Identification and characterization of proteins that protect human telomeres from fragility

Difficulties to replicate telomeres - the ends of our chromosomes - can cause telomere shortening and genome instability. These difficulties are due to the repetitive DNA sequence and distinct structures at telomeres that challenge the semi-conservative DNA replication machinery. Among the proteins that help to overcome the obstacles to telomere replication are factors known to be mutated in genetic diseases (for instance WRN, BLM, RTEL1, CTC1), but we suspect that there are still factors and pathways unknown. DNA replication defects at telomeres manifest as smeary or multiple telomeric fluorescence in situ hybridization (FISH) signals on metaphase chromosomes - commonly called telomere fragility. To identify proteins that protect human telomeres from fragility, we depleted 71 proteins (including 8 controls) from HeLa cells using siRNA pools and analysed their metaphase chromosomes for fragile telomeres. The majority of these proteins had been identified by QTIP-iPOND - a proteomic analysis of chromatin near telomere replication forks. Among the 71 proteins that were depleted in the siRNA screen, we identified 29 novel proteins that prevent telomere fragility and are therefore crucial to maintain genome stability. The telomere fragility screen validated QTIP-iPOND as a technique to identify telomere replication proteins. The hits identified in our telomere fragility screen may for example have functions in regulating telomeric chromatin composition and compaction, regulating transcription of the telomeric long non-coding RNA TERRA and its association with telomeric chromatin, dealing with replication stress and stalled replication forks, modulating DNA damage signalling and repair, modulating sister chromatid cohesion, and preventing oxidative stress. One of the 29 novel proteins identified in the telomere fragility screen is chromobox 8 (CBX8) whose function at telomeres was investigated in further detail in this study. CBX8 is a component of the canonical polycomb repressive complex 1 (PRC1), an epigenetic regulator of gene expression. However, CBX8 also has functions that are independent of the PRC1 complex. We show that human CBX8 prevents telomere fragility caused by RNA-DNA hybrids which could stall replication forks and induce homologous recombination. CBX8 depletion or knockout did however not affect telomeric RNA-DNA hybrid levels. We hypothesize that CBX8 may be involved in repair activities at stalled replication forks that arise when the replisome encounters persistent RNA-DNA hybrids. Moreover, we found that CBX8 localizes to telomeric repeat-binding factor 2 (TRF2)-deficient telomeres in a manner that is dependent on RNA-DNA hybrids and ATM signalling. We demonstrate that H3K27me3 (trimethylated histone 3 lysine 27) marks, which have previously been suggested to help recruitment of CBX proteins to chromatin via the CBX chromodomain, are dispensable for CBX8 recruitment to TRF2-deficient telomeres. Furthermore, we found that CBX8 promotes non-homologous end joining (NHEJ)-mediated telomere fusions of dysfunctional telomeres. We therefore hypothesize that CBX8 is involved in repair activities that influence the DNA repair pathway choice at TRF2- deficient telomeres