Try a new search

Format these results:

Searched for:



Total Results:


Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis

Fu, Yi; Sacco, Olivia; DeBitetto, Emily; Kanshin, Evgeny; Ueberheide, Beatrix; Sfeir, Agnel
Mitochondrial DNA double-strand breaks (mtDSBs) lead to the degradation of circular genomes and a reduction in copy number; yet, the cellular response in human cells remains elusive. Here, using mitochondrial-targeted restriction enzymes, we show that a subset of cells with mtDSBs exhibited defective mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy confirmed the altered mitochondrial membrane and cristae ultrastructure. Intriguingly, mtDSBs triggered the integrated stress response (ISR) via the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by DELE1 and heme-regulated eIF2α kinase (HRI). When ISR was inhibited, the cells experienced intensified mitochondrial defects and slower mtDNA recovery post-breakage. Lastly, through proteomics, we identified ATAD3A-a membrane-bound protein interacting with nucleoids-as potentially pivotal in relaying signals from impaired genomes to the inner mitochondrial membrane. In summary, our study delineates the cascade connecting damaged mitochondrial genomes to the cytoplasm and highlights the significance of the ISR in maintaining mitochondrial homeostasis amid genome instability.
PMID: 37832546
ISSN: 1097-4164
CID: 5604282

POLQ inhibition elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma via cGAS/STING signaling

Oh, Grace; Wang, Annie; Wang, Lidong; Li, Jiufeng; Werba, Gregor; Weissinger, Daniel; Zhao, Ende; Dhara, Surajit; Hernandez, Rosmel E; Ackermann, Amanda; Porcella, Sarina; Kalfakakou, Despoina; Dolgalev, Igor; Kawaler, Emily; Golan, Talia; Welling, Theodore H; Sfeir, Agnel; Simeone, Diane M
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination-repair (HR-repair) proteins in 20%-25% of cases. Defects in HR impart a specific vulnerability to poly ADP ribose polymerase inhibitors and platinum-containing chemotherapy in tumor cells. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we found that POLQ knockdown is synthetically lethal in combination with mutations in HR genes such as BRCA1 and BRCA2 and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates signaling of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING), leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to blocking tumor growth while concurrently activating the cGAS-STING signaling pathway to enhance tumor immune infiltration, highlighting what we believe to be a new role for POLQ in the tumor immune environment.
PMID: 36976649
ISSN: 1558-8238
CID: 5541482

Stem cells at odds with telomere maintenance and protection

Penev, Alex; Markiewicz-Potoczny, Marta; Sfeir, Agnel; Lazzerini Denchi, Eros
Telomeres are distinctive structures that protect the ends of linear chromosomes and ensure genome stability. They are composed of long tracks of repetitive and G-rich DNA that is bound by shelterin, a dedicated six-subunit protein complex. In somatic cells, shelterin protects telomeres from the DNA damage response and regulates telomere length. Telomere repeats are replenished by telomerase, a specialized ribonucleoprotein composed of telomerase reverse transcriptase and an integral RNA component. Telomere protection and telomerase regulation have been primarily studied in somatic cells. However, recent evidence points out striking differences in the context of embryonic stem cells (ESCs). In this review, we discuss insights into telomere protection in ESCs versus somatic cells and summarize findings on telomerase regulation as a function of pluripotency.
PMID: 35063336
ISSN: 1879-3088
CID: 5132012

Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states

Barry, Raymond Mario; Sacco, Olivia; Mameri, Amel; Stojaspal, Martin; Kartsonis, William; Shah, Pooja; De Ioannes, Pablo; Hofr, Ctirad; Côté, Jacques; Sfeir, Agnel
In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.
PMID: 35210222
ISSN: 1549-5477
CID: 5172412

Alternative splicing is a developmental switch for hTERT expression

Penev, Alex; Bazley, Andrew; Shen, Michael; Boeke, Jef D; Savage, Sharon A; Sfeir, Agnel
Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is primarily driven by an alternative splicing event, centered around hTERT exon 2. Skipping of exon 2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon 2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.
PMID: 33852895
ISSN: 1097-4164
CID: 4846132

Nuclear sensing of breaks in mitochondrial DNA enhances immune surveillance

Tigano, Marco; Vargas, Danielle C; Tremblay-Belzile, Samuel; Fu, Yi; Sfeir, Agnel
Mitochondrial DNA double-strand breaks (mtDSBs) are toxic lesions that compromise the integrity of mitochondrial DNA (mtDNA) and alter mitochondrial function1. Communication between mitochondria and the nucleus is essential to maintain cellular homeostasis; however, the nuclear response to mtDSBs remains unknown2. Here, using mitochondrial-targeted transcription activator-like effector nucleases (TALENs)1,3,4, we show that mtDSBs activate a type-I interferon response that involves the phosphorylation of STAT1 and activation of interferon-stimulated genes. After the formation of breaks in the mtDNA, herniation5 mediated by BAX and BAK releases mitochondrial RNA into the cytoplasm and triggers a RIG-I-MAVS-dependent immune response. We further investigated the effect of mtDSBs on interferon signalling after treatment with ionizing radiation and found a reduction in the activation of interferon-stimulated genes when cells that lack mtDNA are exposed to gamma irradiation. We also show that mtDNA breaks synergize with nuclear DNA damage to mount a robust cellular immune response. Taken together, we conclude that cytoplasmic accumulation of mitochondrial RNA is an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including breaks produced by genotoxic agents.
PMID: 33627873
ISSN: 1476-4687
CID: 4806852

In Vivo Analysis of mtDNA Replication at the Single Molecule Level and with High Resolution

Tigano, Marco; Phillips, Aaron Fraser; Sfeir, Agnel
Single molecule analysis of replicating DNA (SMARD) is a powerful methodology that allows in vivo analysis of replicating DNA; identification of origins of replication, assessment of fork directionality, and measurement of replication fork speed. SMARD, which has been extensively used to study replication of nuclear DNA, involves incorporation of thymidine analogs to nascent DNA chains and their subsequent visualization through immune detection. Here, we adapt and fine-tune the SMARD technique to the specifics of human and mouse mitochondrial DNA. The mito-SMARD protocol allows researchers to gain in vivo insight into mitochondrial DNA (mtDNA) replication at the single molecule level and with high resolution.
PMID: 33230762
ISSN: 1940-6029
CID: 4702512

Quantitative Imaging of MS2-Tagged hTR in Cajal Bodies: Photobleaching and Photoactivation

Smith, Michael; Querido, Emmanuelle; Chartrand, Pascal; Sfeir, Agnel
Advances in imaging technologies, gene editing, and fluorescent molecule development have made real-time imaging of nucleic acids practical. Here, we detail methods for imaging the human telomerase RNA template, hTR via the use of three inserted MS2 stem loops and cognate MS2 coat protein (MCP) tagged with superfolder GFP or photoactivatable GFP. These technologies enable tracking of the dynamics of RNA species through Cajal bodies and offer insight into their residence time in Cajal bodies through photobleaching and photoactivation experiments. For complete details on the use and execution of this protocol, please refer to Laprade et al. (2020).
PMID: 33377008
ISSN: 2666-1667
CID: 4936462

Replication stress conferred by POT1 dysfunction promotes telomere relocalization to the nuclear pore

Pinzaru, Alexandra M; Kareh, Mike; Lamm, Noa; Lazzerini-Denchi, Eros; Cesare, Anthony J; Sfeir, Agnel
Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.
PMID: 33122293
ISSN: 1549-5477
CID: 4646842

Imaging of Telomerase RNA by Single-Molecule Inexpensive FISH Combined with Immunofluorescence

Querido, Emmanuelle; Sfeir, Agnel; Chartrand, Pascal
Fluorescent in situ hybridization (FISH) on the RNA moiety of human telomerase (hTR) with 50-mer probes detects hTR RNA accumulated in Cajal bodies. Using both live-cell imaging and single-molecule inexpensive FISH, our published work revealed that only a fraction of hTR localizes to Cajal bodies, with the majority of hTR molecules distributed throughout the nucleoplasm. This protocol is an application guide to the smiFISH method for the dual detection of hTR RNA and telomeres or Cajal bodies by immunofluorescence. For complete details on the use and execution of this protocol, please refer to Laprade et al. (2020).
PMID: 33111129
ISSN: 2666-1667
CID: 4936452