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High Resolution Study of NF-Kb - DNA Interactions Imtiaz Nisar Lone

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High Resolution Study of NF-Kb - DNA Interactions Imtiaz Nisar Lone High Resolution study of NF-kB - DNA Interactions Imtiaz Nisar Lone To cite this version: Imtiaz Nisar Lone. High Resolution study of NF-kB - DNA Interactions. Molecular biology. Ecole normale supérieure de lyon - ENS LYON, 2013. English. NNT : 2013ENSL0802. tel-00947251 HAL Id: tel-00947251 https://tel.archives-ouvertes.fr/tel-00947251 Submitted on 15 Feb 2014 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE En vue d’obtention du grade de Docteur de l’Ecole Normale Supérieure de Lyon-Université de Lyon Discipline: Biologie Laboratoire de Biologie Moléculaire de la Cellule, LBMC Ecole Doctorale de Biologie Moléculaire Intégrative et Cellulaire, BMIC Présentée et soutenue publiquement le 14 Fevrier 2013 par Imtiaz Nisar LONE High Resolution study of NF-kB – DNA Interactions Directeur de thèse: Dr. Dimitar ANGUELOV Apres l’avis de: Dr. Marco BIANCHI Dr. Malcolm BUCKLE Devant la commission d’examen formée de: Dr. Dimitar ANGUELOV Directeur de thèse, ENS de Lyon Dr. Marco BIANCHI Rapporteur, San Rafael, Milan Dr. Malcolm BUCKLE Rapporteur, ENS Cachan Dr. Stefan DIMITROV Examinateur, UJF/IAB, Grenoble Dr. Ali HAMICHE Examinateur, IGBMC, Strasbourg Dr. Gael YVERT Président, ENS de Lyon Thèse préparée au sein du Laboratoire de Biologie Moléculaire de la Cellule École Normale Supérieure 46 allée d'Italie 69364 Lyon cedex 07, FRANCE To my parents Acknowledgements ″Man cannot discover new oceans unless he has the courage to lose sight of the shore. (Andre Gide). This odyssey to discover new oceans requires guidance, support and assistance of others. There are a number of people who I would like to acknowledge for their kind support and encouragement throughout my studies. First and foremost, I would like to thank my supervisor Dr. Dimitar Angelov for providing me an opportunity and space to work in the lab.Dimitar deserves a special recognition because of his wholehearted support to this project and his precious advices. Most critical to the overall effort was the enormous infusion of guidance, encouragement and inspiration contributed by him. I would also like to thank Dr. Stefan Dimitrove for introducing me to Dimitar Angelov and for encouraging and guiding me time to time. I would like to thank my thesis committee members Prof. Hans Geiselman and Dr. Marie Josephe for monitoring my work and for helpful discussions. I would like to extend my gratitude to all the members of Angelov group, past and present. In particular, I would like to thank Dr. Charles Richard for making my stay in Lyon so wonderful. My special thanks to Lama Soueidan - “The Stress buster” for making the lab environment wonderful and also for all the translations she had to do for me. My thanks to Ogi, Ram and Elsa for all the help and for the wonderful time we shared in Angelov team. One of the special advantages I have had is my access to the extraordinary community of research scholars at ENS, Lyon. More often than not, the help I needed to tie down a loose end was right at hand. In this regard, I am especially indebted to Dr. Maria Sirakov for her help and positive criticism and also for giving me an opportunity to collaborate with her. You have been an amazing friend, a constant source of encouragement and of course a wonderful cook. My special thanks to all my best buddies -Dane Georgess, Esber, Mubarak, Jeelani, Faiyaz and Bilal for their friendship. I would specially like to thank “Page layout artist” Mubarak for helping me with the final editing of the manuscript. For chiseling and carving my overall personality my family, especially my parents, my sister and my brother deserve the special mention. They are the special people of my life. My deepest gratitude goes to them as they have always been concerned about me and wished me success and happiness forever in my enterprises. I am particularly thankful to them for patiently putting up with my long absence and for their buoyancy. Imtiaz Nisar i Contents INTRODUCTION ......................................................................................................... 2 1 Introduction ............................................................... Error! Bookmark not defined. 1.1 Specificity of protein-DNA interactions ................................................................... 4 1.2 Tools for determining the specificity of protein-DNA interactions ......................... 5 1.3 Kinetics of DNA-protein interactions ....................................................................... 6 1.4 Accessibility of binding sites ..................................................................................... 8 1.5 Chromatin: The beginning ....................................................................................... 8 1.6 DNA packing and nuclear architecture .................................................................... 9 1.6.1 Nucleosome ...........................................................................................................12 1.6.2 Core Histones ........................................................................................................12 1.6.3 The histone octamer ...............................................................................................13 1.6.4 Nucleosome Core particle ......................................................................................14 1.6.5 Linker Histone .......................................................................................................15 1.6.6 Binding of linker histone to nucleosomes ...............................................................16 1.7 Chromatin structure: ...............................................................................................17 1.7.1 Primary structure: ..................................................................................................18 1.7.2 Secondary Structure: ..............................................................................................18 1.7.3 Tertiary structure ...................................................................................................19 1.8 Chromatin dynamics and its regulation ..................................................................20 1.8.1 Sequence determinants of chromatin accessibility ..................................................20 1.8.2 Chromatin remodeling and DNA accessibility ........................................................24 1.8.3 Chromatin remodeler families ................................................................................25 1.8.4 Mechanism of chromatin remodeling .....................................................................27 1.8.5 Influence of core histone variants ...........................................................................30 1.8.6 Histone modifications and transcription .................................................................32 1.9 Nucleosomes as transcription barriers ....................................................................34 1.9.1 Dynamics of DNA–histone interactions as a mechanism of nucleosome accessibility 35 1.10 NF-κB .......................................................................................................................35 1.10.1 Structures of the NF-κB: DNA complexes ..........................................................38 1.10.2 Selective transactivation by NF-κB ....................................................................39 1.10.3 Selective DNA binding by NF-κB dimers ..........................................................41 1.10.4 Why to study binding specificity of NF-κB ........................................................44 1.10.5 NF-κB and chromatin.........................................................................................45 ii 1.10.6 Binding of NF-κB to nucleosomes .....................................................................46 1.10.7 Linker histones and transcription ........................................................................47 1.11 Techniques used .......................................................................................................48 1.11.1 Electrophoretic mobility shift assay (EMSA)......................................................48 1.11.2 DNase footprinting ............................................................................................48 1.11.3 Hydroxyl radical footprinting .............................................................................49 1.11.4 The UV laser footprinting ..................................................................................49 1.11.5 UV laser footprinting of NF-κB-DNA complexes ...............................................50 1.11.6 Advantages of the UV laser footprinting technique .............................................52 2 Manuscript 1 High resolution study of NF-κB-DNA interactions Objective…………………………………………………………………………………55 Materials and Methods ............................................................................................. 566 2.1 Basis of sequence selection for this study ................................................................56
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