DOCSLIB.ORG

AKT3-Mediated IWS1 Phosphorylation Promotes the Proliferation of EGFR-Mutant Lung Adenocarcinomas Through Cell Cycle-Regulated U2AF2 RNA Splicing ✉ Georgios I

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
AKT3-Mediated IWS1 Phosphorylation Promotes the Proliferation of EGFR-Mutant Lung Adenocarcinomas Through Cell Cycle-Regulated U2AF2 RNA Splicing ✉ Georgios I ARTICLE https://doi.org/10.1038/s41467-021-24795-1 OPEN AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing ✉ Georgios I. Laliotis 1,2,3,11 , Evangelia Chavdoula1,2, Maria D. Paraskevopoulou4, Abdul Kaba1,2, Alessandro La Ferlita1,2,5, Satishkumar Singh2,6, Vollter Anastas 1,2,7, Keith A. Nair II 1,2, Arturo Orlacchio1,2, Vasiliki Taraslia4,12, Ioannis Vlachos8,13, Marina Capece1,2, Artemis Hatzigeorgiou8, Dario Palmieri1,2, 1234567890():,; Christos Tsatsanis3,9, Salvatore Alaimo 5, Lalit Sehgal 2,6, David P. Carbone 2,10, Vincenzo Coppola 1,2 & ✉ Philip N. Tsichlis 1,2,7 AKT-phosphorylated IWS1 regulates alternative RNA splicing via a pathway that is active in lung cancer. RNA-seq studies in lung adenocarcinoma cells lacking phosphorylated IWS1, identified a exon 2-deficient U2AF2 splice variant. Here, we show that exon 2 inclusion in the U2AF2 mRNA is a cell cycle-dependent process that is regulated by LEDGF/SRSF1 splicing complexes, whose assembly is controlled by the IWS1 phosphorylation-dependent deposition of histone H3K36me3 marks in the body of target genes. The exon 2-deficient U2AF2 mRNA encodes a Serine-Arginine-Rich (RS) domain-deficient U2AF65, which is defective in CDCA5 pre-mRNA processing. This results in downregulation of the CDCA5-encoded protein Sororin, a phosphorylation target and regulator of ERK, G2/M arrest and impaired cell proliferation and tumor growth. Analysis of human lung adenocarcinomas, confirmed activation of the pathway in EGFR-mutant tumors and showed that pathway activity correlates with tumor stage, histologic grade, metastasis, relapse after treatment, and poor prognosis. 1 Department of Cancer Biology and Genetics, The Ohio State University, Columbus, OH, USA. 2 The Ohio State University Comprehensive Cancer Center- Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Columbus, OH, USA. 3 School of Medicine, University of Crete, Heraklion, Crete, Greece. 4 Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA, USA. 5 Department of Clinical and Experimental Medicine, Bioinformatics Unit, University of Catania, Catania, Italy. 6 Department of Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA. 7 Tufts Graduate School of Biomedical Sciences, Program in Genetics, Boston, MA, USA. 8 DIANA-Lab, Hellenic Pasteur Institute, Athens, Greece. 9 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Crete, Greece. 10 Department of Internal Medicine, Division of Medical Oncology, The Ohio State University Medical Center, Columbus, OH, USA. 11Present address: Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA. 12Present address: Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece. 13Present address: Department Of Pathology, Beth Israel-Deaconess Medical Center, Harvard ✉ Medical School, Boston, MA, USA. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:4624 | https://doi.org/10.1038/s41467-021-24795-1 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24795-1 he higher complexity of the proteome relative to the gen- genome sequencing of 183 lung adenocarcinomas in one study, ome is due to multiple factors, one of which is RNA identified somatic mutations in the splicing factors U2AF1 and T 1 splicing . At least 97% of the genes in the human genome RBM10 and in several epigenetic factors, which may also regulate have introns2 and more than 95% of them undergo alternative RNA splicing26. Such mutations may render the cancer cells RNA splicing3. RNA splicing therefore plays a critical role in vulnerable to modulators of the core splicing machinery, as determining the biological phenotype4. Several additional obser- suggested by experiments showing that H3B-8800, a recently vations also support the importance of alternative RNA splicing described modulator of SF3B1, preferentially kills cancer cells in biology. First, the pattern of alternative RNA splicing changes with mutations in spliceosomal components. Mechanistically, during differentiation and many of these changes contribute to H3B-8800 may function by promoting the retention of short GC- the differentiation process and/or the phenotype of the differ- rich introns, in the mRNA of mutant cells27. entiated cells5. In addition, cell survival and proliferation signals Activating mutations in epidermal growth factor receptor may function by regulating alternative RNA splicing6. Finally, (EGFR) and Kirsten rat sarcoma (KRAS) are the most common changes in alternative RNA splicing caused by mutations in cis- genetic alterations in NSCLC with 69% of the tumors harboring acting regulatory elements, such as splicing enhancers or silen- mutations in these genes. Both types of mutations activate mul- cers, and changes in the expression and/or post-translational tiple signaling pathways, including the AKT and the ERK path- modification of RNA splicing regulators have been linked to the ways and both carry poor prognosis. Data in this report delineate pathogenesis of several diseases, including cancer7–9. a pathway, which regulates alternative RNA splicing in lung Two complementary molecular mechanisms may contribute to adenocarcinomas, and they show that the influence of the path- alternative RNA splicing, the rate of transcription and chromatin way on the biology of tumors harboring EGFR mutations is more modifications in the body of transcribed genes. Splicing occurs robust, than its influence on the biology of tumors harboring cotranscriptionally and when the rate of transcription is high, it KRAS mutations. increases the probability for exons that are not efficiently spliced, We had previously shown that the transcription elongation to be spliced out of the mature transcript10. Also, chromatin factors IWS1 and AKT play a central role in the regulation of the modifications are recognized by readers of epigenetic marks, alternative splicing of FGFR2, by promoting the skipping of exon which form the nucleus for the assembly of molecular complexes 8 from the mature FGFR2 mRNA transcript. The exclusion of the that bind to, and functionally regulate RNA-associated enhancers FGFR2 exon 8 depends on the phosphorylation of IWS1 by AKT or silencers of splicing, perhaps by altering the rate of assembly (primarily AKT3) on Ser720/Thr721, which recruits SETD2 to an and the composition of spliceosomal complexes11–13. IWS1-containing complex in the C-terminal domain (CTD) of One cellular function, which interfaces with the RNA splicing RNA polymerase II. This results in the trimethylation of histone machinery, is the cell cycle14. The connection between RNA H3 at K36 in the body of the transcribed FGFR2 gene, which splicing and the cell cycle was originally suggested by experiments triggers the skipping of exon 8 from the mature transcript. As a in the yeast Saccharomyces cerevisiae, which showed that the follow-up to this study, we proceeded to address the global effects impact of mutations in the cell cycle gene cdc5, can be suppressed of IWS1 and IWS1 phosphorylation in lung adenocarcinomas. To by removing the intron of the tubulin-encoding gene TUB115. this end, we carried out an RNA-seq experiment in the lung Progression through the cell cycle depends on periodic changes of adenocarcinoma cell line NCI-H522, in which IWS1 was either gene function, which can be achieved by multiple mechanisms, knocked down or replaced by its phosphorylation site mutant one of which is the periodic modulation of RNA splicing and S720A/T721A. The results of this analysis revealed that exon spliceosomal components14. Importantly, some of the changes in inclusion was significantly more common than the exon skipping RNA splicing are regulated by periodic shifts in the expression we observed with the FGFR2 gene. One of the genes undergoing and/or the activity of known cell cycle regulators, such as the exon inclusion was the U2AF2 gene, which encodes the core Aurora kinases16, the large RS domain-containing protein SON17, splicing factor U2AF6528–30. Exon inclusion was also under the and the RNA-recognition motif (RRM)-containing proteins control of SETD2 and histone H3K36 trimethylation. However, TgRRM118 and CLK114. Despite the fact that a connection the reader of the histone H3K36me3 mark was the p52 isoform of between RNA splicing and the regulation of the cell cycle has LEDGF12, which interacts with the RNA-binding protein already been established, our understanding of the mechanisms SRSF131. Therefore, although the chromatin-modification mark and the consequences of RNA splicing during cell cycle pro- promoting exon inclusion is the same as the mark promoting gression, remains rudimentary. Exploring the links between RNA exon exclusion, the effector complexes assembled on H3K36me3 splicing and the cell cycle is likely to yield important information, in the two cases are different. The alternatively spliced exon 2 with a significant impact in our understanding of human disease, encodes the U2AF65 N-terminal serine–arginine-rich domain especially cancer. (RS domain), which is required for the interaction between Shifts in RNA splicing play an important role in many types of U2AF65 and the splicing cofactor Prp1932–35. The binding of human cancer, including non-small-cell lung cancer
Load more

Recommended publications
  • Regulation of SETD2 Stability Is Important for the Fidelity Of
    Bhattacharya and Workman Epigenetics & Chromatin (2020) 13:40 Epigenetics & Chromatin https://doi.org/10.1186/s13072-020-00362-8 RESEARCH Open Access Regulation of SETD2 stability is important for the fdelity of H3K36me3 deposition Saikat Bhattacharya and Jerry L. Workman* Abstract Background: The histone H3K36me3 mark regulates transcription elongation, pre-mRNA splicing, DNA methylation, and DNA damage repair. However, knowledge of the regulation of the enzyme SETD2, which deposits this function- ally important mark, is very limited. Results: Here, we show that the poorly characterized N-terminal region of SETD2 plays a determining role in regulat- ing the stability of SETD2. This stretch of 1–1403 amino acids contributes to the robust degradation of SETD2 by the proteasome. Besides, the SETD2 protein is aggregate prone and forms insoluble bodies in nuclei especially upon proteasome inhibition. Removal of the N-terminal segment results in the stabilization of SETD2 and leads to a marked increase in global H3K36me3 which, uncharacteristically, happens in a Pol II-independent manner. Conclusion: The functionally uncharacterized N-terminal segment of SETD2 regulates its half-life to maintain the requisite cellular amount of the protein. The absence of SETD2 proteolysis results in a Pol II-independent H3K36me3 deposition and protein aggregation. Keywords: Chromatin, Proteasome, Histone, Methylation, Aggregation Background In yeast, the SET domain-containing protein Set2 Te N-terminal tails of histones protrude from the nucle- (ySet2) is the sole H3K36 methyltransferase [10]. ySet2 osome and are hotspots for the occurrence of a variety interacts with the large subunit of the RNA polymerase II, of post-translational modifcations (PTMs) that play key Rpb1, through its SRI (Set2–Rpb1 Interaction) domain, roles in regulating epigenetic processes.
    [Show full text]
  • Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model
    Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 T + is online at: average * The Journal of Immunology , 34 of which you can access for free at: 2016; 197:1477-1488; Prepublished online 1 July from submission to initial decision 4 weeks from acceptance to publication 2016; doi: 10.4049/jimmunol.1600589 http://www.jimmunol.org/content/197/4/1477 Molecular Profile of Tumor-Specific CD8 Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A. Waugh, Sonia M. Leach, Brandon L. Moore, Tullia C. Bruno, Jonathan D. Buhrman and Jill E. Slansky J Immunol cites 95 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2016/07/01/jimmunol.160058 9.DCSupplemental This article http://www.jimmunol.org/content/197/4/1477.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A.
    [Show full text]
  • Identification of Novel Biomarkers and Candidate Small Molecule Drugs in Non-Small-Cell Lung Cancer by Integrated Microarray Analysis
    OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Identification of novel biomarkers and candidate small molecule drugs in non-small-cell lung cancer by integrated microarray analysis This article was published in the following Dove Press journal: OncoTargets and Therapy Qiong Wu1,2,* Background: Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer Bo Zhang1,2,* morbidity and mortality worldwide. In the present study, we identified novel biomarkers Yidan Sun3 associated with the pathogenesis of NSCLC aiming to provide new diagnostic and therapeu- Ran Xu1 tic approaches for NSCLC. Xinyi Hu4 Methods: The microarray datasets of GSE18842, GSE30219, GSE31210, GSE32863 and Shiqi Ren4 GSE40791 from Gene Expression Omnibus database were downloaded. The differential Qianqian Ma5 expressed genes (DEGs) between NSCLC and normal samples were identified by limma Chen Chen6 package. The construction of protein–protein interaction (PPI) network, module analysis and Jian Shu7 enrichment analysis were performed using bioinformatics tools. The expression and prog- Fuwei Qi7 nostic values of hub genes were validated by GEPIA database and real-time quantitative fi Ting He7 PCR. Based on these DEGs, the candidate small molecules for NSCLC were identi ed by the CMap database. Wei Wang2 Results: A total of 408 overlapping DEGs including 109 up-regulated and 296 down- Ziheng Wang2 regulated genes were identified; 300 nodes and 1283 interactions were obtained from the 1 Medical School of Nantong University, PPI network. The most significant biological process and pathway enrichment of DEGs were Nantong 226001, People’s Republic of China; 2The Hand Surgery Research Center, response to wounding and cell adhesion molecules, respectively.
    [Show full text]
  • Multiple Cellular Proteins Interact with LEDGF/P75 Through a Conserved Unstructured Consensus Motif
    ARTICLE Received 19 Jan 2015 | Accepted 1 Jul 2015 | Published 6 Aug 2015 DOI: 10.1038/ncomms8968 Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif Petr Tesina1,2,3,*, Katerˇina Cˇerma´kova´4,*, Magdalena Horˇejsˇ´ı3, Katerˇina Procha´zkova´1, Milan Fa´bry3, Subhalakshmi Sharma4, Frauke Christ4, Jonas Demeulemeester4, Zeger Debyser4, Jan De Rijck4,**, Va´clav Veverka1,** & Pavlı´na Rˇeza´cˇova´1,3,** Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors. 1 Institute of Organic Chemistry and Biochemistry of the ASCR, v.v.i., Flemingovo nam. 2, 166 10 Prague, Czech Republic. 2 Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.
    [Show full text]
  • Recognition of Cancer Mutations in Histone H3K36 by Epigenetic Writers and Readers Brianna J
    EPIGENETICS https://doi.org/10.1080/15592294.2018.1503491 REVIEW Recognition of cancer mutations in histone H3K36 by epigenetic writers and readers Brianna J. Kleina, Krzysztof Krajewski b, Susana Restrepoa, Peter W. Lewis c, Brian D. Strahlb, and Tatiana G. Kutateladzea aDepartment of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA; bDepartment of Biochemistry & Biophysics, The University of North Carolina School of Medicine, Chapel Hill, NC, USA; cWisconsin Institute for Discovery, University of Wisconsin, Madison, WI, USA ABSTRACT ARTICLE HISTORY Histone posttranslational modifications control the organization and function of chromatin. In Received 30 May 2018 particular, methylation of lysine 36 in histone H3 (H3K36me) has been shown to mediate gene Revised 1 July 2018 transcription, DNA repair, cell cycle regulation, and pre-mRNA splicing. Notably, mutations at or Accepted 12 July 2018 near this residue have been causally linked to the development of several human cancers. These KEYWORDS observations have helped to illuminate the role of histones themselves in disease and to clarify Histone; H3K36M; cancer; the mechanisms by which they acquire oncogenic properties. This perspective focuses on recent PTM; methylation advances in discovery and characterization of histone H3 mutations that impact H3K36 methyla- tion. We also highlight findings that the common cancer-related substitution of H3K36 to methionine (H3K36M) disturbs functions of not only H3K36me-writing enzymes but also H3K36me-specific readers. The latter case suggests that the oncogenic effects could also be linked to the inability of readers to engage H3K36M. Introduction from yeast to humans and has been shown to have a variety of functions that range from the control Histone proteins are main components of the of gene transcription and DNA repair, to cell cycle nucleosome, the fundamental building block of regulation and nutrient stress response [8].
    [Show full text]
  • Loss of Function SETD2 Mutations in Poorly Differentiated Metastases
    cancers Article Loss of Function SETD2 Mutations in Poorly Differentiated Metastases from Two Hürthle Cell Carcinomas of the Thyroid 1, 1, 1 2 1 Valeria Pecce y , Antonella Verrienti y, Luana Abballe , Raffaella Carletti , Giorgio Grani , Rosa Falcone 1, Valeria Ramundo 1, Cosimo Durante 1, Cira Di Gioia 2 , Diego Russo 3 , Sebastiano Filetti 1 and Marialuisa Sponziello 1,* 1 Department of Translational and Precision Medicine, “Sapienza” University of Rome, 00161 Rome, Italy; [email protected] (V.P.); [email protected] (A.V.); [email protected] (L.A.); [email protected] (G.G.); [email protected] (R.F.); [email protected] (V.R.); [email protected] (C.D.); sebastiano.fi[email protected] (S.F.) 2 Department of Radiological, Oncological and Pathological Sciences, “Sapienza” University of Rome, 00161 Rome, Italy; raff[email protected] (R.C.); [email protected] (C.D.G.) 3 Department of Health Sciences, “Magna Graecia” University of Catanzaro, 88100 Catanzaro, Italy; [email protected] * Correspondence: [email protected] These authors contributed equally to this work. y Received: 23 June 2020; Accepted: 11 July 2020; Published: 14 July 2020 Abstract: Hürthle cell carcinomas (HCC) are rare differentiated thyroid cancers that display low avidity for radioactive iodine and respond poorly to kinase inhibitors. Here, using next-generation sequencing, we analyzed the mutational status of primary tissue and poorly differentiated metastatic tissue from two HCC patients. In both cases, metastatic tissues harbored a mutation of SETD2, each resulting in loss of the SRI and WW domains of SETD2, a methyltransferase that trimethylates H3K36 (H3K36me3) and also interacts with p53 to promote its stability.
    [Show full text]
  • SETD2 Mutations in Primary Central Nervous System Tumors Angela N
    Viaene et al. Acta Neuropathologica Communications (2018) 6:123 https://doi.org/10.1186/s40478-018-0623-0 RESEARCH Open Access SETD2 mutations in primary central nervous system tumors Angela N. Viaene1, Mariarita Santi1, Jason Rosenbaum2, Marilyn M. Li1, Lea F. Surrey1 and MacLean P. Nasrallah2,3* Abstract Mutations in SETD2 are found in many tumors, including central nervous system (CNS) tumors. Previous work has shown these mutations occur specifically in high grade gliomas of the cerebral hemispheres in pediatric and young adult patients. We investigated SETD2 mutations in a cohort of approximately 640 CNS tumors via next generation sequencing; 23 mutations were detected across 19 primary CNS tumors. Mutations were found in a wide variety of tumors and locations at a broad range of allele frequencies. SETD2 mutations were seen in both low and high grade gliomas as well as non-glial tumors, and occurred in patients greater than 55 years of age, in addition to pediatric and young adult patients. High grade gliomas at first occurrence demonstrated either frameshift/truncating mutations or point mutations at high allele frequencies, whereas recurrent high grade gliomas frequently harbored subclones with point mutations in SETD2 at lower allele frequencies in the setting of higher mutational burdens. Comparison with the TCGA dataset demonstrated consistent findings. Finally, immunohistochemistry showed decreased staining for H3K36me3 in our cohort of SETD2 mutant tumors compared to wildtype controls. Our data further describe the spectrum of tumors in which SETD2 mutations are found and provide a context for interpretation of these mutations in the clinical setting. Keywords: SETD2, Histone, Brain tumor, Glioma, Epigenetics, H3K36me3 Introduction (H3K36me3) in humans.
    [Show full text]
  • Rabbit Anti-CDCA5/FITC Conjugated Antibody
    SunLong Biotech Co.,LTD Tel: 0086-571- 56623320 Fax:0086-571- 56623318 E-mail:[email protected] www.sunlongbiotech.com Rabbit Anti-CDCA5/FITC Conjugated antibody SL7717R-FITC Product Name: Anti-CDCA5/FITC Chinese Name: FITC标记的细胞分裂周期相关蛋白5抗体 Alias: Cell division cycle associated protein 5; MGC16386; p35; Sororin; CDCA5_HUMAN. Organism Species: Rabbit Clonality: Polyclonal React Species: Human,Mouse,Rat,Dog,Pig,Cow,Horse,Rabbit, Flow-Cyt=1:50-200IF=1:50-200 Applications: not yet tested in other applications. optimal dilutions/concentrations should be determined by the end user. Molecular weight: 28kDa Form: Lyophilized or Liquid Concentration: 1mg/ml immunogen: KLH conjugated synthetic peptide derived from human CDCA5 Lsotype: IgG Purification: affinity purified by Protein A Storage Buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibodywww.sunlongbiotech.com is stable at room temperature for at least one month and for greater than a year Storage: when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. background: Sororin, also designated cell division cycle-associated protein 5 (CDCA5) or p35, functions as a regulator of sister chromatid cohesion during mitosis. It interacts with the APC/C complex and is found in a complex consisting of cohesion components SCC- 112, MC1L1, SMC3L1, RAD21 and APRIN. The deduced human and mouse Sororin Product Detail: proteins consist of 252 and 264 amino acid residues, respectively, and both contain a KEN box for APC-dependent ubiquitination.
    [Show full text]
  • Investigation of the Underlying Hub Genes and Molexular Pathogensis in Gastric Cancer by Integrated Bioinformatic Analyses
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423656; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Investigation of the underlying hub genes and molexular pathogensis in gastric cancer by integrated bioinformatic analyses Basavaraj Vastrad1, Chanabasayya Vastrad*2 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423656; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract The high mortality rate of gastric cancer (GC) is in part due to the absence of initial disclosure of its biomarkers. The recognition of important genes associated in GC is therefore recommended to advance clinical prognosis, diagnosis and and treatment outcomes. The current investigation used the microarray dataset GSE113255 RNA seq data from the Gene Expression Omnibus database to diagnose differentially expressed genes (DEGs). Pathway and gene ontology enrichment analyses were performed, and a proteinprotein interaction network, modules, target genes - miRNA regulatory network and target genes - TF regulatory network were constructed and analyzed. Finally, validation of hub genes was performed. The 1008 DEGs identified consisted of 505 up regulated genes and 503 down regulated genes.
    [Show full text]
  • Supplementary Data
    SUPPLEMENTARY DATA A cyclin D1-dependent transcriptional program predicts clinical outcome in mantle cell lymphoma Santiago Demajo et al. 1 SUPPLEMENTARY DATA INDEX Supplementary Methods p. 3 Supplementary References p. 8 Supplementary Tables (S1 to S5) p. 9 Supplementary Figures (S1 to S15) p. 17 2 SUPPLEMENTARY METHODS Western blot, immunoprecipitation, and qRT-PCR Western blot (WB) analysis was performed as previously described (1), using cyclin D1 (Santa Cruz Biotechnology, sc-753, RRID:AB_2070433) and tubulin (Sigma-Aldrich, T5168, RRID:AB_477579) antibodies. Co-immunoprecipitation assays were performed as described before (2), using cyclin D1 antibody (Santa Cruz Biotechnology, sc-8396, RRID:AB_627344) or control IgG (Santa Cruz Biotechnology, sc-2025, RRID:AB_737182) followed by protein G- magnetic beads (Invitrogen) incubation and elution with Glycine 100mM pH=2.5. Co-IP experiments were performed within five weeks after cell thawing. Cyclin D1 (Santa Cruz Biotechnology, sc-753), E2F4 (Bethyl, A302-134A, RRID:AB_1720353), FOXM1 (Santa Cruz Biotechnology, sc-502, RRID:AB_631523), and CBP (Santa Cruz Biotechnology, sc-7300, RRID:AB_626817) antibodies were used for WB detection. In figure 1A and supplementary figure S2A, the same blot was probed with cyclin D1 and tubulin antibodies by cutting the membrane. In figure 2H, cyclin D1 and CBP blots correspond to the same membrane while E2F4 and FOXM1 blots correspond to an independent membrane. Image acquisition was performed with ImageQuant LAS 4000 mini (GE Healthcare). Image processing and quantification were performed with Multi Gauge software (Fujifilm). For qRT-PCR analysis, cDNA was generated from 1 µg RNA with qScript cDNA Synthesis kit (Quantabio). qRT–PCR reaction was performed using SYBR green (Roche).
    [Show full text]
  • The Function and Evolution of C2H2 Zinc Finger Proteins and Transposons
    The function and evolution of C2H2 zinc finger proteins and transposons by Laura Francesca Campitelli A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Laura Francesca Campitelli 2020 The function and evolution of C2H2 zinc finger proteins and transposons Laura Francesca Campitelli Doctor of Philosophy Department of Molecular Genetics University of Toronto 2020 Abstract Transcription factors (TFs) confer specificity to transcriptional regulation by binding specific DNA sequences and ultimately affecting the ability of RNA polymerase to transcribe a locus. The C2H2 zinc finger proteins (C2H2 ZFPs) are a TF class with the unique ability to diversify their DNA-binding specificities in a short evolutionary time. C2H2 ZFPs comprise the largest class of TFs in Mammalian genomes, including nearly half of all Human TFs (747/1,639). Positive selection on the DNA-binding specificities of C2H2 ZFPs is explained by an evolutionary arms race with endogenous retroelements (EREs; copy-and-paste transposable elements), where the C2H2 ZFPs containing a KRAB repressor domain (KZFPs; 344/747 Human C2H2 ZFPs) are thought to diversify to bind new EREs and repress deleterious transposition events. However, evidence of the gain and loss of KZFP binding sites on the ERE sequence is sparse due to poor resolution of ERE sequence evolution, despite the recent publication of binding preferences for 242/344 Human KZFPs. The goal of my doctoral work has been to characterize the Human C2H2 ZFPs, with specific interest in their evolutionary history, functional diversity, and coevolution with LINE EREs.
    [Show full text]
  • Cancer-Driving H3G34V/R/D Mutations Block H3K36 Methylation and H3k36me3–Mutsα Interaction
    Cancer-driving H3G34V/R/D mutations block H3K36 methylation and H3K36me3–MutSα interaction Jun Fanga,b,1, Yaping Huanga,b,1, Guogen Maoc,1, Shuang Yanga,b, Gadi Rennertd, Liya Gue, Haitao Lia,b, and Guo-Min Lic,e,2 aTsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100080, China; bDepartment of Basic Medical Sciences, Tsinghua University, Beijing 100080, China; cDepartment of Toxicology and Cancer Biology, University of Kentucky College of Medicine, Lexington, KY 40506; dDepartment of Community Medicine and Epidemiology, Carmel Medical Center, Clalit National Israeli Cancer Control Center, Haifa 3436212, Israel; and eDepartment of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390 Edited by Paul Modrich, HHMI and Duke University Medical Center, Durham, NC, and approved August 9, 2018 (received for review April 12, 2018) Somatic mutations on glycine 34 of histone H3 (H3G34) cause cancers; however, how H3G34 mutations induce tumorigenesis pediatric cancers, but the underlying oncogenic mechanism re- is unknown. mains unknown. We demonstrate that substituting H3G34 with Since H3G34 is in close proximity to H3K36, we hypothesize that arginine, valine, or aspartate (H3G34R/V/D), which converts the a large side chain created by G34D, G34R, and G34V mutations in non-side chain glycine to a large side chain-containing residue, H3 blocks the interaction between SETD2 and the H3 tail, inhibiting blocks H3 lysine 36 (H3K36) dimethylation and trimethylation by H3K36 trimethylation. Similarly, the large side chains may also in- histone methyltransferases, including SETD2, an H3K36-specific hibit the H3K36me3–MutSα interaction. We tested these hypotheses trimethyltransferase. Our structural analysis reveals that the H3 and found evidence of their validity.
    [Show full text]