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

Vančevska, Aleksandra; Douglass, Kyle M.; Pfeiffer, Verena; Manley, Suliana; Lingner, Joachim

doi:10.1101/gad.294082.116

Cited by 60 publications

(47 citation statements)

References 39 publications

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“…Some generalities emerged from these analyses. As expected, most telomeres appeared spherical, but heterogeneous shapes were also observed 45 . Fig.…”

Section: Trf1hp1α Increases Irregularly-shaped Telomere Structuressupporting

confidence: 79%

Local Enrichment of HP1alpha at Telomeres Alters Their Structure and Regulation of Telomere Protection

Chow

Shi

Wei

et al. 2018

Preprint

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27Enhanced telomere maintenance is evident in malignant cancers. While telomeres are thought 28 to be inherently heterochromatic, detailed mechanisms of how epigenetic modifications impact 29 telomere protection and structures are largely unknown in human cancers. Here we develop a 30 molecular tethering approach to experimentally enrich heterochromatin protein HP1α specifically 31 at telomeres. This results in increased deposition of H3K9me3 at cancer cell telomeres. 32Telomere extension by telomerase is attenuated, and damage-induced foci at telomeres are was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/368464 doi: bioRxiv preprint first posted online Jul. 13, 2018; 56consisting of the telomere nucleosomal chromatin plus shelterin complex, establishes a capping 57 structure to maintain genome integrity 5, 6 . While functions associated with shelterin itself have 58 been widely studied, molecular details of how this peculiar telomere chromatin impacts 59 mammalian telomere maintenance remain largely unexplored. 61Telomere chromatin is thought to be inherently condensed heterochromatin primarily based on 62 findings in yeast 7, 8 , 77All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 95and rather infrequent HP1 are naturally localized at human telomeres 11, 14,[32][33][34][35] . This provides an 96 opportunity to enhance the presence of this naturally occurring component of telomeric 97chromatin to study its role in telomere biology. 99In this report, we present a novel approach to study the consequences of locally altering 100 telomere chromatin properties on the key functions of telomeres. We enrich 101heterochromatinization at telomeres by fusing HP1alpha (HP1α) to the telomere binding 102 shelterin protein TRF1. We find that deposition of heterochromatin marks at telomeres is 103All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 115A model system to study HP1α function at telomeres 116To study how altered telomere chromatin regulates its maintenance, we set up a controlled 117 system to enhance heterochromatin in a locus-specific manner. We fused shelterin TRF1, which 118 confers telomeric locus-specificity, to HP1α, a protein involved in heterochromatin establishment 119 and maintenance. HP1α contains a conserved N-terminal chromo domain (CD) that binds to 120 dimethylated and trimethylated H3K9 (H3K9me2/3) and a C-terminal chromo shadow domain 121 (CSD) for dimerization and ligand binding 31, 36 . These two domains are joined by a flexible hinge 122 domain (Fig. 1a) 31. 124To validate our system, EGFP-tagged TRF1 fused with HP1α ( Fig. 1a) was transiently 125 cotransfected with mCherry-tagged TRF2...

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“…Some generalities emerged from these analyses. As expected, most telomeres appeared spherical, but heterogeneous shapes were also observed 45 . Fig.…”

Section: Trf1hp1α Increases Irregularly-shaped Telomere Structuressupporting

confidence: 79%

Local Enrichment of HP1alpha at Telomeres Alters Their Structure and Regulation of Telomere Protection

Chow

Shi

Wei

et al. 2018

Preprint

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“…The next day cell suspensions were dropped onto slides to prepare metaphase spreads, incubated 1min at 70°C in a wet chamber and dried for 16-24 h before FISH. FISH staining of human telomeric DNA was performed as described (Vancevska et al, 2017). Slides were rehydrated in 1× PBS for 5 min, treated with 4% formaldehyde in PBS for 5 min, washed 3x with 1xPBS and dehydrated with increasing amounts of ethanol (70%, 95%, 100%).…”

Section: Telomeric Pna-fish On Metaphase Spreadsmentioning

confidence: 99%

“…Indirect immunofluorescence detection of human ATM pS981, 53BP1 and γH2AX followed by telomeric FISH staining was performed as described (Vancevska et al 2017). For detection of ATM pS1981 before crosslinking, cells were fractionated with an ice-cold preextraction buffer containing 0.5% Triton X-100, 20mM HEPES-OH pH 7.5, 50mM NaCl, 3mM MgCl2 and 300mM sucrose for 7min.…”

Section: Indirect Immunofluorescence and Telomeric Fish (If-fish)mentioning

confidence: 99%

SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres

Vančevska

Pfeiffer

Feretzaki

et al. 2019

Preprint

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SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing protein 1) has been implicated in X-chromosome inactivation, imprinting and DNA damage repair. Mutations in SMCHD1 can also cause facioscapulohumoral muscular dystrophy. More recently, SMCHD1 has also been detected as component of telomeric chromatin. Here, we identify requirements of SMCHD1 for DNA damage signaling and non-homologous end joining (NHEJ) at unprotected telomeres. Co-depletion of SMCHD1 with TRF2 reduced the rate of 3' overhang removal in time course experiments and the number of telomere end fusions. In SMCHD1 deficient cells, the formation of ATM pS1981, gH2AX and 53BP1 containing telomere dysfunction induced foci (TIFs) were diminished indicating defects in checkpoint signaling. Strikingly, removal of TPP1 and subsequent activation of ATR signaling rescued telomere fusion events in TRF2-depleted SMCHD1 knockout cells. Together, these data indicate that SMCHD1 depletion reduces telomere fusions in TRF2-depleted cells due to defects in ATM-dependent DNA checkpoint signaling. SMCHD1 mediates DNA damage signaling activation upstream of ATM phosphorylation at uncapped telomeres.

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“…These massive telomere-clustering events are specific for ALT cancer cells and mostly contain APBs (ALT-associated promyelocytic leukemia [PML] bodies), damaged DNA, and DNA repair machinery. Moreover, these are also increased in response to double-strand breaks or stalled replication forks at telomeres (11,(40)(41)(42)(43)(44)(45)(46)(47)(48). It has also been previously reported that large telomeric foci contain extrachromosomal telomeric DNAs (49).…”

mentioning

confidence: 91%

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

Min

Wright

Shay

2017

Molecular and Cellular Biology

175

213

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Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

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

Cited by 60 publications

References 39 publications

Local Enrichment of HP1alpha at Telomeres Alters Their Structure and Regulation of Telomere Protection

Local Enrichment of HP1alpha at Telomeres Alters Their Structure and Regulation of Telomere Protection

SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes

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