Identification and characterization of HIRIP3 as a novel histone H2A chaperone Maria Ignatyeva To cite this version: Maria Ignatyeva. Identification and characterization of HIRIP3 as a novel histone H2A chaperone. Ge- nomics [q-bio.GN]. Université de Strasbourg, 2017. English. NNT : 2017STRAJ028. tel-02917943 HAL Id: tel-02917943 https://tel.archives-ouvertes.fr/tel-02917943 Submitted on 20 Aug 2020 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. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE [Institut de génétique et de biologie moléculaire et cellulaire (IGBMC) - UM 41/UMR 7104/UMR_S 964] THÈSE présentée par : [Maria IGNATYEVA] soutenue le : 31 Mai 2017 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Aspects Moleculaires et Cellulaires Biologie Identification et caractérisation de HIRIP3 comme nouveau chaperon d'histone H2A [Identification and characterization of HIRIP3 as a novel histone H2A chaperone] THÈSE dirigée par : [M. HAMICHE Ali] Directeur de recherches, Université de Strasbourg RAPPORTEURS : [Mme. AMEYAR-ZAZOUA Maya] Chercheur statutaire, Institut Necker Enfants Malades [M. THIRIET Christophe] Directeur de recherches, Université de Nantes AUTRES MEMBRES DU JURY : [M. DAVIDSON Irwin] Directeur de recherches, Université de Strasbourg TABLE OF CONTENTS ACKNOWLEDGEMENT ....................................................................................................... 4 LIST OF ABBREVIATIONS .................................................................................................. 5 LIST OF FIGURES ................................................................................................................. 7 LIST OF TABLES ................................................................................................................... 7 1 FOREWORD ......................................................................................................................... 8 2 RÉSUMÉ EN FRANÇAIS ................................................................................................... 9 3 INTRODUCTION ............................................................................................................... 13 3.1 Nucleosome composition........................................................................................................... 13 3.2 Chromatin organisation ........................................................................................................... 14 3.3 Chromatin regulation ............................................................................................................... 16 3.3.1 Histone post-translational modifications ............................................................................. 17 3.3.2 Variant histones incorporation ............................................................................................. 20 3.3.3 Chromatin remodelling complexes ...................................................................................... 24 3.3.3.1 ISWI family ................................................................................................................................ 27 3.3.3.2 SWI-SNF family ......................................................................................................................... 29 3.3.3.3 CHD/Mi-2 family ....................................................................................................................... 30 3.3.3.4 INO80/SWR1 family .................................................................................................................. 32 3.4 Chromatin assembly ................................................................................................................. 35 3.5 Chromatin assembly machinery .............................................................................................. 36 3.5.1 Histone chaperones .............................................................................................................. 36 3.5.1.1 H3-group/H4 histone chaperones................................................................................................ 37 3.5.1.2 H2A-group/H2B histone chaperones .......................................................................................... 38 3.5.2 Chromatin machinery interplay ........................................................................................... 40 4 MATERIALS AND METHODS ....................................................................................... 44 4.1 Protein expression in E.coli ...................................................................................................... 44 4.2 Protein expression in SF9 insect cells ...................................................................................... 46 4.3 Protein expression in HeLa cells .............................................................................................. 47 4.4 Protein methods ........................................................................................................................ 48 4.4.1 Protein purification .............................................................................................................. 48 4.4.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ...................................................... 50 4.4.3 Staining of protein gels ........................................................................................................ 50 4.4.4 Western blotting .................................................................................................................. 50 4.4.5 Mass spectrometry ............................................................................................................... 51 2 4.5 Structural modelling ................................................................................................................. 51 4.6 Phosphorylation assay .............................................................................................................. 51 5 RESULTS AND DISCUSSION ......................................................................................... 52 5.1 PROJECT I. Identification and characterization of HIRIP3 as a novel histone H2A chaperon........................................................................................................................................... 52 5.1.1 Results ................................................................................................................................. 54 5.1.1.1 H2A/H2B dimers are co-purified with HIRIP3 in vivo .............................................................. 54 5.1.1.2 HIRIP3 interacts with H2A/H2B and H2A.Z/H2B dimers in vitro ............................................ 57 5.1.1.3 HIRIP3 CHZ motif is critical for H2A/H2B interaction ............................................................. 58 5.1.1.4 H2A interacts with HIRIP3 through its alphaC domain ............................................................. 62 5.1.1.5 Structural considerations of HIRIP3 and H2A/H2B interaction ................................................. 63 5.1.1.6 CK2 phosphorylates H2A within eHIRIP3 complex .................................................................. 64 5.1.2 Discussion ............................................................................................................................ 65 5.1.2.1 HIRIP3 interaction with H2A/H2B dimers in vivo and in vitro ................................................. 65 5.1.2.2 Structural basis of HIRIP3 interaction with H2A/H2B dimer .................................................... 66 5.1.2.3 CK2 kinase function within HIRIP3 complex ............................................................................ 67 5.1.2.4 HIRIP3 as a novel H2A/H2B histone chaperone ........................................................................ 67 5.2 PROJECT II. Reconstitution of SRCAP complex functional complex ................................ 68 5.2.1 Results ................................................................................................................................. 71 5.2.1.1 YL1 interacts with TIP49A and TIP49B ATPases ..................................................................... 71 5.2.1.2 TIP49A/B, YL1 and H2A.Z/H2B form a complex in vitro ........................................................ 73 5.2.1.3 SRCAP, TIPA49/TIP49B, YL1 and H2A.Z/H2B is a functional sub-complex within SRCAP complex .................................................................................................................................................. 75 5.2.2 Discussion ............................................................................................................................ 78 5.2.2.1 YL1 interaction with TIP49A and TIP49B ................................................................................. 78 5.2.2.2 TIP49A/B ATPases functions within SRCAP complex ............................................................