Exploring the regulation and activation of ALT, the Alternative Lengthening of Telomeres

Cancers and cell lines that utilise the Alternative Lengthening of Telomeres (ALT) to maintain telomere length usually display complex karyotype rearrangements consistent with genome instability that may arise at crisis through dysfunctional telomeres. The absence of the ATRX chromatin remodelling factor is the predominant marker in these cancers and cell lines. It has been shown that specific orphan nuclear receptors (ORs) can bind to (TCAGGG)n repeats in telomeres of some ALT+ cell lines and this binding results in changes to the telomere chromatin that may facilitate the ALT. In this project, the binding site and regulatory roles of ORs were investigated in a panel of five ALT+ cell lines. No relationship was identified between the expression of NR2F2 or NR2C2 proteins and the localisation of these proteins at telomeres in ALT+ cell lines. The frequency of cells with NR2F2 or NR2C2 foci at telomeres varies considerably between the ALT+ cell lines (7- 91% for NR2F2; 1-42% for NR2C2) with only the WI38VA13_2RA ALT+ cell line showing a high frequency for both, consistent with previous work. PCR amplification of XpYp, 12q and 17p telomeres followed by hybridisation to (TTAGGG)n, (TCAGGG)n or (TGAGGG)n variant repeat probes was used to measure the frequency of telomere molecules that contain binding sites for these ORs. This revealed that some telomere alleles in ALT+ cell lines lack binding sites for these ORs, indicating that binding of these ORs is not essential for ALT at some telomeres. Downregulation of NR2F2 in three ALT+ cells resulted in significant changes in the expression of genes involved in DNA replication and repair that resulted in cell cycle arrest in the U2OS, but not in the WV or WI38VA13_2RA ALT+ cell lines. Integrated analyses of whole exome sequence (WES) and RNA-seq analyses were used to uncover the mutation and the gene expression changes associated with ALT activation after ATRX knockout, using CRISPR-Cas9, in SV40- transformed pre-crisis cells. Differential gene expression analysis at early post-crisis time-points in emerging clonal populations showed that downregulation of the JAK-STAT signalling pathway, the ALT-suppressor SP100 and reduced expression of non-homologous end joining (NHEJ) related genes NR4A1 and XLF were the most remarkable events. These gene expression changes were combined with copy number changes in chromosomes 8, 11 and 18 and remarkable level of genome instability. This could suggest that, following the loss of ATRX in SV40-transformed cells, the changes in the expression of the genes required for double strand break (DSB) repair contributed to the activation of ALT.