Biology subjects

Sfeir, A.

Publications and source records attributed to Sfeir, A..

12 recordsLinked to original sources

bioRxiv2026

Random mtDNA mutagenesis maps genotype-phenotype associations across metabolic contexts and lineages

Mitochondrial DNA (mtDNA) mutations cause inherited disease, accumulate with age, and recur in tumors, yet their effects remain unresolved because existing models interrogate one engineered allele at a time. We developed mtDIVE, a random C[->]T mutagenesis platform for pooled mitochondrial forward genetics in human epithelial cells and zebrafish embryos. mtDIVE generates genotypes ranging from single substitutions to >100 mutations per molecule, impairs respiration, and causes lineage-specific developmental delay. Coupling single-cell transcriptomics with mtDNA genotyping revealed that selection on mutant genomes and their transcriptional consequences depend on metabolic state and developmental lineage. Obligate OXPHOS eliminates highly mutated cells by apoptosis, whereas glycolytic conditions promote their competitive displacement. Individual loci exert modest but non-redundant effects that predict cell state beyond aggregate mutation burden, and cancer-recurrent variants are enriched among mutations linked to reduced mitochondrial transcription. Thus, mtDIVE links mtDNA genotypes to cellular phenotypes, separating the consequences of mutation identity from mutational load.

genomics↗

bioRxiv2026

Respiration-Deficient Cells Require Pyruvate Carboxylase to Suppress Asparagine Auxotrophy

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

cell biology↗

bioRxiv2026

MTERF1 loss buffers against pathogenic mtDNA deletions through transcriptional regulation

Large-scale mitochondrial DNA (mtDNA) deletions cripple oxidative phosphorylation once they exceed a critical heteroplasmy threshold, causing incurable mitochondrial pathologies. Using a genome-wide CRISPR/Cas9 screen in an engineered human cell line carrying a large-scale mtDNA deletion at high heteroplasmy, we identified mitochondrial transcription termination factor 1 (MTERF1) as a suppressor of the heteroplasmy burden. Loss of MTERF1 restored mitochondrial function and increased cellular proliferation in cells with a mtDNA deletion burden exceeding the pathogenic threshold, without altering heteroplasmy or mtDNA copy number. MTERF1 binds wild-type and deletion-bearing mitochondrial genomes indiscriminately at a site downstream of the ribosomal RNA genes and curbs transcription. Relieving this constraint broadly increased OXPHOS transcripts, thereby eliciting more respiratory output from the residual wild-type genomes. Notably, the buffering effect of MTERF1 loss extended beyond mtDNA deletions. In a counter-screen, MTERF1 loss could also restore respiratory growth in cells depleted of nuclear-encoded mitochondrial genes such as OPA1 and COX5A. Together, these findings indicate that by relieving a transcriptional constraint, MTERF1 loss compensates for reduced genome dosage, defining a strategy to enhance residual mitochondrial function in mtDNA deletion disorders and related conditions.

molecular biology↗

bioRxiv2026

POLQ-driven repair scars shape the immunogenic landscape of homologous recombination-deficient pancreatic cancer

Pancreatic cancer (PC) is broadly resistant to immune checkpoint blockade, although a subset of homologous recombination-deficient (HRD) tumors exhibits durable immune engagement. The genomic features that distinguish these immune-responsive tumors from immune-inert HRD tumors remain poorly understood. Here we identify a microhomology-mediated end joining (MMEJ) repair scar, the MMEJ Deletion Footprint (MDF), as a genomic readout of POLQ-associated error-prone repair that enriches for frameshift indels. Across the multi-omic discovery cohort integrating tumor genomics, single-nucleus transcriptomics and spatial immune profiling, MDF-high HRD PC exhibited increased frameshift-indel-derived neoantigens and interferon programs. MDF was further associated with remodeling of the myeloid compartment toward MHC II-high dendritic cell-like antigen-presenting macrophage states and the immune synapse architecture marked by increased spatial interaction between APC-like macrophages and cytotoxic CD8+ T cells. These tissue-level features aligned with a functional trajectory shift of CD8+ T cells, consistent with effective anti-tumor immunity and was associated with favorable clinical outcomes of patients. Together, our findings position MMEJ-linked repair scarring as actionable biology that connects an HRD genotype to immune organization and suggests rational immunotherapy combinations that may enhance antigen presentation and myeloid activation to extend durable benefit in HRD-lineage cancers.

cancer biology↗

bioRxiv2023

RHINO restricts MMEJ activity to mitosis

DNA double-strand breaks (DSBs) are toxic lesions that can lead to genome instability if not properly repaired. Breaks incurred in G1 phase of the cell cycle are predominantly fixed by non-homologous end-joining (NHEJ), while homologous recombination (HR) is the primary repair pathway in S and G2. Microhomology-mediated end-joining (MMEJ) is intrinsically error-prone and considered a backup DSB repair pathway that becomes essential when HR and NHEJ are compromised. In this study, we uncover MMEJ as the major DSB repair pathway in M phase. Using CRISPR/Cas9-based synthetic lethal screens, we identify subunits of the 9-1-1 complex (RAD9A-HUS1-RAD1) and its interacting partner, RHINO, as critical MMEJ factors. Mechanistically, we show that the function of 9-1-1 and RHINO in MMEJ is inconsistent with their well-established role in ATR signaling. Instead, RHINO plays an unexpected and essential role in directing mutagenic repair to M phase by directly binding to Polymerase theta (Pol{theta}) and promoting its recruitment to DSBs in mitosis. In addition, we provide evidence that mitotic MMEJ repairs persistent DNA damage that originates in S phase but is not repaired by HR. The latter findings could explain the synthetic lethal relationship between POLQ and BRCA1/2 and the synergistic effect of Pol{theta} and PARP inhibitors. In summary, our study identifies MMEJ as the primary pathway for repairing DSBs during mitosis and highlights an unanticipated role for RHINO in directing mutagenic repair to M phase.

molecular biology↗

bioRxiv2022

mtDNA breaks compromise mitochondrial membrane ultrastructure and trigger an integrated stress response

Double-strand breaks in mitochondrial DNA (mtDSBs) lead to the degradation of the circular genomes and a reduction in copy number. However, it is unclear how mtDSBs are sensed and what signaling pathways are activated in response to mtDNA damage. In this study, we used mitochondrial-targeted restriction enzymes to investigate the cellular response to mtDSBs. Our results showed that a subset of cells with mtDSBs exhibited defects in mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy revealed compromised mitochondrial membrane and cristae ultrastructure. We also found that mtDSBs activated the integrated stress response (ISR) through the phosphorylation of eIF2 by DELE1 and HRI. Notably, inhibition of the ISR exacerbated the mitochondrial import defect and delayed the recovery of mtDNA copy number following break formation. These findings suggest that the ISR plays a role in mitigating mitochondrial dysfunction following mtDNA damage and is critical to promoting mtDNA repopulation. Last, we used proteomics to survey the proteins present in the nucleoids shortly after mtDSBs and identified ATAD3A, a membrane-anchored protein interacting with nucleoids, as a potential factor in transmitting the signal from damaged genomes to the inner mitochondrial membrane. In summary, our study reveals the sequence of events linking damaged mitochondrial genomes with the cytoplasm and highlights the unexpected role of the ISR in reestablishing homeostasis in response to mitochondrial genome instability.

cell biology↗

bioRxiv2021

RAP1 regulates TIP60 function during fate transition between 2 cell-like and pluripotent states

In mammals, the conserved telomere binding protein RAP1 serves a diverse set of non- telomeric 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 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 2-cell-like cells. Specifically, RAP1 enhances the repressive activity of Tip60/p400 across a subset of 2-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential upregulation of genes proximal to MERVL elements in Rap1 deficient settings implicate these endogenous retroviral elements in the de- repression of proximal genes. Altogether, our study reveals an unprecedented link between RAP1 and TIP60/p400 complex in the regulation of totipotency.

molecular biology↗

bioRxiv2020

Alternative splicing is a developmental switch for hTERT expression

High telomerase activity is restricted to the blastocyst stage of embryonic development when telomere length is reset, and is characteristic of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, the pathways involved in telomerase regulation as a function of pluripotency remain unknown. To explore hTERT transcriptional control, we compare genome-wide interactions (4C-seq) and chromatin accessibility (ATAC-seq) between human ESCs and epithelial cells and identify several putative hTERT cis-regulatory elements. CRISPR/Cas9-mediated deletion of candidate elements in ESCs reduces the levels of hTERT mRNA but does not abolish telomerase expression, thus implicating post-transcriptional processes in telomerase regulation. In agreement with this hypothesis, we find an hTERT splice variant lacking exon-2 and prone to degradation, to be enriched in differentiated cells but absent from ESCs. In addition, we show that forced retention of exon-2 prevents telomerase silencing during differentiation. Lastly, we highlight a role for the splicing co-factor SON in hTERT exon-2 inclusion and identify a SON mutation in a Dyskeratosis congenita patient with short telomeres and decreased telomerase activity. Altogether, our data uncover a novel alternative splice switch that is critical for telomerase activity during development.

molecular biology↗

bioRxiv2020

Single-molecule imaging of telomerase RNA reveals a Recruitment-Retention model for telomere elongation

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatio-temporal coordination of telomerase and telomeres, and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combined CRISPR genome editing and MS2 RNA-tagging to image single-molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, then base-pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life-cycle of telomerase RNA and provides a framework to understand how cancer-associated mutations mechanistically drive defects in telomere homeostasis.

molecular biology↗

bioRxiv2020

Nuclear sensing of mitochondrial DNA breaks enhances immune surveillance

Mitochondrial double-strand breaks (mtDSBs) are toxic lesions that compromise mitochondrial function. Mito-nuclear communication is essential to maintain cellular homeostasis, however, the nuclear response to mtDSBs remains unknown. Using mitochondrial-targeted TALENs, we show that mtDSBs activate a type I interferon response evidenced by phosphorylation of STAT1 and activation of interferon stimulated genes (ISG). Following mtDNA break formation, BAK-BAX mediated herniation releases mitochondrial RNA to the cytoplasm and trigger a RIG-I/MAVS-dependent immune response. In an independent set of experiments, we investigate the role of mtDSBs in interferon signaling due to genotoxic stress. Our data reveal that activation of ISGs is greatly diminished when cells lacking mtDNA are exposed to ionizing radiation. Furthermore, we show that mtDNA breaks synergize with nuclear DNA damage to mount a robust interferon response. In conclusion, cytoplasmic accumulation of mitochondrial RNA is as an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including ones inflicted by genotoxic agents.

molecular biology↗

bioRxiv2020

Telomere relocalization to the nuclear pore complex in response to replication stress

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 investigated the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR-interference and biotin-based proximity labelling, respectively. These screens revealed that replication stress is a major vulnerability in cells expressing mutant POT1 and manifest in increased mitotic DNA synthesis (MiDAS) at telomeres. Our study also unveiled a role for the nuclear pore complex (NPC) in resolving replication defects at telomeres. Depletion of NPC subunits in the context of POT1 dysfunction increased DNA damage signaling and telomere fragility. 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.

molecular biology↗

bioRxiv2017

The helicase domain of Polθ counteracts RPA to promote alt-NHEJ

Mammalian polymerase theta (Pol{theta}; encoded by POLQ) is a unique multifunctional enzyme that promotes error-prone DNA repair by alternative-NHEJ (alt-NHEJ). Here we perform structure-function analyses and report that, in addition to the polymerase domain, the helicase activity plays a central role during Pol{theta}-mediated double-stranded break (DSB) repair. Our results show that Pol{theta}-helicase promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells. In addition, the helicase activity suppresses CRISPR/Cas9 mediated gene targeting by homologous recombination (HR). In vitro experiments reveal that Pol{theta}-helicase displaces RPA to facilitate the annealing of complementary DNA during alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, we show that the inhibition of RPA1 subunit enhances end-joining and suppresses recombination at telomeres. Taken together, our results reveal that the balance between HR and alt-NHEJ is regulated by opposing activities of Pol{theta} and RPA, providing critical insight into the mechanism that control DSB repair pathway choice in mammalian cells.

genetics↗