Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

在进化上保守的Cse4，着丝粒的组蛋白H3变体(人类的CENP-A)及其伴侣Scm3(人类的HJURP)是染色体分离所必需的，已被证明在许多癌症中过表达。HJURP在肺癌和乳腺癌细胞中的过度表达和错误定位已有报道，HJURP表达升高的患者存活率降低。目前尚不清楚HJURP过表达是否会导致肿瘤的发生。我们发现HJURP和SCM3的化学计量不平衡导致人类和酵母细胞中染色体分离和动粒完整性的缺陷，从而在HJURP过表达和癌症中的有丝分裂缺陷之间提供了联系。全基因组筛查现在将使我们能够识别抑制或增强与SCM3/HJURP过度表达相关的表型的基因/途径，从而可能推断为癌症。在这些研究的继续中，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，我们使用了一株pat1缺失菌株来定义酵母动粒上的Cse4分子的数量。对PAT1的研究将帮助我们理解着丝粒染色质的拓扑结构如何调节染色体分离，这是目前很大程度上未被探索的研究领域。组蛋白的翻译后修饰(PTM)是包括染色体分离在内的许多染色质活动的关键。组蛋白脱乙酰酶(HDAC)抑制剂被用于某些癌症的治疗，然而，我们并不完全了解这些抑制剂的分子靶点。我们研究了发芽酵母中着丝粒组蛋白的PTM的性质和作用，长期目标是靶向组蛋白的PTM用于抗癌治疗。我们首次发现发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)上的乙酰化增加会导致染色体的错误分离。我们还发现，染色体分离需要H4K16乙酰转移酶SAS2和H4K16去乙酰基酶Sir2的平衡。值得注意的是，SAS2和Sir2都有人类同源基因。我们现在将确定H4的乙酰化模式是否受细胞周期调节，H4乙酰化是否影响着丝粒染色质的结构以及组蛋白脱乙酰酶(HDAC)在染色体分离中的作用。我们建议，将HDAC抑制剂与损害动粒功能的药物联合使用可能对癌症治疗更有效，而对正常细胞的影响最小。为了研究Cse4的PTM，我们设计了一种新的生化纯化方法，这有助于首次对Cse4的PTMS进行全面的分析。确定了Cse4中乙酰化、甲基化和磷酸化的保守位点。我们制备了一种磷酸化特异性抗体，证明了磷酸化的Cse4与着丝粒的结合，并确定了Ipl1在体内和体外磷酸化Cse4以实现忠实的染色体分离。我们的研究表明，Cse4的磷酸化和甲基化调控着酿酒酵母的染色体分离。在结直肠癌中观察到CENP-A的过度表达和错误定位，并导致果蝇的非整倍体。我们发现酿酒酵母spt4突变体表现出cse4的错误定位和染色体分离缺陷，而人类spt4补充了这些缺陷。我们通过证明组蛋白剂量的改变和Cse4对非着丝粒基因的错误定位与染色体丢失相关，从而确定了Cse4错误定位的原因和影响。我们的研究明确了Cse4的N端在其Ub介导的蛋白质分解中的新作用，以实现忠实的染色体分离。我们正在与查理·布恩合作，确定调节Cse4蛋白分解的途径，以实现忠实的染色体分离。长期目标是确定具体导致过度表达CENP-A的癌细胞被杀死的途径。我们的实验室最近报道了对基因组稳定性具有“单倍性不足”(HI)的基因的鉴定和表征。HI是指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情况。HI可导致较高的肿瘤发生率，许多肿瘤表现为非整倍体。我们设计了一种新的筛选方法，使用代表几乎所有基因的半合子酵母缺失文库(6500)来识别和表征对基因组稳定性而言“单倍体不足”(HI)的基因。我们将BCY1和进化上保守的Gamma微管蛋白复合体的新角色定义为HI，用于染色体分离。我们的研究确定了伽马微管蛋白复合体在纺锤体组织中的新角色。

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.