Yuan Guo RNA Polymerase I regulation by chromatin remodelling RNA Polymerase I regulation by chromatin remodelling chromatin by I regulation Polymerase RNA Yuan Guo Yuan Guo ISBN 978-91-7911-210-3 Department of Molecular Biosciences, The Wenner-Gren Institute Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2020 RNA Polymerase I regulation by chromatin remodelling Yuan Guo Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Friday 25 September 2020 at 09.00 in Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20. Abstract Cell proliferation and growth is correlated with effective protein synthesis and ribosome biogenesis. The transcription of the 47S pre-ribosomal RNA by RNA Polymerase I (RNA Pol I) machinery is the rate-limiting step of ribosome biogenesis and can accounts for more than 50% of total cellular transcription. RNA Polymerase I transcription is a highly energy- consuming process which requires regulation at various stages. In the work presented in this thesis, we have investigated the regulation of RNA Pol I transcription, and investigated the stress response triggered by impaired RNA Pol I transcription. We showed in study I that the ATP dependent chromatin remodelling complex B-WICH is required to maintain an open chromatin landscape at the ribosomal DNA (rDNA) gene promoter in order to allow for transcription initiation by RNA Pol I. In absence of B-WICH, the NuRD complex reconfigures the chromatin landscape to an inaccessible state. We showed in study II that impairment of RNA Pol I transcription by deleting WSTF, a core subunit of B-WICH resulted in cell cycle arrest and apoptosis. More severe inhibition of RNA Pol I transcription through chemical compounds resulted in activation of cellular stress response cascades including but not limited to cell cycle arrest, unfolded protein response and oxidative stress response. We showed in study III that RNA Pol I transcription was increased during epithelial-mesenchymal transition (EMT) in the context of development and disease. The association of the EMT-driving transcription factor SNAIL1 with the rDNA gene promoter was shown to be essential in EMT triggered RNA Pol I transcription. The work presented in this thesis demonstrates the importance of RNA Pol I transcription regulation in maintaining cellular homeostasis. Keywords: RNA Pol I, ribosome genes, chromatin remodelling, WSTF, CHD4. Stockholm 2020 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-184045 ISBN 978-91-7911-210-3 ISBN 978-91-7911-211-0 Department of Molecular Biosciences, The Wenner-Gren Institute Stockholm University, 106 91 Stockholm RNA POLYMERASE I REGULATION BY CHROMATIN REMODELLING Yuan Guo RNA Polymerase I regulation by chromatin remodelling Yuan Guo ©Yuan Guo, Stockholm University 2020 ISBN print 978-91-7911-210-3 ISBN PDF 978-91-7911-211-0 All previously published papers were reproduced with permission from the publishers. Printed in Sweden by Universitetsservice US-AB, Stockholm 2020 “Do. Or do not. There is no try.” Yoda Populärvetenskaplig sammanfattning Det sker komplexa processer i alla celler, och majoriteten av dessa utförs helt eller delvis med hjälp av proteiner. Protein består av tiotal till tiotusentals av små byggstenar, aminosyror som sätts samman med hjälp av ribosomer. I varje cell finns det upp mot miljontals av ribosomer, som är makromolekyler bestående av proteiner och ribonukleinsyror (RNA) och ansvarar för omvandling av budbärande RNA till proteiner. Produktionen av RNA sker genom stora proteinkomplex, RNA polymeraser som läser av gensekvenser i form av deoxiribonukleinsyror (DNA) och sekvensen det till RNA. I människoceller består varje ribosom av fyra olika ribosomala RNA (rRNA), som kommer från två gener och kräver varsin separata former av RNA polymeras komplex (I och III). Cellens tillväxt står i direkt relation till fungerande syntes av ribosomer som begränsas av produktionen av rRNA. Processen är extremt energikrävande och kan stå för över 50% av all RNA-produktion i cellen. RNA polymeras I (RNA Pol I) ansvarar för 47S rRNA genen som ger upphov till tre av fyra rRNA som ingår i ribosomen. Regleringen av RNA Pol I produktionen kan ske på många olika sätt, via ändringar på proteinkomplexet eller på 47S rRNA genen och är svar på diverse signaleringsvägar. Denna avhandling fokuserar på att utöka förståelse för regleringen av RNA Pol I: Studie 1 visar att proteinkomplexet B-WICH kan omvandla DNA landskapet vid 47S rRNA genen för att tillåta RNA Pol I att binda till 47S rRNA genen vid tillförsel av glukos. Studie 2 visar att normal funktion av B-WICH är viktig för att upprätthålla cellulär homeostas. Vi visar även att flertal signaleringsvägar relaterad till cellstress sätts igång vid hämmande av RNA Pol I funktion, samt att dessa kan skifta beroende på typ och grad av hämmande. Studie 3 visar att RNA Pol I aktivitet ökas under celltransformation till mesenkymala celler, en mer migrerande celltyp som är viktig under t.ex. embryoutveckling eller metastasering av tumörer. Dessutom visar vi att denna process kan stoppas med hjälp av RNA Pol I hämmaren CX-5461. Sammanfattningsvis bidrar studier i denna avhandling till en ökad förståelse av reglering av ribosomal transkription som svar på förändringar i omgivningen och dess roll i att bevara cellulär homeostas. I List of articles included in this thesis I. The chromatin-remodeling complexes B-WICH and NuRD regulate ribosomal transcription in response to glucose. Anna Rolicka*, Yuan Guo*, Antoni Gañez Zapater, Kanwal Tariq, Jaclyn Quin, Anna Vintermist, Fatemeh Sadeghifar, Marie Arsenian-Henriksson, Ann-Kristin Östlund Farrants. FASEB Journal, 2020 Jun 29. doi: 10.1096/fj.202000411R II. Responses induced by B-WICH deficiency. Yuan Guo, Kanwal Tariq, Lukas Habering, Michaela Keuper, Xin Xie, Pergiorgio Percipalle, Martin Jastroch, Sabrina Büttner, Marie Arsenian-Henriksson, Ann-Kristin Östlund Farrants. Manuscript III. Ribosome biogenesis during cell cycle arrest fuels EMT in development and disease. Varsha Prakash, Brittany B Carson, Jennifer M Feenstra, Randall A Dass , Petra Sekyrova , Ayuko Hoshino, Julian Petersen, Yuan Guo, Matthew M Parks, Chad M Kurylo, Jake E Batchelder, Kristian Haller, Ayako Hashimoto, Helene Rundqivst, John S Condeelis, C David Allis, Denis Drygin, M Angela Nieto, Michael Andäng, Piergiorgio Percipalle, Jonas Bergh, Igor Adameyko, Ann-Kristin Östlund Farrants, Johan Hartman, David Lyden, Kristian Pietras, Scott C Blanchard, C Theresa Vincent. Nature communications, 2019 May 8;10(1):2110. doi: 10.1038/s41467-019-10100-8 Work not included in this thesis: IV. The SWI/SNF subunit BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA. Antoni Gañez Zapater, Sebastian D. Mackowiak*, Yuan Guo*, Antonio Jordan-Plá, Marc R. Friedländer, Neus Visa, Ann-Kristin Östlund Farrants. Epigenetics & Chromatin (under revision) *Equal contribution II Abbreviations ActD Actinomycin D AMPK AMP-activated Protein Kinase B-WICH WSTF-ISWI chromatin remodelling complex B BAZ1B/WSTF William syndrome transcription factor BAZ2A/TIP5 TTF-I-interacting protein 5 CHD3/4 Chromodomain-helicase DNA binding protein 3/4 CpG Cytosine-phosphate-Guanine EMT Epithelial-Mesenchymal Transition HAT Histone acetyltransferase HDAC Histone deacetylase HDM Histone demethylase HMT Histone methyltransferase HDM2 Mouse double minute 2 homolog HP1 Heterochromatin protein 1 ISWI Immitation SWItch KAT2A/GCN5 Lysine acetyltransferase 2A KAT2B/PCAF p300/CBP-associated factor KAT3A/CBP CREB-binding protein KAT3B/p300 Lysine acetyltransferase 3B KAT8/MOF Lysine acetyltransferase 8 MBD3 Methylated CpG-binding protein 3 MYC v-myc avian myelocytomatosis viral oncogene homolog NM1 Nuclear myosin 1 NuRD Nucleosome remodelling deacetylase p53 Tumour protein p53 RNA Pol I RNA Polymerase I RNA Pol II RNA Polymerase II RNA Pol III RNA Polymerase III SIRT1 Sirtuin 1 III SIRT7 Sirtuin 7 SNF2h Sucrose Non-Fermentable 2 homolog TAF TBP-associated factor TAFI110/TAF1C TBP-associated factor 1C TAFI68/TAF1B TBP-associated factor 1B TBP TATA-box Binding Protein TFIIIA/B/C Transcription Factor IIIA/B/C TGF-β (Transforming growth factor beta) TIF-IA/hRRN3 Transcription initiation factor IA / rDNA-binding RNA Polymerase I transcription factor homolog TIF-IB/SL1 Transcription initiation factor IB / Selective factor 1 UBF Upstream binding factor WICH WSTF-ISWI chromatin remodelling complex IV Table of contents Populärvetenskaplig sammanfattning ......................................................................................... I List of articles included in this thesis ........................................................................................ II Abbreviations ........................................................................................................................... III Introduction ................................................................................................................................ 1 Chapter 1 – Chromatin ............................................................................................................... 1 1.1. Histone modifications ...................................................................................................... 2 1.2. ATP-dependent chromatin remodelling complexes ........................................................ 3 1.2.1. Mi-2/NuRD complexes ............................................................................................