The role of histone chaperone Asf1 in Alternative Lengthening of Telomeres

描述(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。端粒在每一次细胞分裂过程中缩短，代表细胞钟，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，以抵消端粒缩短并赋予细胞永生能力。90%的癌症是通过激活端粒酶来做到这一点的，端粒酶是一种将端粒重复添加到染色体末端的逆转录酶复合体。剩下的10%的癌症利用一种基于重组的机制来维持端粒长度，称为ALT(端粒替代延长)。虽然端粒酶激活是最常用和被广泛研究的机制，但ALT通路可以在端粒酶抑制后被激活变得越来越明显，强调在端粒延长被成功地作为癌症治疗的靶点之前，必须了解这两种端粒维持机制。ALT依赖的端粒延长是基于长和短端粒之间的重组，但其机制目前尚不清楚。长期以来，ALT一直被认为是细胞重组途径缺陷的结果，但尽管进行了大量的努力，仍未在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，而且可能是不正确的。我们发现，ALT可能是组蛋白在重复区域(如端粒)放置不当的结果。我们可以通过抑制组蛋白伴侣蛋白ASF1的异构体来诱导依赖ALT的端粒重组。ASF1下调后，ALT的所有特征都出现在原代和转化细胞中，包括端粒姐妹染色单体互换、端粒长度不均、单链端粒C环的形成以及PML、RPA和TTAGGG重复序列在ALT相关的PML小体中的共存。这一提议的三个智力上相互联系但又相互独立的目的是为了研究我们关于ALT机制的新概念，该机制认为核小体在端粒上的不当放置会导致端粒上的单链环，然后这些单链环很容易相互重组。在AIM1中，我们将研究ASF1抑制的细胞的端粒结构，以及ASF1抑制的细胞的核小体放置和DNA损伤信号在端粒和整个细胞核中的分布。我们还将讨论ASF1依赖的ALT激活是否能够长期维持端粒。AIM2旨在研究ASF1抑制具有端粒特异性效应的原因，以及它是否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是否可以被ASF1表达抑制，以及ALT是否是一种表观遗传状态，可以通过结构性ASF1抑制来诱导。

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.