The telomeric DNA damage response occurs in the absence of chromatin decompaction

Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.

The telomeric DNA damage response: Insights into telomere compaction and identification of novel damage signaling factors

Aleksandra Vancevska

Telomeres are dynamic nucleo-protein structures capping the ends of all eukaryotic chromosomes. Together with telomerase, they counteract replication-dependent telomere attrition. Additionally, they disguise the linear ends of the chromosome from the DNA damage response (DDR) machinery. Otherwise, the chromosome end would be recognized as a DSB and would elicit a deleterious DDR signal. Both of these functions contribute greatly to the maintenance of genome stability. Telomeres are composed of repetitive DNA sequences, protein complex dubbed shelterin and telomeric repeat-containing RNAs. In addition to the core protein complex, other proteins are important for proper telomere structure and function. Our laboratory has a longstanding interest in the discovery of novel factors that have indispensable roles in telomere biology. For that purpose, previous lab members developed a Quantitative Telomeric Chromatin Isolation protocol (QTIP) and detected binding of new proteins to long telomeres, SMCHD1 and LRIF1. Their function at telomeres is not described but they are shown to function in higher-order chromatin organization and genome-wide DDR. Understanding their role in telomere biology is important because the list of players involved in DDR at telomeres is far from complete and we know very little about how chromatin structure affects DDR activation. Thus, this thesis provides insights into the DDR at telomeres by studying the functions of SMCHD1 and LRIF1 and describes the implementation of a novel microscopy-based method to study the role of these proteins and shelterins in early steps of the DDR. Firstly, we describe crucial roles for SMCHD1 and LRIF1 in DDR activation at telomeres lacking shelterin protein TRF2. Removal of TRF2 leads to activation of the ATM kinase and elicits a DDR giving rise to persistent chromosome fusions. We show that LRIF1 and SMCHD1 removal leads to attenuation of ATM activation and subsequent DDR defect as well as impairment in non-homologous end joining (NHEJ) at unprotected telomeres. Considering that these phenotypes are rescued by removal of TPP1 and activation of the ATR kinase we propose that these proteins mainly participate in DDR signaling operating at uncapped telomeres. They are among the rare ones described to act early in the DDR cascade upon TRF2 removal. Stimulated by the discovery that SMCHD1 and LRIF1 function in X-chromosome compaction, we sought to test if they would function in remodeling telomeric chromatin. To do that, we have implemented super-resolution microscopy method (STORM) to measure sizes and shapes of normal human telomeres and ones lacking SMCHD, LRIF1, and shelterin. We have shown, by examination of thousands of telomeres, that removal of shelterin proteins leads to decompaction of only a small subset of telomeres. This decompaction is not required for efficient DDR activation, as the DDR was also efficiently elicited from compacted TRF2-depleted telomeres. Thus, we propose that DDR is triggered by changes at the molecular level in protein recruitment upon telomere deprotec-tion. In addition, transient removal of SMCHD1 and LRIF1 did not affect the compaction state of telo-meres as they do in the X-chromosome. Overall, we have described two novel factors that are important for DDR at uncapped telomeres and excluded the need for decompaction as the initial step of DDR at uncapped telomeres and excluded the need for decompaction as the initial step of DDR activation.

EPFL2018

Telomerase is essential to alleviate pif1-induced replication stress at telomeres

Joachim Lingner

Pif1, an evolutionarily conserved helicase, negatively regulates telomere length by removing telomerase from chromosome ends. Pif1 has also been implicated in DNA replication processes Such as Okazaki fragment maturation and replication fork pausing. We find that overexpression of Saccharomyces cervisiae results in dose-dependent. growth inhibition. Strong overexpression causes relocalization of the DNA damage response factors Rfa1 and Mre11 into nuclear foci and activation of the Rad53 DNA damage checkpoint kinase, indicating that. the toxicity is caused by accumulation of DNA-damage. We screened the complete set of similar to 4800 haploid gene deletion mutants and found that moderate overexpression of PIF1, which is only mildly toxic oil its own, causes growth defects in strains with Mutations in genes involved in DNA replication and the DNA damage response. Interestingly, we find that telomerase-deficient strains are also sensitive to PIF1 overexpression. Cur data are consistent with a model whereby increased levels of Pif1 interfere with DNA replication, causing collapsed replication forks. At chromosome ends, Collapsed forks result in truncated telomeres that must be rapidly elongated by telomerase to maintain viability.

2009

Insight into the Regulation of Telomerase Access to Telomeres by Shelterin Proteins

Elena Aritonovska

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase. Impaired telomerase function can lead to premature organismal aging, development of cancer and multisystem disorders such as dyskeratosis congenita. Telomerase access to telomeres may be regulated by a telomere-binding complex termed shelterin, which is composed of TRF1, TRF2, RAP1, TIN2, TPP1 and POT1 proteins. However the molecular basis for human telomerase recruitment to telomeres is not known. Here, we have directly investigated the process of telomerase recruitment via chromatin immunoprecipitation (ChIP) and fluorescence in situ hybridization (FISH). We find that depletion of two components of the shelterin complex – TPP1 and the protein that tethers TPP1 to the complex, TIN2 – results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Furthermore, we find that the double-stranded telomere binding protein, TRF2 is dispensable for telomerase association with telomeres. Deletion of the oligonucleotide/oligosaccharide-binding fold (OB-fold) of TPP1 further disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that the recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere. We propose that the loss of TRF2 can be compensated by the second double-stranded telomere binding protein, TRF1 that anchors TIN2/TPP1-recruited telomerase onto the telomeres. Dyskeratosis congenita (DC) is a multisystem disorder characterized with bone marrow failure, cancer predisposition and defective telomere maintenance. The TINF2 gene that encodes for the TIN2 shelterin protein is one of seven mutated genes identified in DC patients. Here, we have examined the effects of TIN2 DC mutants on telomere protection and shelterin protein stability. We find that exogenous expression of TIN2 DC mutants can rescue the TIN2-depleted phenotype, restoring TPP1 protein levels and telomere protection. Our findings indicate that TIN2 DC mutants preserve the intact telomere structure and protection. We propose that defective telomere elongation may be the underlying cause of impaired telomere maintenance in TIN2 DC patients.