Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Abstract

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging-associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA-mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long-term viability of normal somatic mammalian cells.

Telomere dysfunction promotes transdifferentiation of human fibroblasts into myofibroblasts

“…Since ROS, generated through the TGF‐β1/Smad3/NOX4‐signaling pathway, caused telomere dysfunction and proliferative arrest of human somatic fibroblasts that lack detectable telomerase activity, but did not affect proliferation rates of fibroblasts expressing hTERT, our data suggest that opposing effects of TGF‐β1 on cell proliferation depend on the levels of telomerase activity in a cell. The insensitivity of hTERT‐expressing fibroblasts to TGF‐β1‐induced growth arrest and myofibroblast transdifferentiation likely is due to the ability to telomerase to suppress the formation of dysfunctional telomeres in cells encountering ROS‐induced telomeric replication stress, as demonstrated previously (Boccardi et al, 2015). As hTERT expression reduced formation of both telomeric and to some extent also nontelomeric DDR foci, it is also possible, however, that noncanonical functions of telomerase, such as its ability to reduce cellular ROS production (Saretzki, 2014), contribute to suppressing activation of a DDR and myofibroblast transdifferentiation of human fibroblasts.…”

“…Similarly, using human fibroblasts in which dysfunctional telomeres can be repaired using a doxycycline inducible hTERT expression system (Patel et al, 2016), we demonstrate that induction of hTERT expression in TGF‐β1 transdifferentiated myofibroblasts again reduced the levels of α‐SMA expressed in cells (Supporting Information Figure S3f). While the primary function of telomerase is to elongate telomeres and suppress formation of dysfunctional telomeres generated due to telomere shortening and ROS‐induced telomeric replication stress, (Boccardi et al, 2015; Patel et al, 2016), telomerase also possesses noncanonical functions that promote DNA repair and cell survival (Martinez & Blasco, 2011). To determine whether noncanonical functions of hTERT contribute to the suppression of myofibroblast transdifferentiation, we overexpressed in human BJ fibroblasts DN‐hTERT, a dominant defective mutant of hTERT that inactivates the catalytic activity of hTERT while leaving its noncanonical functions intact (Hahn et al, 1999).…”

“…For example, genotoxic stresses that cause DSB formation in telomeric repeats can rapidly activate TDIS, as telomeric breaks resist DNA repair activities (Fumagalli et al, 2012; Hewitt et al, 2012; Suram et al, 2012). Stresses that have been demonstrated to cause TDIS include oncogene expression (Patel, Suram, Mirani, Bischof, & Herbig, 2016; Suram et al, 2012), DNA replication stress (Boccardi et al, 2015; Fumagalli et al, 2012; Hewitt et al, 2012), and reactive oxygen species (ROS) (Boccardi et al, 2015), among others. Whether generated due to progressive telomere erosion or due to DSB formation in telomeric repeats, telomere dysfunction activates a persistent DDR that is mediated by and dependent on p53, a transcription factor that not only trans‐activates DDR genes (Molchadsky, Rivlin, Brosh, Rotter, & Sarig, 2010), but also regulates a wide range of other biological activities including cell differentiation (Rivlin, Koifman, & Rotter, 2015).…”

DNA Replication Controls Volume 2

“…One of the first reports on the effect of transcription on genetic stability in a eukaryotic system showed that HOT1, a segment of the ribosomal DNA locus that is actively transcribed by RNA polymerase I, could function as a cis-acting enhancer of recombination in S. cerevisiae [229]. A similar increase in recombination and spontaneous mutations was observed with high transcription levels by RNA Pol II [230,231]. Two different hypotheses explain the stimulation of spontaneous recombination by transcription: the first one centers around the increased accessibility of homologous recombination proteins to the DNA during transcription; the second hypothesis suggests that collisions between the transcription and replication machineries, the presence of stalled replication forks during transcription, or the formation of transcription-associated DNA:RNA hybrids (R-loops) can be significant sources of DNA damage and TAR ( Figure 8).…”

“…Because replication forks stall naturally at mammalian telomeres, an ATR-dependent fork restart mechanism is needed to complete DNA replication [227][228][229]. Knockdown of expression of CST components decreases bromodeoxyuridine incorporation at telomeres after release from hydroxyurea-induced replication fork arrest, and elicits telomere fragility [212,226,[230][231][232][233][234][235][236]. Several lines of evidence suggest that CST contributes to fork restart not only in telomeres but also genome-wide under conditions of replication stress [232,233,235,237].…”