ALT Telomeres Get Together with Nuclear Receptors

Nuclear receptors bind chromosome ends in "alternative lengthening of telomeres" (ALT) cancer cells that maintain their ends by homologous recombination instead of telomerase. Marzec et al. now demonstrate that, in ALT cells, nuclear receptors not only trigger distal chromatin associations to mediate telomere-telomere recombination events, but also drive chromosome-internal targeted telomere insertions (TTI).

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

Expression and differential regulation of human TERRA at several chromosome ends

Marianna Feretzaki, Joachim Lingner, Patricia Renck Nunes

The telomeric long noncoding RNA TERRA has been implicated in regulating telomere maintenance by telomerase and homologous recombination, and in influencing telomeric protein composition during the cell cycle and the telomeric DNA damage response. TERRA transcription starts at subtelomeric regions resembling the CpG islands of eukaryotic genes extending toward chromosome ends. TERRA contains chromosome-specific subtelomeric sequences at its 5' end and long tracts of UUAGGG-repeats toward the 3' end. Conflicting studies have been published as to whether TERRA is expressed from one or several chromosome ends. Here, we quantify TERRA species by RT-qPCR in normal and several cancerous human cell lines. By using chromosome-specific subtelomeric DNA primers, we demonstrate that TERRA is expressed from a large number of telomeres. Deficiency in DNA methyltransferases leads to TERRA up-regulation only at the subset of chromosome ends that contain CpG-island sequences, revealing differential regulation of TERRA promoters by DNA methylation. However, independently of the differences in TERRA expression, short telomeres were uniformly present in a DNA methyltransferase deficient cell line, indicating that telomere length was not dictated by TERRA expression in cis. Bioinformatic analyses indicated the presence of a large number of putative transcription factors binding sites at TERRA promoters, and we identified a subset of them that repress TERRA expression. Altogether, our study confirms that TERRA corresponds to a large gene family transcribed from multiple chromosome ends where we identified two types of TERRA promoters, only one of which is regulated by DNA methylation.

COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT2019

The makings of TERRA R-loops at chromosome ends

Marianna Feretzaki, Joachim Lingner, Rita Valador Fernandes

Telomeres protect chromosome ends from nucleolytic degradation, uncontrolled recombination by DNA repair enzymes and checkpoint signaling, and they provide mechanisms for their maintenance by semiconservative DNA replication, telomerase and homologous recombination. The telomeric long noncoding RNA TERRA is transcribed from a large number of chromosome ends. TERRA has been implicated in modulating telomeric chromatin structure and checkpoint signaling, and in telomere maintenance by homology directed repair, and telomerase - when telomeres are damaged or very short. Recent work indicates that TERRA association with telomeres involves the formation of DNA:RNA hybrid structures that can be formed post transcription by the RAD51 DNA recombinase, which in turn may trigger homologous recombination between telomeric repeats and telomere elongation. In this review, we describe the mechanisms of TERRA recruitment to telomeres, R-loop formation and its regulation by shelterin proteins. We discuss the consequences of R-loop formation, with regard to telomere maintenance by DNA recombination and how this may impinge on telomere replication while counteracting telomere shortening in normal cells and in ALT cancer cells, which maintain telomeres in the absence of telomerase.

TAYLOR & FRANCIS INC2021

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

Anna-Sophia Briod

EPFL2019