用，我们设计了一种创新的Cse4生化纯化方法，这有助于首次对Cse4的PTM进行全面分析。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以调节染色体分离。在我们对PTM的继续研究中，我们发现Cse4的n端泛素化以调节其蛋白水解和定位。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。在与Charlie Boone的合作中，我们使用全基因组筛选确定了几个进化保守的Cse4蛋白水解的新调控因子。Cse4人类同源基因（CENP-A）的过表达和错误定位在结直肠癌中被观察到，并导致果蝇的非整倍体。我们研究的长期目标是确定特异性杀死过度表达CENP-A的癌细胞的途径。考虑到基因组稳定性途径的进化保守性，我们决定使用出芽酵母来研究“单倍不足”（HI）的作用。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全基因组筛选方法，使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高的基因。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。总之，我们对出芽酵母及其人类同源物的研究为许多癌症中经常观察到的染色体分离错误的原因和后果提供了重要的见解。

Evolutionarily conserved Cse4 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 have provided important insights into how topological structure of centromeric chromatin regulates chromosome segregation an area of research that is largely unexplored at the present time. Faithful chromosome segregation is also regulated by post-translational modifications (PTM) of centromeric histones and kinetochore proteins. We investigated the nature and role of PTM of centromeric histone H4 and Cse4 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 Histone Deacetylase (HDAC), Sir2, is required for chromosome segregation. Notably, both Sas2 and Sir2 have human homologs. We are now investigating if acetylation pattern of H4 is cell cycle regulated, if altered H4 acetylation affect the structure of centromeric chromatin and the role of HDAC's in chromosome segregation. HDAC inhibitors are used for treatment of certain cancers, however, we do not fully understand the molecular targets of these inhibitors. 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. In order to investigate the role of 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 to regulate chromosome segregation. In continuation of our studies with PTM, we have shown that the N-terminus of Cse4 is ubiquitinated to regulate its proteolysis and localization. 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. In collaboration with Charlie Boone we have used genome-wide screens to identify several new regulators for Cse4 proteolysis that are evolutionarily conserved. Overexpression and mis-localization of human homolog of Cse4 (CENP-A) is observed in colorectal cancers and leads to aneuploidy in flies. The long-term objective of our research is to identify pathways that will specifically lead to killing of cancer cells overexpressing CENP-A. Given the evolutionary conservation of pathways for genome stability we decided to use budding yeastinvestigate the role of "haploinsufficiency" (HI). 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 genome-wide screen using the hemizygous yeast deletion library representing nearly all genes (6500) to identify and characterize genes that are HI for genome stability. We defined novel roles for BCY1 and the evolutionarily conserved Gamma Tubulin complex as HI for chromosome segregation. Taken together, our studies with budding yeast and its human homologs are providing critical insights into the causes and consequences of errors in chromosome segregation that are frequently observed in many cancers.