Chromatin remodeling in the UV-induced DNA damage response Özge Zelal Aydın Printed by: Proefschriftmaken.nl || Uitgeverij BOXPress Published by: Uitgeverij BOXPress, ’s-Hertogenbosch Cover adapted and modified by: Özge Zelal Aydın from www.health-news.com ISBN: 978-90-8891-984-8 © Copyright 2014 by Özge Zelal Aydın. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without prior written permission of the author. Chromatin remodeling in the UV-induced DNA damage response Remodellering van chromatine in de UV-geïnduceerde DNA-schade respons Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. H.A.P. Pols en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 29 oktober 2014 om 13.30 uur door Özge Zelal Aydın geboren te Ankara, Turkije Promotiecommissie Promotor: Prof.dr. J.H.J. Hoeijmakers Overige leden: Prof.dr. A.B. Houtsmuller Dr. R.A. Poot Dr. H. van Attikum Copromotor: Prof.dr. W. Vermeulen Dr. H. Lans Aşkım da değişebilir gerçeklerim de Pırıl pırıl dalgalı bir denize karşı Yangelmişim diz boyu sulara Hepinize iyi niyetle gülümsüyorum Hiçbirinizle dövüşemem Siz ne derseniz deyiniz Benim bir gizli bildiğim var Sizin alınız al inandım Sizin morunuz mor inandım Ben tam kendime göre Ben tam dünyaya göre Ama sizin adınız ne Benim dengemi bozmayınız Turgut Uyar Contents Page Scope of the Thesis 9 Chapter I Introduction 11 Chapter II ISWI chromatin remodeling complexes in the DNA 31 damage response Chapter III Human ISWI complexes are targeted by SMARCA5 45 ATPase and SLIDE domains to help resolve lesion-stalled transcription Chapter IV Quantitative proteomics analysis of SMARCA5 67 suggests a role in mRNA metabolism after DNA damage Chapter V SWI/SNF facilitates Nucleotide Excision Repair 97 Chapter VI General Discussion & Perspectives 117 References 124 Chapter VII Summary 148 Nederlandse Samenvatting 150 List of Abbreviations 152 Curriculum Vitae 154 List of Publications 155 PhD Portfolio 156 Acknowledgements 158 Scope of the Thesis Scope of the Thesis DNA damage interferes with transcription and replication, causing cell death, chromosomal aberrations or mutations, eventually leading to aging and tumorigenesis. The integrity of DNA is protected by a network of DNA repair and associated signalling pathways, collec- tively called the DNA damage response (DDR). Chromatin poses a barrier for DNA repair and as such plays a critical role in controlling DDR efficiency. Chromatin is modified to regulate access of repair proteins to DNA and also needs to be restored to its original configuration afterwards. Chromatin also serves as an optimal regulation platform for DNA repair by mediating signalling events, providing docking sites for signaling proteins and controlling their activity. The work that we describe in this thesis is focused on the role of chromatin remodelling in DDR, specifically the Nucleotide Excision Repair (NER) pathway. To provide the necessary background on this emerging field, Chapter I summarizes the current state-of-art of chromatin modifications and ATP-dependent chromatin remodeling associated with the DDR. Chapter II describes the mammalian ISWI family of ATP-depen- dent chromatin remodeling complexes and its versatile roles in the DDR. We describe vari- ous essential functions through which ISWI complexes regulate the DDR and highlight how targeting of ISWI complexes to sites of DNA repair improves understanding of how chro- matin remodeling complexes identify and associate with substrate nucleosomes in vivo. In Chapter III, we identify a novel function for two distinct mammalian ISWI ATP-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI iso- form SMARCA5 and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 recruitment to UV-C damage depends on transcription and histone modifications and requires functional ATPase and SLIDE domains. Intriguingly, after initial recruitment to UV damage, SMARCA5 relocalizes away from the center of DNA damage which requires its HAND domain. This peripheral relocalization may be indicative of actual chromatin remodeling. Our data support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis dependent scanning and proofreading mechanism, highlighting how ISWI chromatin remodelers identify and bind nucleosomes containing damaged DNA. After analysing the role of ISWI in the UV-DDR, Chapter IV aims to identify the proteins with which SMARCA5 interacts in mammalian cells to have a deeper understanding of the mechanisms underlying SMARCA5 function in the UV-DDR. This chapter focuses on two proteomics experiments which give detailed insight in the interactome of SMARCA5, both before and after UV irradiation, and describes a characterization of the pathways in which SMARCA5 functions upon UV damage. Functional classification of interacting pro- teins revealed associations with transcription and translation machinery, RNA processing and export pathways, and chromatin reorganisation. Thus, our proteomics based data indi- cate that SMARCA5 is part of complex networks of proteins, which modulate transcription, translation and chromatin reorganization in response to UV. Chapter V discusses the function of SWI/SNF, another ATP-dependent chromatin remodel- ing family, in the UV-DDR. Several subunits of SWI/SNF complexes were identified in aC. elegans screen for genes that protect against UV irradiation. Functional analysis in human cells confirms that the SWI/SNF BRM and BRG1 ATPases and several subunits are also essential for UV survival in mammals. Our results show that BRG1 interacts with NER-initi- ation factor DDB2 and regulates its mobility after UV irradiation. BRM interacts with DDB2 in a UV dependent manner. Both BRG1 and BRM are recruited to local UV-C damage and also appear to be essential for transcription recovery after UV. These data suggest that BRM and BRG1 function to regulate the initial steps of Global Genome NER by interacting with DDB2 and possibly also TC-NER. Finally, Chapter VI concludes all results and discusses the future direction of chromatin remodeling involvement in the UV-DDR. 9 10 Chapter I Introduction 1) DNA damage DNA is continuously damaged by various endogenous and environmental factors. Damage in DNA interferes with transcription and replication, causing aging and mutations and/or 1 chromosomal aberrations which result in tumorigenesis (Hoeijmakers, 2009). There are various endogenous physiological processes that lead to DNA aberrations in organisms, such as DNA replication, abortive topoisomerase I and topoisomerase II activ- ities, hydrolytic reactions and methylations. Moreover, reactive oxygen compounds which are produced as byproducts of oxidative respiration or through redox events (Jackson & Bartek, 2009), or reactive oxygen and nitrogen compounds that are produced by the im- mune system at sites of inflammation and infections (Kawanishi et al, 2006) can attack DNA, leading to adducts that impair base-pairing, cause base loss, DNA single-strand breaks and/or interfere with DNA replication and transcription (Jackson & Bartek, 2009). Ultraviolet (UV) light is one of the most toxic environmental DNA damaging agents. UVC has the shortest wavelength (<290 nm) and is the most harmful part of the solar UV spec- trum for DNA. UVC is fortunately absorbed by the ozone layer and even ordinary air. How- ever, UVA (320-400 nm) and UVB (290-320 nm) are not completely blocked by the at- mosphere and residual UVA and UVB in strong sunlight can still induce large amounts of DNA lesions in the cell (Ikehata & Ono, 2011). UVA creates oxygen radicals which pre- dominantly and indirectly produces oxidative DNA damage. In contrast, UVB is directly absorbed by DNA and induces the chemical conjugation between two adjacent pyrimi- dines: cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoprod- ucts (6-4PPs). Another very toxic environmental DNA damaging agent is ionizing radiation (IR) causing different forms of damage, the most toxic of which are double strand breaks (DSBs). There are different sources of IR including the sun, naturally-occurring radioactive compounds or the radioisotopes used during cancer radiotherapy. Other important DNA damaging agents are various industrial chemicals like vinyl chloride and hydrogen peroxide and environmental chemicals such as polycyclic aromatic hydrocarbons found in smoke, soot and tar, creating a huge diversity of DNA adducts including ethenobases, oxidized bases, alkylated phosphotriesters and DNA crosslinks. These chemicals trigger various cancers, most notably those of the lung, oral cavity and adjacent tissues (Wogan et al, 2004). Cancer-causing DNA-damaging chemicals are also found in different foods, such as aflatoxins found in contaminated peanuts and heterocyclic amines in over-cooked meat. 2) DNA damage response mechanisms To protect against the adverse effects of DNA damage, organisms are equipped with diverse complementary mechanisms of DNA repair, DNA damage tolerance processes to allow replica- tion over damaged DNA and associated DNA damage signaling processes to induce cell cycle arrest, collectively called the DNA damage response (DDR) (Fig.1) (Hoeijmakers,