Telomeric states in cancer development

Telomere dysfunction can unleash genome instability or permanently arrest cell proliferation, and thereby contribute to the processes of cancer and aging. Proper telomere function relies on many proteins that form part of its structure. Some telomeric proteins mediate telomere replication whereas others suppress the DNA damage response and DNA repair activities avoiding chromosome end-to-end fusions. However, our knowledge of the telomeric proteome and its pathological perturbations remains incomplete. Progress in telomere research has been partially hampered by a lack of systematic approaches and sensitive tools to analyze telomere composition and function. This thesis provides methodological improvements and applications that have helped to expand the repertoire of proteins involved in protection of telomere integrity. Firstly, this work offers a detailed implementation guide for the Quantitative Telomeric Chromatin Isolation Protocol (QTIP), a mass-spectrometry based technique, which enables identification of telomeric proteins and their quantitative changes between different cell states. Secondly, QTIP is used to characterize the alterations that occur in the telomeric proteome during cellular transformation. I have discovered that proteins involved in telomere DNA replication and DNA damage repair, as well as the telo-meric RNA TERRA are upregulated during cellular transformation. Thirdly, I demonstrate that several of the upregulated proteins counteract telomere fragility, which can be induced by oncogenic stress. SAMHD1 is one of the proteins induced upon cellular transformation. It is frequently mutated in cancer and Aicardi-Goutières syndrome. SAMHD1 association with telomeres is con-firmed and its function explored. I show that SAMHD1 deficiency leads to telomere breakage events when telomere replication is compromised by TRF1 depletion. These results suggest that telomeres break during replication when deprived of TRF1 and that SAMHD1 is required for telomeric DNA damage repair. Finally, the molecular basis of the telomere syndrome caused by CTC1 mutations is examined. Overall, SAMHD1 and CTC1 functions are characterized and added to the conundrum of proteins that facilitate faithful telomere replication.

RADX: A novel single-stranded DNA-binding protein regulating telomere recombination

Anna-Sophia Briod

Telomeres are specialized nucleoprotein structures present at the ends of linear chromosome. The telomeric DNA part is comprised of 5-15 kilo base pairs of double stranded TTAGGG repeats and it contains at the 3' end a single-stranded G-rich overhang of 50-400 nucleotides. Telomeric DNA associates with a large number of proteins, which inhibit DNA damage checkpoint activation, DNA repair activities and nucleolytic degradation. To better understand the complex roles which telomeres play in genome stability, premature aging and cancer development we explored the telomeric proteomic environment with BioID. We employed a CRISPR/Cas9 based knock-in approach to integrate the promiscuous biotin ligase BirA at the genomic loci of the telomeric components TRF1, TRF2 and POT1. Thus, we expressed the fusion proteins from native promoters aiming at retaining native expression levels. Upon incubation with Biotin, the promiscuous biotinylase can label proximal proteins, which can be selectively purified and identified via mass spectrometry. Our BioID results indicated that TRF1, TRF2 and POT1 share a large number of common protein partners. In addition to already known telomere components, we identified a significant number of novel telomeric proteins. Among those newly identified proteins, we found RADX, an RPA-like single-stranded DNA-binding-protein, which counteracts RAD51 at stalled replication forks. Even though RADX had so far only been characterized for its role in DNA replication we observed colocalization of ectopically expressed 3xFlag-RADX with telomeres throughout the cell cycle. The colocalization decreased when the RADXs single-stranded (ss)DNA-binding domain was mutated. We therefore hypothesize that RADX binds the single-stranded G-rich telomeric strand either as displaced strand in the t-loop or as single-stranded telomeric overhang. Chromatin-immunoprecipitation experiments suggest that RADX plays a distinct role in telomere replication, since upon treatment with the replication stress inducing reagent hydroxyurea we observed increased binding of RADX specifically to telomeres. Notably, RADX depletion on its own did not alter telomere integrity and telomere length but upon co-depletion with POT1, telomere fragility, sister-chromatid associations and telomere length increased strikingly. POT1 and POT1/RADX depletions lead to increased associations of the recombinase RAD51 with telomeres and consequently silencing of RAD51 rescued telomere integrity and elongation implying the regulatory role of RADX for RAD51. In summary my thesis describes the proteomic microenvironment of different telomeric proteins, identifies the novel telomeric protein RADX and characterizes its role at telomeres. My findings provide important insights into how homologous recombination and RAD51 loading is suppressed at telomeres.

EPFL2019

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.

EPFL2011

TIN2-tethered TPP1 recruits human telomerase to telomeres in vivo

Elena Aritonovska, Joachim Lingner

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres--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. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 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 recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.