CTC1 deletion results in defective telomere replication, leading to catastrophic telomere loss and stem cell exhaustion

Abstract

The proper maintenance of telomeres is essential for genome stability. Mammalian telomere maintenance is governed by a number of telomere binding proteins, including the newly identified CTC1-STN1-TEN1 (CST) complex. However, the in vivo functions of mammalian CST remain unclear. To address this question, we conditionally deleted CTC1 from mice. We report here that CTC1 null mice experience rapid onset of global cellular proliferative defects and die prematurely from complete bone marrow failure due to the activation of an ATR-dependent G2/M checkpoint. Acute deletion of CTC1 does not result in telomere deprotection, suggesting that mammalian CST is not involved in capping telomeres. Rather, CTC1 facilitates telomere replication by promoting efficient restart of stalled replication forks. CTC1 deletion results in increased loss of leading C-strand telomeres, catastrophic telomere loss and accumulation of excessive ss telomere DNA. Our data demonstrate an essential role for CTC1 in promoting efficient replication and length maintenance of telomeres.

CTC1‐STN1 coordinates G‐ and C‐strand synthesis to regulate telomere length

“…However, a reliable antibody against endogenous CTC1 is not commercially available, and we were unsuccessful in our multiple attempts to generate antibodies against both human and mouse CTC1 (data not shown). To circumvent this difficulty, we performed IF microscopy using an anti‐STN1 antibody to visualize endogenous STN1, which we have shown previously to be a reliable marker to detect the endogenous CST complex (Gu et al., 2012). We found that STN1 is present exclusively in the nuclei of WT HCT116 cells, but in HCT116 CTC1 L1142H mutants, nuclear levels of STN1 are reduced (Figure 1d).…”

“…Telomere‐FISH revealed progressive increase in the percentage of sister chromatids with greatly reduced or missing telomere signals, a phenomenon termed sister telomere loss (STL), on metaphase spreads from late passage R‐46‐5 mutant cells, but not WT cells (Crabbe, Verdun, Haggblom & Karlseder, 2004) (Figure 3f, g). Fragile telomeres, prominent in CTC1 knockout mouse cells and suggestive of telomere replicative defects, were not significantly increased above background levels in CTC1 L1142H RPE mutants (data not shown) (Gu et al., 2012). While telomere length also shortened progressively in serially passaged WT RPE cells, sister telomere loss was infrequent and did not significantly increase with serial passages.…”

“…CTC1 and STN1 were originally discovered as proteins that stimulate DNA Pol‐α: primase activity, suggesting an essential role for CST in DNA replication (Casteel et al., 2009; Miyake et al., 2009; Surovtseva et al., 2009). Targeted deletion of CTC1 in the mouse, as well as depletion of individual CST components in human cells, all results in telomere replication defects and global telomere attrition, suggesting that the CST complex is required for multiple steps of telomere replication (Feng, Hsu, Kasbek, Chaiken & Price, 2017; Gu et al., 2012; Wu, Takai & de Lange, 2012). After DNA replication, leading‐strand telomeres are initially blunt‐ended, requiring nucleolytic processing of the leading‐strand termini to generate the 3′‐overhang needed for telomerase extension of the G‐strand.…”

“…The recruitment of CST to telomeres, by POT1b in mouse cells and TPP1 in human cells, in turn promotes DNA Pol‐α to perform C‐strand fill‐in reactions (Wu et al., 2012). CST: DNA Pol‐α‐mediated C‐strand fill‐in is absolutely required for telomere length maintenance, as telomerase by itself is insufficient to generate the proper duplex telomere (Feng et al., 2017; Gu et al., 2012). Defects in telomere replication due to disruption of the CST complex lead to replication fork stalling, as telomeres can adopt secondary structures that are difficult to replicate (Gu et al., 2012; Stewart et al., 2012).…”

“…CST: DNA Pol‐α‐mediated C‐strand fill‐in is absolutely required for telomere length maintenance, as telomerase by itself is insufficient to generate the proper duplex telomere (Feng et al., 2017; Gu et al., 2012). Defects in telomere replication due to disruption of the CST complex lead to replication fork stalling, as telomeres can adopt secondary structures that are difficult to replicate (Gu et al., 2012; Stewart et al., 2012). Stalled replication forks and the failure of stalled fork restart at telomeres initiate aberrant homologous recombination events that in part account for the catastrophic loss of total telomeric DNA observed in mouse cells devoid of CTC1 (Gu et al., 2012), or the activation of a DDR in mammalian cells (Wang et al., 2012).…”