Biology subjects

Yigit, G.

Publications and source records attributed to Yigit, G..

6 recordsLinked to original sources

bioRxiv2026

Distinct mitochondrial DNA single-nucleotide variant signatures in TOP3A-deficient cardiomyocytes

The human heart has a continuous and exceptionally high demand for energy, which is met primarily through mitochondrial oxidative phosphorylation. This dependence places the maintenance and integrity of mitochondrial DNA (mtDNA) for proper mitochondrial function at the center of cardiac health, as mtDNA instability has been shown to cause mitochondrial dysfunction, which can ultimately impair cardiac function leading to cardiomyopathy (CM) and heart failure. However, the contribution of mtDNA instability to cardiac disease remains poorly understood. A growing number of nuclear-encoded proteins have emerged as essential regulators of mtDNA maintenance, organization and segregation. DNA Topoisomerase 3 (TOP3A) is expressed as two isoforms: one is a nuclear-related isoform involved in nuclear genome maintenance while the other localizes to the mitochondria to preserve mtDNA integrity. Recently, individuals bearing biallelic loss-of-function variants in TOP3A manifested phenotypic traits, including CM, typical for mitochondrial dysfunction, supporting a potential mechanistic link between mitochondrial genome instability and TOP3A-related cardiac disease. Here, we investigated the effects of TOP3A deficiency on mtDNA maintenance and stability in the context of TOP3A-associated CM. We employed isogenic wild-type, TOP3A-knockout and BLM-knockout induced pluripotent stem cells (iPSCs) to generate cardiomyocytes (iPSC-CMs) and established a high-throughput, ultra-deep mtDNA sequencing strategy achieving approximately 500,000X coverage to characterize low-frequency mtDNA mutational patterns. Loss of TOP3A triggered an early burst of low-frequency de novo mtDNA single-nucleotide variants during cardiac differentiation, with a striking enrichment within the mitochondrial ribosomal RNA genes, accompanied by a progressive increase in the heteroplasmy of low-frequency mtDNA variants inherited from the common isogenic background. These unique mtDNA signatures were associated with defective mtDNA copy-number expansion and impaired mitochondrial respiration in mature iPSC-CMs. Together, our approach uncovered a previously uncharacterized consequence of TOP3A deficiency and established a link between impaired mtDNA maintenance and mitochondrial dysfunction in TOP3A-associated CM.

genetics↗

bioRxiv2025

BTRR complex deficiency is a driver for genomic instability in Bloom syndrome

Biallelic loss-of-function (LoF) variants in the BTRR complex members BLM, TOP3A, RMI1, and RMI2 cause Bloom syndrome (BSyn). The BTRR complex mainly acts on DNA replication and DNA repair processes, and dysfunction of this complex underlies the increased genomic instability and cancer predisposition associated with the BSyn phenotype. Here, we report CRISPR/Cas9-based genome-edited isogenic induced pluripotent stem cell (iPSC) models with compound heterozygous LoF variants in BLM, TOP3A, and RMI1. The cellular phenotype of all three BTRR-deficient iPSC lines included chromosome segregation defects, increased sister chromatid exchange rates, and impaired DNA single-strand template repair. Using single-cell whole genome sequencing, we showed that BTRR complex deficiency causes increased genome copy number alterations (CNAs) and, therefore, is a driver for genomic instability. CNA load was further induced by applying replication stress, and we observed that BTRRKO iPSCs acquired fewer de novo CNA events compared to wild-type cells, suggesting a possible selection loss against cells with high levels of DNA damage. Importantly, stress-induced and non-stress-induced CNAs in single-cell genomes were not stochastically distributed throughout the genome, but instead enriched at fragile sites. This finding might offer an opportunity for the development of novel NGS-based approaches to measure rates of genomic instability in disease conditions.

genetics↗

bioRxiv2021

A novel single-cell RNA-sequencing platform and its applicability connecting genotype to phenotype in ageing-disease

Advances in single cell multi-omics analysis such as the ability of a genotype to show phenotypic diversity lead to a comprehensive understanding of cellular events in human diseases. The cardinal feature to access this information is the technology used for single-cell isolation, barcoding, and sequencing. While numerous platforms have been reported for single-cell RNA-sequencing, technical enhancements are needed in terms of highly purified single cell selection, cell documentation or limitations in cell sizing and chemistry. In this study, we present a novel high-throughput platform using a full-length RNA-sequencing approach that offers substantial technical improvements of all parameters. We applied this platform to dermal fibroblasts derived from six patients with different segmental progeria syndromes and defined phenotype-associated pathway signatures and variant-associated expression modifiers. These results validate the applicability of our platform to highlight genotype-expression relationships for molecular phenotyping in ageing disorders.

genomics↗

bioRxiv2021

TRAIP regulates DNA double-strand break-induced ATM activation

DNA double-strand breaks (DSBs) affect cell survival and genomic integrity. They are repaired by a highly coordinated process called the DNA damage response. Here, we report that the ubiquitously expressed nucleolar E3 ubiquitin ligase TRAF-interacting protein (TRAIP), previously shown to regulate the spindle assembly checkpoint, has an essential role during the DNA damage response. A biotinylation proximity screening assay (BioID) identified Ku80, Ku70, SMARCA5 (SNF2H) and DNA-PKcs as novel TRAIP interactors. Co-immunoprecipitations demonstrated that the interaction of TRAIP with Ku80 was transiently increased while the one with SMARCA5 was strongly decreased after treatment of HeLa cells with neocarzinostatin (NCS). Treatment of fibroblasts from a microcephalic primordial dwarfism patient carrying a hypomorphic TRAIP mutation or shRNA-mediated knockdown of TRAIP in HeLa cells with NCS impaired the activation of ataxia-telangiectasia mutated (ATM), a protein kinase crucial for the DNA damage response. As consequence, the maintenance of {gamma}H2AX and Chk2-T68 phosphorylation, two downstream targets of ATM, was significantly abrogated after NCS-inflicted DSBs. DNA repair assays showed that TRAIP inhibits incorrect end utilization during non-homologous end joining. These observations highlight TRAIP as novel regulator of ATM activity in DNA damage signaling.

cell biology↗

bioRxiv2021

Single-cell transcription profiles in Bloom syndrome patients link BLM deficiency with altered condensin complex expression signatures

Bloom syndrome (BS) is an autosomal recessive disease clinically characterized by primary microcephaly, growth deficiency, immunodeficiency, and predisposition to cancer. It is mainly caused by biallelic loss-of-function mutations in the BLM gene, which encodes the BLM helicase, acting in DNA replication and repair processes. Here, we describe the gene expression profiles of three BS fibroblast cell lines harboring causative, biallelic truncating mutations obtained by single-cell (sc) transcriptome analysis. We compared the scRNA transcription profiles from three BS patient cell lines to two age-matched wild-type controls and observed specific deregulation of gene sets related to the molecular processes characteristically affected in BS, such as mitosis, chromosome segregation, cell cycle regulation, and genomic instability. We also found specific upregulation of genes of the Fanconi anemia pathway, in particular FANCM, FANCD2, and FANCI, which encode known interaction partners of BLM. The significant deregulation of genes associated with inherited forms of primary microcephaly observed in our study might explain in part the molecular pathogenesis of microcephaly in BS, one of the main clinical characteristics in patients. Finally, our data provide first evidence of a novel link between BLM dysfunction and transcriptional changes in condensin complex I and II genes. Overall, our study provides novel insights into gene expression profiles in BS on a single-cell level, linking specific genes and pathways to BLM dysfunction.

genetics↗

bioRxiv2020

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers critically implicated in human pathologies, with CHD6 being one of its least studied members. Here, we discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. We show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS-mutation affects CHD6 protein folding and impairs its ability to recruit co-factors in response to DNA damage or autophagy stimulation. This leads to an accumulation of DNA damage burden and to senescence-like phenotypes. By combining genomics and functional assays, we describe for the first time a molecular mechanism for the chromatin control of autophagic flux and genotoxic stress surveillance that applies broadly to human cell types and explains HSS onset.

cell biology↗