Alternative lengthening of telomeres: remodelling the telomere architecture

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The vast majority of human cancers utilise a telomere maintenance mechanism (TMM) to compensate for the gradual telomere shortening that accompanies cellular proliferation, and thereby obtain an unlimited replicative capacity. This can be accomplished by activation of the ribonucleoprotein telomerase which adds telomeric repeats onto the ends of linear chromosomes by reverse transcription of an RNA template molecule, or by the homologous recombination (HR)-mediated alternative lengthening of telomeres (ALT) pathway. Telomere length maintenance is a characteristic of almost all cancers, which means there is considerable interest in the use of telomere maintenance inhibitors as a broad-spectrum cancer therapy. Therefore successful therapeutic targeting of telomere maintenance in cancers will encompass the development of ALT inhibitors. This will be facilitated by insights into the molecular details of ALT and how this mechanism is activated. Telomeres in cells that utilise ALT elicit a DNA damage response (DDR) that is partly independent of telomere length, suggesting the existence of underlying structural defects. The aim of this thesis was to investigate whether ALT telomeres contain structural alterations that contribute to ALT activity, and may be targeted by potential ALT inhibitors. It has previously been demonstrated that the proximal 2 kb regions of normal human telomeres contain a non-random distribution of canonical (TTAGGG) and variant repeats (TCAGGG, TGAGGG and TTGGGG). In addition, ALT-mediated telomere exchange events have been detected within these variant repeat-dense proximal regions. Here we used next generation sequencing to analyse the DNA content of ALT telomeres. We discovered that variant repeats were interspersed throughout the telomeres of ALT cells. We propose that variant repeat interspersion is specific to ALT telomeres, and results from HR-mediated telomere copying and spreading of variant repeats that are normally restricted to the proximal regions. We found that the C-type (TCAGGG) variant repeat predominated and created a high-affinity binding site for the nuclear receptors COUP-TF2 and TR4. Nuclear receptors were directly recruited to telomeres following incorporation of the C-type variant repeat using a mutant telomerase, and resulted in an undersaturation of shelterin binding and the induction of ALT-associated characteristics. We propose that nuclear receptor binding to variant repeats within ALT telomeres alters the telomeric architecture to facilitate further recombination. We then demonstrated that the nucleosome remodelling and histone deacetylation (NuRD) complex was recruited to nuclear receptors by an N-terminal RRK motif within ZNF827, a zinc finger protein of unknown function. We found that recruitment of the NuRD-ZNF827 complex resulted in histone deacetylation indicative of chromatin compaction. NuRD-ZNF827 also recruited HR proteins, and promoted ALT activity. Finally, NuRD-ZNF827 binding caused telomeres to interact with each other as well as ALT-associated PML bodies (APBs). ZNF827 depletion elevated the telomeric DDR followed by the onset of senescence/apoptosis in ALT cells. We propose that NuRD-ZNF827 provides a molecular scaffold for telomeric HR, by simultaneously enabling telomeric interactions, recruiting HR proteins, and protecting telomeres from the DDR. Together these data implicate NuRD-ZNF827 as a potential target in ALT-specific cancer therapeutics.