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Publication Open Access A proximity mapping journey into the biology of the mammalian centrosome/cilium complex(Multidisciplinary Digital Publishing Institute (MDPI), 2020) Department of Molecular Biology and Genetics; Arslanhan, Melis Dilara; Gülensoy, Dila; Karalar, Elif Nur Fırat; Faculty Member; Department of Molecular Biology and Genetics; Graduate School of Sciences and Engineering; College of Sciences; N/A; N/A; 206349The mammalian centrosome/cilium complex is composed of the centrosome, the primary cilium and the centriolar satellites, which together regulate cell polarity, signaling, proliferation and motility in cells and thereby development and homeostasis in organisms. Accordingly, deregulation of its structure and functions is implicated in various human diseases including cancer, developmental disorders and neurodegenerative diseases. To better understand these disease connections, the molecular underpinnings of the assembly, maintenance and dynamic adaptations of the centrosome/cilium complex need to be uncovered with exquisite detail. Application of proximity-based labeling methods to the centrosome/cilium complex generated spatial and temporal interaction maps for its components and provided key insights into these questions. In this review, we first describe the structure and cell cycle-linked regulation of the centrosome/cilium complex. Next, we explain the inherent biochemical and temporal limitations in probing the structure and function of the centrosome/cilium complex and describe how proximity-based labeling approaches have addressed them. Finally, we explore current insights into the knowledge we gained from the proximity mapping studies as it pertains to centrosome and cilium biogenesis and systematic characterization of the centrosome, cilium and centriolar satellite interactomes.Publication Open Access Aurora kinase A proximity map reveals centriolar satellites as regulators of its ciliary function(Wiley, 2021) Rauniyar, N.; Yates, J. R. III; Department of Molecular Biology and Genetics; Karalar, Elif Nur Fırat; Arslanhan, Melis Dilara; Faculty Member; Department of Molecular Biology and Genetics; College of Sciences; Graduate School of Sciences and Engineering; 206349; N/AAurora kinase A (AURKA) is a conserved kinase that plays crucial roles in numerous cellular processes. Although AURKA overexpression is frequent in human cancers, its pleiotropic functions and multifaceted regulation present challenges in its therapeutic targeting. Key to overcoming these challenges is to identify and characterize the full range of AURKA interactors, which are often weak and transient. Previous proteomic studies were limited in monitoring dynamic and non-mitotic AURKA interactions. Here, we generate the proximity interactome of AURKA in asynchronous cells, which consists of 440 proteins involving multiple biological processes and cellular compartments. Importantly, AURKA has extensive proximate and physical interactions to centriolar satellites, key regulators of the primary cilium. Loss-of-function experiments identify satellites as negative regulators of AURKA activity, abundance, and localization in quiescent cells. Notably, loss of satellites activates AURKA at the basal body, decreases centrosomal IFT88 levels, and causes ciliogenesis defects. Collectively, our results provide a resource for dissecting spatiotemporal regulation of AURKA and uncover its proteostatic regulation by satellites as a new mechanism for its ciliary functions.Publication Open Access BRD9-containing non-canonical BAF complex maintains somatic cell transcriptome and acts as a barrier to human reprogramming(Elsevier, 2022) Philpott, M.; Cribbs, A.P.; Dunford, J.E.; Sigua, L.H.; Qi, J.; Oppermann, U.; Department of Molecular Biology and Genetics; N/A; Sevinç, Kenan; Cavga, Ayşe Derya; Kelekçi, Simge; Can, Hazal; Yıldız, Abdullah Burak; Yılmaz, Alperen; Ayar, Enes Sefa; Ata, Deniz; Önder, Tamer Tevfik; Faculty Member; Department of Molecular Biology and Genetics; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); School of Medicine; Graduate School of Sciences and Engineering; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; 42946Epigenetic reprogramming to pluripotency requires extensive remodeling of chromatin landscapes to silence existing cell-type-specific genes and activate pluripotency genes. ATP-dependent chromatin remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of recently identified Bromodomain-containing protein 9 (BRD9) and the associated non-canonical BRG1-associated factors (ncBAF) complex in reprogramming remains unknown. Here, we show that genetic or chemical inhibition of BRD9, as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase human somatic cell reprogramming efficiency and can replace KLF4 and c-MYC. We find that BRD9 is dispensable for human induced pluripotent stem cells under primed but not under naive conditions. Mechanistically, BRD9 inhibition downregulates fibroblast-related genes and decreases chromatin accessibility at somatic enhancers. BRD9 maintains the expression of transcriptional regulators MN1 and ZBTB38, both of which impede reprogramming. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.Publication Open Access CCDC57 cooperates with microtubules and microcephaly protein CEP63 and regulates centriole duplication and mitotic progression(Elsevier, 2020) Lince Faria, Mariana; Department of Molecular Biology and Genetics; Gürkaşlar, Hazal Kübra; Culfa, Efraim; Arslanhan, Melis Dilara; Karalar, Elif Nur Fırat; Master Student; Department of Molecular Biology and Genetics; Graduate School of Sciences and Engineering; College of Sciences; N/A; N/A; N/A; 206349Centrosomes function in key cellular processes ranging from cell division to cellular signaling. Their dysfunction is linked to cancer and developmental disorders. Here, we identify CCDC57 as a pleiotropic regulator of centriole duplication, mitosis, and ciliogenesis. Combining proximity mapping with superresolution imaging, we show that CCDC57 localizes to the proximal end of centrioles and interacts with the microcephaly protein CEP63, centriolar satellite proteins, and microtubules. Loss of CCDC57 causes defects in centriole duplication and results in a failure to localize CEP63 and CEP152 to the centrosome. Additionally, CCDC57 depletion perturbs mitotic progression both in wild-type and centriole-less cells. Importantly, its centrosome-targeting region is required for its interaction with CEP63 and functions during centriole duplication and cilium assembly, whereas the microtubule-targeting region is required for its mitotic functions. Together, our results identify CCDC57 as a critical interface between centrosome and microtubule-mediated cellular processes that are deregulated in microcephaly.Publication Open Access Centriolar satellites are required for efficient ciliogenesis and ciliary content regulation(Wiley, 2019) Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Odabaşı, Ezgi; Karalar, Elif Nur Fırat; Gül, Şeref; Kavaklı, İbrahim Halil; Other; Researcher; Faculty Member; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Graduate School of Sciences and Engineering; N/A; 206349; N/A; 40319Centriolar satellites are ubiquitous in vertebrate cells. They have recently emerged as key regulators of centrosome/cilium biogenesis, and their mutations are linked to ciliopathies. However, their precise functions and mechanisms of action remain poorly understood. Here, we generated a kidney epithelial cell line (IMCD3) lacking satellites by CRISPR/Cas9-mediated PCM1 deletion and investigated the cellular and molecular consequences of satellite loss. Cells lacking satellites still formed full-length cilia but at significantly lower numbers, with changes in the centrosomal and cellular levels of key ciliogenesis factors. Using these cells, we identified new ciliary functions of satellites such as regulation of ciliary content, Hedgehog signaling, and epithelial cell organization in three-dimensional cultures. However, other functions of satellites, namely proliferation, cell cycle progression, and centriole duplication, were unaffected in these cells. Quantitative transcriptomic and proteomic profiling revealed that loss of satellites affects transcription scarcely, but significantly alters the proteome. Importantly, the centrosome proteome mostly remains unaltered in the cells lacking satellites. Together, our findings identify centriolar satellites as regulators of efficient cilium assembly and function and provide insight into disease mechanisms of ciliopathies.Publication Open Access Generation of integration-free induced pluripotent stem cells from a patient with Familial Mediterranean Fever (FMF)(Elsevier, 2015) Gül, Ahmet; Department of Molecular Biology and Genetics; Fidan, Kerem; Kavaklıoğlu, Gülnihal; Ebrahimi, Ayyub A.; Özlü, Can; Ay, Nur Zeynep; Ruacan, Ayşe Arzu; Önder, Tamer Tevfik; Master Student; Faculty Member; Faculty Member; Department of Molecular Biology and Genetics; School of Medicine; Graduate School of Sciences and Engineering; N/A; N/A; N/A; N/A; N/A; 38250; 42946Fibroblasts from a Familial Mediterranean Fever (FMF) patient were reprogrammed with episomal vectors by using the Neon Transfection System for the generation of integration-free induced pluripotent stem cells (iPSCs). The resulting iPSC line was characterized to determine the expression of pluripotency markers, proper differentiation into three germ layers, the presence of normal chromosomal structures as well as the lack of genomic integration. A homozygous missense mutation in the MEFV gene (p.Met694Val), which lead to typical FMF phenotype, was shown to be present in the generated iPSC line.Publication Open Access Human genetics and neuropathology suggest a link between miR-218 and amyotrophic lateral sclerosis pathophysiology(American Association for the Advancement of Science (AAAS), 2019) Reichenstein,I.; Eitan, C.; Diaz-Garcia, S.; Haim, G.; Magen, I.; Siany, A.; Hoye, M.L.; Rivkin, N.; Olender, T.; Toth, B.; Ravid, R.; Mandelbaum, A.D.; Yanowski, E.; Liang, J.; Rymer, J.K.; Levy, R.; Beck, G.; Ainbinder, E.; Farhan,S.M.K.; Lennox, K.A.; Bode, N.M.; Behlke, M.A.; Möller, T.; Saxena, S.; Moreno, C.A.M.; Costaguta, G.; van Eijk, K.R.; Phatnani, H.; Al-Chalabi, A.; van den Berg, L.H.; Hardiman, O.; Landers, J.E.; Mora, J.S.; Morrison, K.E.; Shaw, P.J.; Veldink, J.H.; Pfaff S.L.; Yizhar, O.; Gross, C.; Brown, R.H. Jr.; Ravits, J.M.; Harms, M.B.; Miller, T.M.; Hornstein, E.; Başak, Ayşe Nazlı; Faculty Member; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); 1512Motor neuron–specific microRNA-218 (miR-218) has recently received attention because of its roles in mouse development. However, miR-218 relevance to human motor neuron disease was not yet explored. Here, we demonstrate by neuropathology that miR-218 is abundant in healthy human motor neurons. However, in amyotrophic lateral sclerosis (ALS) motor neurons, miR-218 is down-regulated and its mRNA targets are reciprocally up-regulated (derepressed). We further identify the potassium channel Kv10.1 as a new miR-218 direct target that controls neuronal activity. In addition, we screened thousands of ALS genomes and identified six rare variants in the human miR-218-2 sequence. miR-218 gene variants fail to regulate neuron activity, suggesting the importance of this small endogenous RNA for neuronal robustness. The underlying mechanisms involve inhibition of miR-218 biogenesis and reduced processing by DICER. Therefore, miR-218 activity in motor neurons may be susceptible to failure in human ALS, suggesting that miR-218 may be a potential therapeutic target in motor neuron disease.Publication Open Access Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells(Elsevier, 2022) Planes, R.; Pinilla, M.; Santoni, K.; Hessel, A.; Passemar, C.; Lay, K.; Paillette, P.; Valadao, A.C.; Robinson, K.S.; Bastard, P.; Lam, N.; Fadrique, R.; Rossi, I.; Pericat, D.; Bagayoko, S.; Leon-Icaza, S.A.; Rombouts, Y.; Perouzel, E.; Tiraby, M.; COVID Human Genetic Effort; Zhang, Q.; Cicuta, P.; Jouanguy, E.; Neyrolles, O.; Bryant, C.E.; Floto, A.R.; Goujon, C.; Lei, F.Z.; Martin-Blondel, G.; Silva, S.; Casanova, J.L.; Cougoule, C.; Marcoux, J.; Ravet, E.; Meunier, E.; Reversade, Bruno; Faculty Member; School of MedicineInflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 in-flammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.Publication Open Access Hypoxia-induced endoplasmic reticulum stress characterizes a necrotic phenotype of pancreatic cancer(Impact Journals, 2015) Kong, Bo; Cheng, Tao; Wu, Weiwei; Regel, Ivonne; Raulefs, Susanne; Friess, Helmut; Esposito, Irene; Kleeff, Joerg; Michalski, Christoph W.; N/A; Erkan, Murat Mert; Faculty Member; School of Medicine; 214689Stromal fibrosis and tissue necrosis are major histological sequelae of hypoxia. The hypoxia-to-fibrosis sequence is well-documented in pancreatic ductal adenocarcinoma (PDAC). However, hypoxic and necrotic PDAC phenotypes are insufficiently characterized. Recently, reduction of tuberous sclerosis expression in mice together with oncogenic Kras demonstrated a rapidly metastasizing phenotype with histologically eccentric necrosis, transitional hypoxia and devascularisation. We established cell lines from these tumors and transplanted them orthotopically into wild-type mice to test their abilities to recapitulate the histological features of the primary lesions. Notably, the necrotic phenotype was reproduced by only a subset of cell lines while others gave rise to dedifferentiated tumors with significantly reduced necrosis. In vitro analysis of the necrotic tumor-inducing cell lines revealed that these cells released a significant amount of vascular endothelial growth factor A (Vegfa). However, its release was not further increased under hypoxic conditions. Defective hypoxia-induced Vegfa secretion was not due to impaired Vegfa transcription or hypoxia-inducible factor 1-alpha activation, but rather a result of hypoxia-induced endoplasmic reticulum (ER) stress. We thus identified hypoxia-induced ER stress as an important pathway in PDACs with tissue necrosis and rapid metastasis.Publication Open Access Kdm2b, an h3k36-specific demethylase, regulates apoptotic response of gbm cells to trail(Nature Publishing Group (NPG), 2017) Gumus, Zeynep H.; Kurt, İbrahim Çağrı; Sur, İlknur Erdem; Kaya, Ezgi; Cingöz, Ahmet; Kazancıoğlu, Selena; Kahya, Zeynep; Toparlak, Ömer Duhan; Şenbabaoğlu, Filiz; Kaya, Zeynep; Özyerli, Ezgi; Karahüseyinoğlu, Serçin; Lack, Nathan Alan; Önder, Tamer Tevfik; Önder, Tuğba Bağcı; PhD Student; Undergraduate Student; Other; PhD Student; Faculty Member; Faculty Member; Faculty Member; Graduate School of Health Sciences; School of Medicine; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; 110772; 120842; 42946; 184359Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.