The role of histone chaperone Asf1 in Alternative Lengthening of Telomeres

组蛋白伴侣 Asf1 在端粒选择性延长中的作用

• 批准号: 8824891
• 负责人: Jan Karlseder
• 金额: $ 40.26万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824891
• 关键词: Address; Affinity; Antibodies; Biological Assay; Cell Line; Cell Nucleus; Cell division; Cells; Characteristics; Chromatin; Chromosomes; Complex; DNA; DNA Damage; DNA Packaging; Data; Defect; Dependence; Diagnostic; Down-Regulation; Electrophoresis; Epigenetic Process; Event; Exhibits; Gene Mutation; Genetic Recombination; Genome Stability; Goals; Health; Heterochromatin; Heterogeneity; Histone Acetylation; Histones; Interphase; Length; Link; Longevity; Maintenance; Malignant Neoplasms; Metabolism; Metaphase; Micrococcal Nuclease; Molecular; Molecular Chaperones; Nucleosomes; Pathway interactions; Phenotype; Protein Isoforms; RNA-Directed DNA Polymerase; Repetitive Sequence; Role; Signal Transduction; Sister Chromatid Exchange; Structure; Telomerase; Telomerase inhibition; Telomere Length Maintenance; Telomere Maintenance; Telomere Pathway; Telomere Shortening; Testing; base; cancer cell; cancer therapy; cell transformation; chromatin immunoprecipitation; design; gel electrophoresis; histone methylation; histone modification; neoplastic cell; novel; research study; response; small hairpin RNA; telomere; tissue culture; tool; tumor

DESCRIPTION (provided by applicant): Telomeres are the natural ends of linear chromosomes and crucial for genome stability, cellular viability and chromosome integrity. Telomeres shorten during each cell division, representing a cellular clock and limiting the replicative lifespan. To overcome this limit, cancer cells have to activate telomere maintenance mechanisms, which counteract telomere shortening and endow the cells with immortality. Ninety percent of cancers do so by activating telomerase, a reverse transcriptase complex that adds telomeric repeats to chromosome ends. The remaining 10% of cancers take advantage of a recombination-based mechanism for telomere length maintenance, called ALT (Alternative Lengthening of Telomeres). While telomerase activation is the more frequently used mechanism and widely investigated, it is becoming clear that ALT pathways can be activated upon telomerase inhibition, emphasizing that both telomere maintenance mechanisms have to be understood before telomere elongation can be successfully targeted as cancer therapy. ALT-dependent telomere lengthening is based on recombination between long and short telomeres, but the mechanisms are not currently understood. ALT has long been considered the result of defects in cellular recombination pathways, but despite intense efforts no deficiencies in recombination regulators have been identified in ALT-cells. Recent data suggest the hypothesis that ALT relies on aberrant recombination pathways needs to be reexamined and is likely incorrect. We discovered that ALT is likely a consequence of poor histone placement at repetitive regions, such as telomeres. We can induce ALT dependent telomeric recombination by suppression of isoforms of the histone chaperone Asf1. Upon Asf1 down regulation all characteristics of ALT emerge in primary and transformed cells, which include telomere sister chromatid exchange, telomere length heterogeneity, the formation of single stranded telomeric C-circles and the colocalization of PML, RPA and TTAGGG repeats in ALT associated PML bodies. The three intellectually connected but independent aims of this proposal are designed to investigate our novel concept for the ALT mechanism, which suggests that improper nucleosome placement at telomeres leads to single stranded loops at telomeres, which then readily recombine with each other. In AIM1 we will investigate telomere structure in cells with suppressed Asf1, as well as nucleosome placement and DNA damage signaling at telomeres and throughout the nucleus in Asf1 suppressed cells. We will also address whether Asf1 dependent ALT activation is capable of long-term telomere maintenance. AIM2 is designed to investigate why Asf1 suppression has telomere-specific effects and whether it leads to changes in telomeric chromatin in primary and transformed cells, therefore defining an ALT specific epigenetic signature. In AIM3 we will investigate the Asf1 status of ALT tumor cells, whether ALT can be suppressed by Asf1 expression, and whether ALT is an epigenetic state that can be induced by constitutive Asf1 suppression.

描述（由申请人提供）：端粒是线性染色体的天然末端，对于基因组稳定性、细胞活力和染色体完整性至关重要。端粒在每次细胞分裂过程中都会缩短，代表细胞时钟并限制复制寿命。为了克服这一限制，癌细胞必须激活端粒维持机制，从而抵消端粒缩短并赋予细胞永生性。百分之九十的癌症是通过激活端粒酶来实现的，端粒酶是一种逆转录酶复合物，可将端粒重复序列添加到染色体末端。其余 10% 的癌症利用基于重组的端粒长度维持机制，称为 ALT（端粒替代延长）。虽然端粒酶激活是更常用的机制并得到广泛研究，但越来越清楚的是，端粒酶抑制可以激活 ALT 途径，这强调在端粒延长能够成功地作为癌症治疗的目标之前，必须了解两种端粒维持机制。 ALT 依赖性端粒延长是基于长端粒和短端粒之间的重组，但目前尚不清楚其机制。长期以来，ALT 一直被认为是细胞重组途径缺陷的结果，但尽管付出了巨大的努力，但尚未在 ALT 细胞中发现重组调节因子的缺陷。最近的数据表明 ALT 依赖于异常重组途径的假设需要重新检验，并且可能是错误的。我们发现 ALT 可能是重复区域（例如端粒）组蛋白放置不良的结果。我们可以通过抑制组蛋白伴侣 Asf1 的亚型来诱导 ALT 依赖性端粒重组。 Asf1下调后，原代细胞和转化细胞中出现 ALT 的所有特征，包括端粒姐妹染色单体交换、端粒长度异质性、单链端粒 C 环的形成以及 ALT 相关 PML 小体中 PML、RPA 和 TTAGGG 重复的共定位。该提案的三个相互关联但独立的目标旨在研究我们关于 ALT 机制的新概念，该概念表明端粒处核小体放置不当会导致端粒处形成单链环，然后很容易彼此重组。在 AIM1 中，我们将研究 Asf1 被抑制的细胞中的端粒结构，以及 Asf1 被抑制的细胞中端粒和整个细胞核的核小体位置和 DNA 损伤信号传导。我们还将探讨 Asf1 依赖性 ALT 激活是否能够长期维持端粒。 AIM2 旨在研究为什么 Asf1 抑制具有端粒特异性效应，以及它是否会导致原代细胞和转化细胞中端粒染色质的变化，从而定义 ALT 特异性表观遗传特征。在AIM3中，我们将研究ALT肿瘤细胞的Asf1状态，ALT是否可以被Asf1表达抑制，以及ALT是否是可以通过组成性Asf1抑制诱导的表观遗传状态。