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自然末端，对基因组稳定性，细胞活力和染色体完整性至关重要。在每个细胞分裂过程中，端粒缩短，代表一个细胞时钟并限制了复制寿命。为了克服这一限制，癌细胞必须激活端粒维持机制，这抵消了端粒缩短并赋予细胞的永生。 90％的癌症通过激活端粒酶（一种逆转录酶复合酶，可在染色体末端增加端粒重复量）来做到这一点。其余10％的癌症利用了基于重组的端粒长度维持的机制，称为ALT（端粒的替代性延长）。虽然端粒酶激活是更常用的机制，并且已经广泛研究了，但很明显，在端粒酶抑制时可以激活ALT途径，这强调必须在端粒伸长延伸之前能够成功地将两种端粒维持机制理解为癌症治疗。依赖Alt依赖性的端粒延长是基于长端粒和短端粒之间的重组，但目前尚不了解这些机制。长期以来，ALT一直被认为是细胞重组途径缺陷的结果，但是尽管迫切努力，在Alt细胞中尚未发现重组调节剂缺陷。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查，并且可能是不正确的。我们发现ALT可能是重复区域（例如端粒）中组蛋白放置不佳的结果。我们可以通过抑制组蛋白伴侣ASF1的同工型来诱导依赖性端粒重组。在ASF1下调后，在原代和转化的细胞中出现了ALT的所有特征，其中包括端粒姐妹染色单体交换，端粒长度异质性，单链端粒C-Circles的形成以及PML，RPA和TTAGGG在Alt相关PML体内的共同定位。该提案的三个智力联系但独立的目的旨在研究我们对Alt机制的新颖概念，这表明端粒的核小体位置不当会导致端粒的单个绞线环，然后很容易重新组合。在AIM1中，我们将研究抑制ASF1的细胞中的端粒结构，以及端粒的核小体放置和DNA损伤信号传导，以及整个ASF1抑制细胞中的整个核。我们还将解决ASF1依赖性ALT激活是否能够进行长期端粒维护。 AIM2旨在研究为什么ASF1抑制具有特异性效应，以及它是否导致原代和转化细胞中端粒染色质的变化，因此定义了ALT特定的表观遗传学特征。在AIM3中，我们将研究ALT肿瘤细胞的ASF1状态，ALT是否可以通过ASF1表达抑制ALT以及ALT是否是由组成型ASF1抑制诱导的表观遗传态。