Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

进化上保守的Cse 4及其伴侣Scm 3（人类中的HJURP）对于染色体分离是必需的，已经显示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误定位，HJURP表达升高的患者显示存活率降低。HJURP过表达是否诱导肿瘤发生尚不清楚。我们发现，HJURP和SCM 3的不平衡化学计量导致人类和酵母细胞中染色体分离和动粒完整性的缺陷，从而提供了HJURP过表达和癌症中有丝分裂缺陷之间的联系。全基因组筛选现在将允许我们鉴定抑制或增强与SCM 3/HJURP过表达相关的表型的基因/途径，以便可能外推到癌症。在这些研究的继续中，我们已经表明Pat 1（拓扑异构酶II相关蛋白）与Scm 3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帕特1。在与Kerry Bloom的合作中，我们使用pat 1无效菌株来定义酵母动粒处的Cse 4分子的数量。Pat 1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动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Cse 4的PTM在芽殖酵母中的性质和作用，其长期目标是靶向组蛋白的PTM用于抗癌治疗。我们首次发现芽殖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（H4 K16）上的乙酰化增加导致染色体错误分离。我们还发现，H4 K16乙酰转移酶Sas 2和组蛋白脱乙酰酶（HDAC）Sir 2的平衡是染色体分离所必需的。值得注意的是，Sas 2和Sir 2都有人类同源物。我们正在研究H4的乙酰化模式是否受细胞周期调控，H4乙酰化的改变是否影响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。我们认为，HDAC抑制剂与损害动粒功能的药物相结合可能对癌症治疗更有效，对正常细胞的影响最小。为了研究Cse 4的PTM的作用，我们设计了一种用于Cse 4的生化纯化的创新方法，这促进了Cse 4的PTM的首次全面分析。Cse 4中乙酰化、甲基化和磷酸化的保守位点被鉴定。我们产生了磷酸化特异性抗体，并显示磷酸化Cse 4与着丝粒的关联，并确定Ipl 1在体内和体外磷酸化Cse 4以调节染色体分离。在继续我们对PTM的研究中，我们已经表明Cse 4的N-末端被泛素化以调节其蛋白水解和定位。我们通过显示改变的组蛋白剂量和Cse 4到非着丝粒位点的错误定位与染色体丢失相关来确定Cse 4错误定位的原因和影响。在与Charlie Boone的合作中，我们使用全基因组筛选来鉴定几种进化上保守的Cse 4蛋白水解的新调节剂。在结直肠癌中观察到Cse 4（CENP-A）的人同源物的过表达和错误定位，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。鉴于基因组稳定性途径的进化保守性，我们决定使用芽殖酵母研究“单倍不足”（HI）的作用。HI是基因的单个功能拷贝不足以维持正常活性并导致突变表型的病症。HI导致更高的肿瘤发生率，并且许多肿瘤显示非整倍体。我们设计了一种新的全基因组筛选，使用代表几乎所有基因（6500）的半合子酵母缺失文库来鉴定和表征基因组稳定性HI的基因。我们定义了BCY 1和进化上保守的γ微管蛋白复合物作为染色体分离的HI的新作用。总之，我们对芽殖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.