PhD degree in Systems Medicine (curriculum in Molecular Oncology) European School of Molecular Medicine (SEMM), University of Milan and University of Naples “Federico II” Settore disciplinare: bio/11 Structure-function analysis of Myc/Max-DNA binding Paola Pellanda IIT, Milan Matricola n. R10314 Supervisor: Dr. Bruno Amati IEO, Milan Added Supervisor: Dr. Arianna Sabò IEO, Milan Anno accademico 2016-2017 Table of Contents List of abbreviations-------------------------------------------------------------------------------------------- 4 List of figures---------------------------------------------------------------------------------------------------- 6 List of tables------------------------------------------------------------------------------------------------------7 Abstract----------------------------------------------------------------------------------------------------------- 8 1. Introduction -------------------------------------------------------------------------------------------------------- 10 1.1 Myc protein -------------------------------------------------------------------------------------------------- 10 1.1.1 Myc discovery ---------------------------------------------------------------------------------------- 10 1.1.2 Myc protein family ---------------------------------------------------------------------------------- 11 1.2 Myc functions ----------------------------------------------------------------------------------------------- 12 1.2.1 Proliferation and metabolism --------------------------------------------------------------------- 13 1.2.2 Apoptosis ---------------------------------------------------------------------------------------------- 14 1.2.3 Cell adhesion and morphology ------------------------------------------------------------------- 14 1.2.4 DNA and RNA biology ---------------------------------------------------------------------------- 15 1.3 Myc regulation ---------------------------------------------------------------------------------------------- 15 1.4 Myc structure and functional domains ---------------------------------------------------------------- 18 1.4.1 Myc N-terminal region ----------------------------------------------------------------------------- 18 1.4.2 Myc central region ---------------------------------------------------------------------------------- 20 1.4.3 Myc C-terminal region ----------------------------------------------------------------------------- 21 1.5 Myc-DNA binding ----------------------------------------------------------------------------------------- 23 1.5.1 The E-box sequence -------------------------------------------------------------------------------- 23 1.5.2 In vivo genome recognition ----------------------------------------------------------------------- 27 1.5.3 Regulatory models: selective transcription versus general transcriptional amplification ------------------------------------------------------------------------------------------------------- 30 1.6 Targeting Myc in cancer --------------------------------------------------------------------------------- 32 1.7 Aim of the project ------------------------------------------------------------------------------------------ 33 2. Materials and methods ------------------------------------------------------------------------------------------ 34 2.1 Cell culture -------------------------------------------------------------------------------------------------- 34 2.2 Pymol --------------------------------------------------------------------------------------------------------- 34 2.3 Myc-Max co-Immunoprecipitation -------------------------------------------------------------------- 34 2.4 Western Blot ------------------------------------------------------------------------------------------------ 35 2.5 Antibodies --------------------------------------------------------------------------------------------------- 36 2.6 Transcriptional Factor Assay Kits: TransAMTM c-Myc ------------------------------------------ 36 2.7 Cycloheximide treatment -------------------------------------------------------------------------------- 36 2.8 Myc immunofluorescence ------------------------------------------------------------------------------- 37 2.9 Proliferation assays ---------------------------------------------------------------------------------------- 37 2.10 Genome editing: CRISPR/Cas9 ------------------------------------------------------------------------ 38 2 2.10.1 cb9 MycΔb fibroblasts ----------------------------------------------------------------------------- 40 2.10.2 cb9 MycHEA clones ---------------------------------------------------------------------------------- 41 2.11 RNA extraction and qPCR analysis ------------------------------------------------------------------- 42 2.12 Chromatin immunoprecipitation (ChIP) -------------------------------------------------------------- 42 2.13 Primers and oligos sequences --------------------------------------------------------------------------- 44 2.14 Computational analysis ----------------------------------------------------------------------------------- 45 2.14.1 Next generation sequencing data filtering and quality evaluation ----------------------- 45 2.14.2 ChIP-seq data analysis ----------------------------------------------------------------------------- 45 3. Results --------------------------------------------------------------------------------------------------------------- 47 3.1 Design of Myc mutants compromised in DNA binding ------------------------------------------ 47 3.2 MycHEA and MycRA retain normal dimerization with Max --------------------------------------- 49 3.3 Assessment of Myc mutants DNA-binding activities in vitro ----------------------------------- 50 3.4 Re-expression of Mycwt and Myc mutant proteins in rat c-myc null fibroblasts ------------ 51 3.4.1 Determination of Myc mutant proteins localization and stability ------------------------ 51 3.4.2 Assessment of the proliferative potential of Myc mutants -------------------------------- 53 3.5 Generation of a cellular model for the phenotypic characterization of Myc mutants ------ 56 3.5.1 Overexpression of Myc mutants in cb9 MycΔb fibroblasts ------------------------------- 58 3.5.2 Myc mutants show no proliferative activity -------------------------------------------------- 61 3.5.3 Genome-wide analysis of DNA-binding activities ------------------------------------------ 63 3.6 Generation of MycHEA knock-in cell clones --------------------------------------------------------- 74 3.7 MycHEA is impaired in activating gene expression ------------------------------------------------- 84 4. Discussion ---------------------------------------------------------------------------------------------------------- 91 4.1 Mutations in the Myc DNA binding domain impair its ability to sustain cellular growth 91 4.2 E-box recognition is required for stabilization of Myc binding to DNA --------------------- 93 4.3 Myc binding to chromatin is not predictive of gene regulation --------------------------------- 94 4.4 Myc genome recognition in vivo ----------------------------------------------------------------------- 95 3 List of abbreviations Aa Amino acid BAX BCL2 Associated X Protein Bcl-2 B cell lymphoma 2 BCL-2A1 Bcl-2-related protein A1 bHLH-LZ Basic-Helix-Loop-Helix Leucine Zipper Bim Bcl-2 interacting mediator of cell death BrdU 5'-Bromo-2'Deoxyuridine CDK7 cyclin-dependent kinase 7 CTD C-terminal domain DNA Deoxyribonucleic acid Doxy Doxycycline E-box Enhancer box eIF4E Eukaryotic translation initiation factor 4E ELISA Enzyme-linked immunosorbent assay EMSA Electrophoretic mobility shift assay FACS Fluorescence-activated cell sorting Fbw7 F-box/WD repeat-containing protein 7 gcPBM Genomic context protein binding microarray GSEA Gene Set Enrichment Analysis GTF2H1 General Transcription Factor IIH Subunit 1 GTF2H4 General Transcription Factor IIH Subunit 4 HCF-1 Host cell factor 1 HDAC3 Histone deacetylase 3 IRES Internal ribosomal entry sites LPS Lipopolysaccharide Max Myc-associated factor X MbI Myc box I MbII Myc box II MbIIIa Myc box IIIa MbIIIb Myc box IIIb MbIV Myc box IV Mcm Mini-chromosome maintenance proteins Mdm2 Mouse double minute 2 homolog MDSR Myc-dependent serum response Miz1 MYC- interacting zinc finger protein 1 Myc Myelocytomatosis oncogene MycER Myc estrogen receptor 4 Nmi N-Myc interactor OD Optical Density OHT Synthetic steroid 4-hydroxytamoxifen ORC Origin Replication Complex PBM Protein Binding Microarray PUMA p53-upregulated modulator of apoptosis Pus7 Pseudouridylate Synthase 7 Ras Rat sarcoma virus oncogene RBP RNA-binding protein Reep6 Receptor Accessory Protein 6 REF Rat embryonic fibroblasts Rrp9 Ribosomal RNA Processing 9 SCLC Small cell lung cancer SELEX Systematic Evolution of Ligands by Exponential Enrichment SHMT Serine hydroxymethyltransferase Sin3a SIN3 Transcription Regulator Family Member A Sin3b SIN3 Transcription Regulator Family Member B Skp2 S-phase kinase-associated protein 2 Smpdl3b Sphingomyelin Phosphodiesterase Acid Like 3B SUMO Small ubiquitin-like modifier TAD Transcriptional activation domain TAF1 TATA-Box Binding Protein Associated Factor 1 TFIIH Transcription factor II Human TIAR T-cell internal antigen-1 (TIA-1)–related protein TRRAP Transformation/transcription domain-associated protein v-myc Viral myelocytomatosis WDR5 WD repeat-containing protein 5 5 List of figures Figure 1. Schematic representation of some of Myc-dependent cellular processes. ......................... 13 Figure 2. Myc family functional