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.
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