Functional analysis of the telomeric long non-coding RNA TERRA

Telomeres are nucleoprotein structures present in the end of linear chromosomes. In humans, a core complex of 6 proteins cap and protect the chromosome ends from being recognized as double-strand breaks and eliciting unwanted DNA damage and repair responses, which lead to chromosomal end-to-end fusions and genomic instability. In addition to the shelterins, another 200 proteins are detected at telomeres by mass spectrometry. Their functions at telomere are not yet completely understood. Telomeres are transcribed into a long non-coding RNA called TERRA. It is believed that subtelomeric CpG islands act as TERRA promoters. TERRA is suggested to regulate telomerase activity, to participate in the processing of uncapped telomeres, in telomeric heterochromatin formation and in promoting recombination-mediated telomere elongation. TERRA levels are increased in patients affected by the ICF (immunodeficiency, centromeric instability, facial anomalies) syndrome. Since these patients also present very short telomeres, it has been suggested that TERRA regulates telomere length, either by inhibiting telomerase or by promoting access to end-attacking nucleases. We used HCT116 DNMT1 (deletion of exons3-5/exons3-5) DNMT3B (-/-) (DKO) cells as a model for the ICF syndrome to try to comprehend the relationship between TERRA expression and telomere length. In this thesis, we try to understand (1) if there is a direct correlation between TERRA expression and telomere length, (2) what drives TERRA transcription, (3) what are the consequences of TERRA overexpression, and (4) what could cause the short telomere phenotype in DKO cells. We show that not all telomeres overexpress TERRA in DKO cells, but even telomeres with normal TERRA expression are short. By analyzing subtelomeric sequences, we found that the presence and density of subtelomeric CpG islands determine if its transcript will be upregulated in DKO cells or not. We used absolute quantification methods to demonstrate that TERRA is expressed from multiple chromosome ends, disproving a recent proposal that TERRA stems only from one locus. We also discovered that CpG-negative subtelomeres express TERRA in levels similar to CpG-positive ones, suggesting that the presence of subtelomeric CpG-islands is not a requirement for high TERRA expression. We next evaluated the telomere length dynamics in DKO cells. We found that DKO cells show lower telomere elongation rates than the WT. Moreover, DKO cells fail to downregulate TERRA in S-phase, in contrast to WT HCT116 and HeLa cells. The downregulation of TERRA in late S-phase has been proposed to alleviate TERRA-dependent telomerase inhibition and thereby allow the enzyme to extend short telomeres. To better understand TERRA transcription, we performed a siRNA screen to discover new TERRA transcription regulators. We found ZNF148, ZFX, PLAG1 and EGR1 to be new repressors of TERRA transcription. Finally, we established a CRISPR activation system to induce overexpression of endogenous TERRA. Overexpression of 10q and 13q TERRA in HCT116 did not lead to telomere shortening in cis. In HeLa cells, overexpression of multiple TERRA species led to deposition of the heterochromatin marks H3K9me3 and H4K20me3 in the telomeres. These marks were also enriched in subtelomeric sequences in cis. Since TERRA has been shown to interact with SUV39H1 to ensure H3K9me3 deposition, we speculate that TERRA also recruits SUV420H2 to induce H4K20me3 accumulation.

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

Identification and characterization of proteins that protect human telomeres from fragility

Anna Christina Näger

Difficulties to replicate telomeres - the ends of our chromosomes - can cause telomere shortening andgenome instability. These difficulties are due to the repetitive DNA sequence and distinct structures at telomeresthat challenge the semi-conservative DNA replication machinery. Among the proteins that help toovercome the obstacles to telomere replication are factors known to be mutated in genetic diseases (forinstance 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 proteinsthat protect human telomeres from fragility, we depleted 71 proteins (including 8 controls) from HeLa cellsusing siRNA pools and analysed their metaphase chromosomes for fragile telomeres. The majority of theseproteins had been identified by QTIP-iPOND - a proteomic analysis of chromatin near telomere replicationforks. Among the 71 proteins that were depleted in the siRNA screen, we identified 29 novel proteins thatprevent telomere fragility and are therefore crucial to maintain genome stability. The telomere fragilityscreen validated QTIP-iPOND as a technique to identify telomere replication proteins. The hits identified inour telomere fragility screen may for example have functions in regulating telomeric chromatin compositionand compaction, regulating transcription of the telomeric long non-coding RNA TERRA and its associationwith telomeric chromatin, dealing with replication stress and stalled replication forks, modulating DNAdamage 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 functionat telomeres was investigated in further detail in this study. CBX8 is a component of the canonical polycombrepressive complex 1 (PRC1), an epigenetic regulator of gene expression. However, CBX8 also hasfunctions that are independent of the PRC1 complex. We show that human CBX8 prevents telomere fragilitycaused 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 thatCBX8 may be involved in repair activities at stalled replication forks that arise when the replisome encounterspersistent RNA-DNA hybrids. Moreover, we found that CBX8 localizes to telomeric repeat-binding factor2 (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 beensuggested to help recruitment of CBX proteins to chromatin via the CBX chromodomain, are dispensable forCBX8 recruitment to TRF2-deficient telomeres. Furthermore, we found that CBX8 promotes non-homologousend joining (NHEJ)-mediated telomere fusions of dysfunctional telomeres. We therefore hypothesizethat CBX8 is involved in repair activities that influence the DNA repair pathway choice at TRF2-deficient telomeres.

EPFL2022

A Quantitative Telomeric Chromatin Isolation Protocol (Q-TIP) to Characterize Telomere Composition Changes

Larissa Grolimund

Telomeres protect linear ends of eukaryotic chromosomes by preventing chromosomal end-to-end fusions and telomere attrition. They consist of repetitive DNA sequences, telomeric repeat containing RNAs (TERRAs) and proteins. Telomeres play a crucial role in chromosome stability, cancer biology, and ageing. With each cell division, telomeres shorten, as the replication machinery of the cell is not capable to fully copy the very terminal part of chromosomes. Therefore, after a certain number of cell divisions, telomeres get critically short and elicit signals in order to stop the proliferation. A cell can circumvent the signal for critically short telomeres by activating mechanisms that elongate telomeres. In most of the cases, telomere elongation is achieved by the expression of the telomerase enzyme. In this case, cells gain the ability to divide indefinitely, which can, in combination with other events, eventually lead to cancer. The telomeric protein composition changes during tumorigenesis, aging and in telomere syndromes are poorly defined. Here, we develop a quantitative telomeric chromatin isolation protocol (q-TIP) for human cells in which chromatin is crosslinked, immunopurified and analyzed by mass spectrometry. Q-TIP involves stable isotope labeling by amino acids in cell culture (SILAC) in order to compare and identify quantitative differences in telomere protein composition of cells from various states. With q-TIP, we specifically enrich telomeric DNA and all shelterin components. We identify and validate known and novel telomere associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply q-TIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterin components TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate q-TIP to study telomeric states during normal development and in disease.

EPFL2013