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Clustered Telomeres in Phase-Separated Nuclear Condensates Engage Mitotic DNA Synthesis Through BLM and RAD52

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Clustered Telomeres in Phase-Separated Nuclear Condensates Engage Mitotic DNA Synthesis Through BLM and RAD52 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52 Jaewon Min, Woodring E. Wright, and Jerry W. Shay Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, 75390, USA Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in pro- myelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-inter- acting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors. [Keywords: ALT; MiDAS; phase separation; biomolecular condensates; break-induced replication] Supplemental material is available for this article. Received January 29, 2019; revised version accepted April 24, 2019. Telomeres are composed of TTAGGG repeats at the ends repeat-containing RNA (TERRA) is also localized in APBs of chromosomes. Cell divisions are accompanied by telo- (Arora et al. 2014). PML bodies are one of the nuclear mere length shortening; therefore, cancer cells almost membrane-less organelles that are formed by liquid– universally acquire a telomere maintenance mechanism liquid phase separation (LLPS) and are organized by (TMM) during neoplastic transformation. Most cancers multivalent interactions between small ubiquitin-like are of epithelial origin (carcinomas) and reactivate telome- modification (SUMO) sites and SUMO-interacting motifs rase activity (Kim et al. 1994). However, cancers of mesen- (SIMs) in PML and other associated proteins (Banani et al. chymal origin, such as sarcomas and soft tissue tumors, 2016). The relative stoichiometry of the scaffold (SUMO frequently acquire a telomerase-independent TMM, and SIM ratio) determines the recruitment of client pro- which has been termed alternative lengthening of telo- teins containing SUMOylation sites and SIMs (Ditlev meres (ALT) (Bryan et al. 1997). ALT is a recombination- et al. 2018). LLPS drives the formation of membrane-less mediated telomere elongation process, but the underlying compartments (known as biomolecular condensates) mechanism by which the ALT pathway is initially en- through liquid–liquid demixing from the surrounding nu- gaged and the molecular mechanisms underlying ALT in cleoplasm or cytoplasm and is involved in the assembly of human cancer are still undetermined. nuclear bodies, such as PML bodies and Cajal bodies One of the hallmarks of ALT-positive cancer specimens (Banani et al. 2017; Wheeler and Hyman 2018). In addition is large bright telomere signals revealed by telomere fluo- to the role of LLPS in normal cellular function, aberrant rescence in situ hybridization (FISH) (Heaphy et al. 2011a). LLPS is involved in the pathogenesis of diseases, such as These bright telomere signals are clustered in promyelo- aggregation-related neurodegenerative disease and cancer cytic leukemia (PML) bodies (Heaphy et al. 2011b), known (Aguzzi and Altmeyer 2016; Bouchard et al. 2018). as ALT-associated PML bodies (APBs). APBs contain not PML bodies are disassembled when cells enter mitosis only telomeric DNA but also many proteins involved in (Bernardi and Pandolfi 2007; Chung et al. 2012). However, DNA replication and repair processes (Yeager et al. 1999). Also, the long noncoding RNA (lncRNA) telomeric © 2019 Min et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publi- Corresponding author: [email protected], jaewon.min@ cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After utsouthwestern.edu six months, it is available under a Creative Commons License (Attribu- Article published online ahead of print. Article and publication date are tion-NonCommercial 4.0 International), as described at http://creative- online at http://www.genesdev.org/cgi/doi/10.1101/gad.324905.119. commons.org/licenses/by-nc/4.0/. 814 GENES & DEVELOPMENT 33:814–827 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/19; www.genesdev.org Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Telomere clustering promotes ALT-mediated by MiDAS APBs are not disassembled until later in mitosis, possibly condensates through LLPS and functionally mimic the due to their hyper-SUMOylated status. Thus, they appear PML bodies in vivo (Fig. 1A). SUMO-abundant scaffold; as APB-like foci in metaphase spreads in a subset of ALT (SUMO)10-(SIM)6 selectively recruits SIM-containing cli- cancer cell lines (Cesare et al. 2009). Recently, it has ent proteins, whereas SIM-abundant scaffold;(SUMO)6- been shown that telomeres can be elongated during (SIM)10 recruits SUMOylated client proteins (Ditlev mitosis in these APB-like foci, known as mitotic DNA et al. 2018). We engineered the original scaffold protein synthesis (MiDAS) at telomeres (Min et al. 2017b; Özer to make it (1) form the condensates in the nucleus by et al. 2018). However, the molecular mechanism for adding nuclear localization signals (NLS) and (2) target how telomeres cluster in PML bodies and are processed the telomeres to these scaffolds by adding the RAP1 C ter- and participate in the ALT pathway are not known. minus (RCT) domain, which directly binds to TRF2 pro- Two ALT mechanisms have been identified in the yeast teins with strong affinity (Fig. 1B; Li et al. 2000; Chen Saccharomyces cerevisiae and are termed type I and type et al. 2011). We transfected plasmid DNAs containing cy- II ALT (Lundblad and Szostak 1989; Lundblad and Black- tomegalovirus (CMV) promoter-driven scaffold proteins burn 1993). Type I ALT in yeast is mediated by Rad51- in 293FT simian virus (SV40) T-antigen transformed dependent recombination, whereas type II ALT in yeast is mediated by the Rad51-independent break-induced rep- lication process (Teng and Zakian 1999; Teng et al. 2000; A Chen et al. 2001; Lydeard et al. 2007). Based on recent studies, it is now believed that these two distinct ALT mechanisms in yeast are also conserved in human ALT cancers (Verma and Greenberg 2016; Sobinoff and Pickett 2017). The type I ALT-like mechanism in human cancer is initiated by RAD51-dependent recombination and elon- gated by the BLM–TOP3A–RMI (BTR) dissolvase complex during S/G2 phases (Cho et al. 2014; Ramamoorthy and Smith 2015; Min et al. 2017a; Sobinoff et al. 2017). In con- B trast, the type II ALT-like mechanism in human cancer is mediated by a RAD51-independent pathway during G2/M phases (Henson et al. 2009; Muntoni et al. 2009; Nabetani and Ishikawa 2009; Oganesian and Karlseder 2011; O’Sul- livan et al. 2014; Dilley et al. 2016; Root et al. 2016; Verma et al. 2019; Zhang et al. 2019), typically observed in APB- C like foci in metaphase spreads (Min et al. 2017b). Here, we present a biophysical model system that can reconstitute PML bodies from minimal components and generate telomere-clustered nuclear condensates and thus artificially engineered APB-like condensates in vivo. We found that the ALT-like phenotypes (i.e., a small frac- tion of heterogeneous telomere lengths and formation of C circles) can be triggered rapidly by the reconstitution of APB-like condensates in the presence of BLM overexpres- sion. Persistent telomere clustering in nuclear conden- sates leads to MiDAS at APB-like foci in metaphase through RAD52. We provide evidence that the clustering D of telomeres promotes the ALT pathway mediated by mi- totic telomere synthesis. Results Induction of telomere clustering in nuclear polySUMO/polySIM condensates can mimic Figure 1. Engineering poly(SUMO)/poly(SIM) scaffolds to in- the APBs in ALT cancer cells duce telomere clustering in the nucleus. (A) Illustration of poly To test whether the clustering of large amounts of telo- (SUMO)/poly(SIM) scaffolds [(SUMO)6-(SIM)10 or (SUMO)6- (SIM)10] mimicking PML bodies in vivo. (B) Strategy for the gen- meres in PML bodies per se is sufficient to engage the eration of telomeres targeting nuclear condensates using NLS and ALT pathway, we decided to use the recently developed RCT that bind to the RAP1-binding domain in TRF2. (C) Repre- multivalent scaffold proteins that consist of 10 or six re- sentative images showing the telomere localization of engineered peats of human SUMO3 (polySUMO) and six or 10 repeats poly(SUMO)/poly(SIM) scaffolds. (D) Representative images of the SIM from PIASx (polySIM) (Banani et al. 2016). The showing a telomere clustering event induced by telomere cluster- polySUMO/polySIM scaffolds can form biomolecular ing scaffolds. GENES & DEVELOPMENT 815 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Min et al. human embryonic kidney cells that are telomerase- Telomere clustering induces the ALT-like phenotypes positive. The original scaffold proteins only formed the in the presence of BLM overexpression condensates in the cytoplasm due to its big size [Fig. 1C, top panel, mCherry-(SUMO)10/6-(SIM)6/10]. Adding two We hypothesized that additional factors were required to NLSs derived from c-Myc and SV40 to the original scaffold trigger the ALT pathway in addition to the telomere clus- proteins allowed them to form condensates in the nucleus tering scaffold.
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