The role of chromatin structure in Alternative Lengthening of Telomeres (ALT): lessons from the Heterochromatin Protein 1 Binding Partner 3 (HP1BP3).

端粒存在于染色体的两端，保护基因组免受退化、不必要的重组和染色体内融合的影响。端粒是由重复序列组成的。人类端粒的平均长度从10kb到15kb不等。每次细胞进行复制时，染色体末端都会缩短25-200个碱基对，这一过程称为末端复制问题。这一过程起到了细胞时钟的作用，发出细胞增殖结束的信号。癌细胞通过两条途径克服这一点：端粒酶重新激活途径(TERT)和交替延长端粒(ALT)。虽然许多癌症使用TERT途径，但大约4%到11%的癌症使用ALT，这是一种基于端粒重复序列之间同源重组的途径，依赖于DNA损伤反应蛋白(DDR)的招募。这些肿瘤更具侵袭性，预后较差。ALT阳性细胞具有端粒酶阳性细胞所没有的许多特征。它们有未附着在染色体末端的端粒DNA(额外的染色体DNA)，端粒DNA可以是线性的，也可以是圆形的(c-环)。它们还进行端粒姐妹染色单体交换，并在不同的染色体上改变端粒长度。虽然ALT的激活是相当一部分肿瘤的特征，但对ALT机制缺乏完全的了解阻碍了选择性地杀死ALT肿瘤的策略的发展，尽管ALT肿瘤具有侵袭性和较差的预后。端粒含有高度紧密的异染色质。染色质结构如何有助于保持ALT表型，以及它是否可以用于治疗ALT肿瘤，目前尚不清楚。由于在ALT肿瘤中观察到了组蛋白变体H3.3及其伴侣ATRX和DAXX的突变，这是异染色质的标志，因此普遍认为ALT受到染色质压缩的抑制。然而，最近的研究表明，ALT端粒上异染色质的形成与ALT表型有关。在本研究中，我们将使用异染色质相关蛋白HP1BP3作为模型来研究染色质结构在维持ALT中的作用。我们将首先描述它在促进端粒稳定性和重组中的作用，并剖析促进每种功能的蛋白质结构域以及染色质压缩是如何参与的。然后，我们将使用无偏见的方法来鉴定在HP1BP3基因消融时它们结合有缺陷的蛋白质。最后，为了扩大分析范围，我们将筛选其他表观遗传因素和选择性杀死ALT癌细胞的药物。这些方法将使我们能够仔细剖析使ALT表型永久化的表观遗传机制，并确定我们如何设计染色质状态或重新使用当前的表观遗传药物来选择性治疗ALT肿瘤。我们的研究将推动关于染色质在基因组维持中作用的既定知识的界限，并将为个性化癌症治疗的选择性干预奠定基础。

Telomeres are present at both ends of chromosomes and protect the genome form degradation, unnecessary recombination and intrerchromosomal fusion. Telomeres are made up of repetitive sequences. The average length of human telomeres varies from 10 to 15 Kilobase (Kb). Every time the cells go through replication, the chromosome ends are shortened by 25-200 base pairs, a process termed end replication problem. This process acts as a cellular clock and signals end of cell proliferation. Cancer cells overcome this by using two pathways: the telomerase reactivation pathway (TERT) and Alternative Lengthening of Telomeres (ALT). Although many cancers use the TERT pathway, around 4 to 11% of cancers use ALT a pathway that is based on homologous recombination between telomeric repeats and relies on the recruitment of DNA damage response proteins (DDR). These tumours are more aggressive and have poor prognosis. ALT positive cells have a number of characteristics that are not seen in the Telomerase positive cells. They have telomeric DNA that is not attached to the chromosome ends (extra chromosomal DNA) which can be linear or circular (c-circles). They also exert telomeric sister chromatid exchanges and varied telomere length from chromosome to chromosome. Although activation of ALT characterises a substantial fraction of tumours, the lack of complete understanding of the mechanisms of ALT has hindered the development of strategies to selectively kill ALT tumours, despite their aggressive nature and poor prognosis. Telomeres contain highly compacted chromatin called heterochromatin. How chromatin structure contributes to perpetuate the ALT phenotype and whether it can be exploited for treating ALT tumours is unclear. Since mutations in the histone variant H3.3 and its chaperones ATRX and DAXX which are marks of heterochromatin have been observed in ALT tumours, the prevailing view was that ALT is inhibited by chromatin compaction. Recent studies, however, have demonstrated that heterochromatin formation at ALT telomeres contributes to ALT phenotype.In this proposal, we will use the heterochromatin related protein HP1BP3 as a model to study the role of chromatin structure in maintenance of ALT. We will first characterise its role in promoting telomeric stability and recombination and dissect the protein domains that promote each function and how chromatin compaction is involved. Then, we will use unbiased approaches to identify the proteins that their binding is defective upon genetic ablation of HP1BP3. Finally, to broaden the analysis, we will screen for additional epigenetic factors and drugs that selectively kills ALT cancer cells. These approaches will allow us to carefully dissect the epigenetic mechanisms that perpetuate the ALT phenotype and determine how we can engineer a chromatin state or repurpose current epigenetic drugs for selectively treating ALT tumours. Our research will push forward the boundaries of established knowledge about the role of chromatin in genome maintenance and will lay the basis for selective interventions in personalized cancer therapy.