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

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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.