the MYC GENE : methods and protocols.

Description

Preface Acknowledgments Contents Contributors Chapter 1: An ``-omycs´´ Toolbox to Work with MYC References Chapter 2: Methods of Expression, Purification, and Preparation of the c-Myc b-HLH-LZ for Its Biophysical Characterization 1 Introduction 2 Materials 2.1 Expression of c-Myc b-HLH-LZ Domain 2.2 Purification of the c-Myc b-HLH-LZ 3 Methods 3.1 Expression of the c-Myc b-HLH-LZ Domain 3.2 Purification of the c-Myc b-HLH-LZ 4 Notes References Chapter 3: Biophysical and Structural Methods to Study the bHLHZip Region of Human c-MYC 1 Introduction 2 Materials 2.1 Expression of the c-MYC bHLHZip and c-MYC:MAX bHLHZip Complex Constructs 2.2 Purification of c-MYC bHLHZip Protein and c-MYC:MAX bHLHZip Complexes 2.3 NMR Studies 2.4 Crystallization Studies 3 Methods 3.1 Expression of c-MYC bHLHZip and c-MYC:MAX bHLHZip Constructs in Rich or in Minimal Media 3.2 Purification of Cleavable His-Tag c-MYC bHLHZip Protein 3.3 Purification of c-MYC:MAX bHLHZip Complex Constructs 3.4 Production of Selectively Labeled c-MYC:MAX bHLHZip Complexes for NMR Studies 3.5 NMR Protocols to Study c-MYC bHLHZip by Itself and c-MYC:MAX bHLHZip Complex 3.6 Crystallization Protocol for c-MYC:MAX bHLHZip Complex 4 Notes References Chapter 4: Identifying and Validating MYC:Protein Interactors in Pursuit of Novel Anti-MYC Therapies 1 Introduction 1.1 Identifying and Validating MYC Interactors 1.2 BioID 1.3 Validation of BioID Results 2 Materials 2.1 Components of BioID 2.2 Components of In Vitro GST-Pull-Down Assay 2.3 Components of Proximity Ligation Assay 2.4 Components of Biolayer Interferometry 3 Methods 3.1 MYC-BioID (See Notes 1-3) 3.1.1 Preparation of Cells for BioID 3.1.2 Purification of Biotinylated Proteins 3.1.3 Mass Spectrometry Analysis 3.1.4 High-Confidence Interactome Network Analysis 3.2 In Vitro GST-Pull-Down Assay 3.2.1 Recombinant GST-MYC Purification 3.2.2 GST-Pull-Down 3.3 Proximity Ligation Assay 3.3.1 Immunofluorescence 3.3.2 Proximity Ligation Assay (Duolink In Situ Red Starter Kit Mouse/Rabbit) 3.4 BLI Performed on Octet System 3.4.1 Biotinylated MYC Sample Preparation 3.4.2 Running a Kinetic Experiment 4 Notes References Chapter 5: Detection of Post-translational Modifications on MYC 1 Introduction 2 Materials 2.1 Tissue Culture 2.2 Antibody-Bead Conjugation 2.3 Immunoprecipitation 2.4 Western-Blot Analysis 2.5 Primary and Secondary Antibodies 2.6 Cell Transfection 2.7 Cell Ubiquitination and SUMOylation Assays 2.8 Formalin-Fixed Paraffin-Embedded Tissue Staining 3 Methods 3.1 Antibody to Bead Conjugation 3.2 Immunoprecipitation for Detection of c-Myc Serine 62 Phosphorylation 3.3 Detection of c-Myc Phosphorylation by Commercially Available Phospho-Specific Antibodies 3.4 Detection of c-Myc Ubiquitination in Cells 3.5 Detection of c-Myc SUMOylation in Cells 3.6 Immunofluorescence Staining of Formalin-Fixed Paraffin-Embedded Tissue 3.7 Detection of c-Myc in Formalin-Fixed Paraffin-Embedded Tissue 4 Notes References Chapter 6: MYC Analysis in Cancer and Evolution 1 Introduction 2 Materials 3 Methods 3.1 Preparation and Cultivation of Avian Embryo Fibroblasts 3.2 DNA Transfection of MYC-Carrying Retroviral Vector Plasmids 3.3 Focus Assay of MYC-Transformed Cells 3.4 Testing MYC Inhibitors by Agar Colony Assay and Cell Proliferation Measurement 3.5 MYC-Protein Interactions Monitored by Protein Pull-Down Analysis 3.5.1 Detection by Radioactive Protein Labeling 3.5.2 Detection by Immunoblotting Using ECL 3.6 MYC Protein Interactions Monitored by Co-immunoprecipitation (CoIP) Analysis 3.6.1 Detection by Radioactive Protein Labeling and Fluorography 3.6.2 Detection by Immunoblotting Using ECL 3.7 Analysis of MYC Expression by In Situ Hybridization in Hydra 3.8 Analysis of MYC Expression by Northern Analysis 3.9 Protein-DNA Interaction Analysis in Hydra by Chromatin Immunoprecipitation (ChIP) 3.10 MYC-DNA Interaction Analysis by Electrophoretic Mobility Shift Assay (EMSA) 4 Notes References Chapter 7: Genome-Wide Analysis of c-MYC-Regulated mRNAs and miRNAs and c-MYC DNA-Binding by Next-Generation Sequencing 1 Introduction 2 Materials 2.1 RNA-Seq 2.2 miRNA-Seq 2.3 ChIP-Seq 2.3.1 ChIP 2.3.2 ChIP-Seq Library Generation 3 Methods 3.1 RNA-Seq 3.1.1 mRNA Library Preparation Quality and Quantity Control of Total RNA Input mRNA Purification and Fragmentation RNA Bead Plate Preparation, Washing, and Elution Generation/Incubation of RNA Fragmentation Plate (RFP) First Strand cDNA Synthesis Second Strand Synthesis cDNA Cleanup End Repair 3′-end Adenylation Adapter Ligation DNA Fragment Enrichment 3.1.2 Library Quality Control 3.1.3 mRNA Library Sequencing 3.2 miRNA-Seq 3.2.1 miRNA Library Preparation 3′-Adapter Ligation 5′-Adapter Ligation Sample Electrophoresis and RNA Gel Extraction Reverse Transcription and Amplification Template Preparation PCR miRNA Library Purification Recovery of miRNA Library from Gel 3.2.2 miRNA Library Validation 3.2.3 miRNA Library Sequencing 3.3 ChIP-Seq 3.3.1 ChIP Experimental Design of ChIP for Subsequent NGS Analysis Pre-blocking of Sepharose Beads Cross-Linking and Sonication Immunoprecipitation Cross-Link Removal and Purification Quality and Quantity Control of Immunoprecipitated DNA 3.3.2 ChIP-Seq Library ChIP-Seq Library Generation End Repair Addition of ``A´´ Bases to the 3′ End of the DNA Fragments Adapter to DNA Fragment Ligation Size Selection of the Library Enrichment of Adapter-Modified DNA Fragments by PCR ChIP-Seq Library Validation 3.3.3 Library Sequencing 3.4 Bioinformatics Analyses of NGS Results 3.4.1 ChIP-Seq Bioinformatics 3.4.2 mRNA-Seq Bioinformatics 3.4.3 miR-Seq Bioinformatics 3.5 Validation of NGS Results 3.5.1 qPCR Validation of c-MYC-Regulated Transcripts 3.5.2 qPCR Validation of c-MYC-Regulated miRNAs 3.5.3 qChIP Validation of Occupancy by c-MYC 4 Notes References Chapter 8: Analysis of Myc Chromatin Binding by Calibrated ChIP-Seq Approach 1 Introduction 2 Materials 2.1 Reagents 2.2 Buffers 2.3 Equipment 2.4 Software 3 Methods 3.1 Cell Harvest 3.2 Chromatin Immunoprecipitation 3.3 DNA Purification 3.4 Library Preparation and Sequencing 3.5 Data Analysis 3.6 Peak Calling 3.7 GSEA for Myc-Binding Sites 3.8 Metagenome Analysis 3.9 Intersecting RNA-Seq and ChIP-Seq Data 4 Notes References Chapter 9: A High-Throughput Chromatin Immunoprecipitation Sequencing Approach to Study the Role of MYC on the Epigenetic Land... 1 Introduction 2 Materials 2.1 Reagents 2.2 Buffers 2.3 Equipment 2.4 Software and Bioinformatic Tools 3 Methods 3.1 Cell Harvest and Fixation 3.2 Cell Lysis and Chromatin Sonication 3.3 Chromatin Immunoprecipitation and De-crosslinking 3.4 DNA Purification with SPRI Beads and HT-ChIP Validation by qPCR 3.5 Library Preparation 3.6 Reads Quality Control, Mapping, and Visualization 3.7 Peak Calling 4 Notes References Chapter 10: Methods for Determining Myc-Induced Apoptosis 1 Introduction 2 Materials 2.1 In Vitro Assays 2.1.1 Cell Culture and Microscopy 2.1.2 Flow Cytometry for Annexin V 2.1.3 Western Blotting for Cleaved Caspase 3 2.2 In Vivo Assays 2.2.1 Histology 2.2.2 Haematoxylin and Eosin (H&E) Staining 2.2.3 TUNEL Staining 2.2.4 Immunohistochemistry for Cleaved Caspase 3 2.2.5 Western Blotting for Cleaved Caspase 3 2.2.6 RNA Analysis 3 Methods 3.1 Phase Contrast Microscopy 3.2 Detection of Exposed Annexin V by Flow Cytometry 3.3 Western Blotting for Cleaved Caspase 3 3.4 Haematoxylin and Eosin (H&E) Stain 3.5 Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) 3.6 Immunohistochemistry for Activated Caspase 3 3.7 Western Blotting of Tissue Samples for Cleaved Caspase 3 3.8 Isolation of RNA and Determination of RNA Expression of Selected Proapoptotic Proteins 3.9 Conclusions 4 Notes References Chapter 11: Measuring MYC-Mediated Metabolism in Tumorigenesis 1 Introduction 2 Materials 2.1 Polar Metabolite Extraction 2.2 Lipid Extraction 3 Methods 3.1 Extraction of Polar Metabolites for LC-MS/MS Analysis 3.2 Extraction of Nonpolar Lipids for LC-MS/MS Analysis 4 Notes References Chapter 12: Methods to Study Myc-Regulated Cellular Senescence: An Update 1 Introduction 2 Materials 2.1 Reagents and Solutions for SA-β-gal Detection In Situ 2.2 Reagents and Solutions for EdU Labeling 2.3 Reagents and Solutions for Phalloidin and DAPI Staining 2.4 Reagents and Solutions for SA-β-gal Detection in Cell Lysates 2.5 Reagents and Solutions for Sudan Black B (SBB) Analogue GL13 Detection in Tissue Sections 3 Methods 3.1 Simultaneous Detection of Multiple Senescence Markers In Situ 3.2 Fluorogenic SA-β-gal Assay (MUG Assay) 3.3 Detection of Senescent Cells in Formalin-Fixed Paraffin-Embedded Tissues with Sudan Black B (SBB) Analogue GL13 4 Notes References Chapter 13: Examining Myc-Dependent Translation Changes in Cellular Homeostasis and Cancer 1 Introduction 2 Materials 2.1 35S Metabolic Labeling in Cultured Cells 2.2 O-Propargyl-Puromycin (OP-Puro) Incorporation In Vivo 2.3 Investigating Specific Alterations in mRNA Translation by Fractionating Polysome-Associated mRNAs 2.4 Toeprint Assay to Visualize the Position of Translating Ribosomes 2.5 Measuring Translation Initiation and Start Codon Usage Using a Reporter Assay 3 Methods 3.1 35S Metabolic Labeling in Cultured Cells 3.2 O-Propargyl-Puromycin (OP-Puro) Incorporation In Vivo 3.3 Investigating Specific Alterations in mRNA Translation by Fractionating Polysome-Associated mRNAs 3.4 Toeprint Assay to Visualize the Position of Translating Ribosomes 3.5 Measuring Translation Initiation and Start Codon Usage Using a Reporter Assay 4 Notes References Chapter 14: A Versatile In Vivo System to Study Myc in Cell Reprogramming 1 Introduction 2 Materials 2.1 AAV Vectors (see Note 2) 2.2 Mice and Injection Material 2.3 Immunochemistry Material 2.4 iPS Cells Isolation 3 Methods 3.1 AAV Vector Injection 3.2 Mouse Monitoring 3.3 Histopathological Analysis of Teratomas 3.3.1 Hematoxylin and Eosin Staining 3.3.2 Immunohistochemistry 3.4 Histopathological Evaluation of Organs 3.5 In Vivo Induced Pluripotent Stem Cells Isolation 3.5.1 iPS Cells Isolation from Teratomas 3.5.2 iPS Cells Isolation from Peripheral Blood 3.5.3 iPS Cells Isolation from Bone Marrow 4 Notes References Chapter 15: Using Flow Cytometry to Study Myc´s Role in Shaping the Tumor Immune Microenvironment 1 Introduction 2 Materials 2.1 Mouse Necropsy and Sample Collection 2.2 Reagents for Tumor Single-Cell Preparation 2.3 Reagents for T Cell Activation 2.4 Reagents and Antibodies for Cell Surface and Intracellular Antigen Staining 3 Methods 3.1 Mouse Necropsy and Sample Collection 3.2 Dissociation of Mouse Tumors and Single-Cell Suspension Preparation 3.3 T Cell Activation 3.4 Surface and Intracellular Flow Cytometry Staining 3.4.1 Dead Cell Labeling 3.4.2 Surface Antigen Staining 3.4.3 Intracellular Antigen Staining Transcription Factor Staining Protocol Intracellular Cytokines Staining Protocol 4 Notes References Chapter 16: Generation of a Tetracycline Regulated Mouse Model of MYC-Induced T-Cell Acute Lymphoblastic Leukemia 1 Introduction 2 Materials 2.1 Generation of the Tet-O-MYC and EμSRα-tTA Constructs 2.2 In Vitro Validation of Conditional Expression of the Tet-O-MYC Construct 2.3 Genotypic Analysis of Founders and Progeny 2.4 Identification of Founders with Conditional Gene Expression 2.5 Mouse Necroscopy and Sample Collection 2.6 FACS Analysis of Primary Tumor to Identify the Phenotype of Cells Comprising the Tumor 2.7 Generating Cell Lines from Primary Tumor Tissue 2.8 Evaluating Conditional Expression of Tet System In Vivo 2.9 In Vivo Tumor Transplantation Studies 3 Methods 3.1 Generation of the Tet-O-MYC and EμSRα-tTA Constructs 3.1.1 Preparation of the Tet-O-MYC Transgenic Construct 3.1.2 Preparation of the EμSRα-tTA Transgenic Construct 3.1.3 To Prepare the Transgenic Constructs for Microinjection 3.2 In Vitro Validation of Conditional Expression of the Tet-O-MYC Construct 3.3 Genotypic Analysis of Founders and Progeny 3.4 Identification of Founders with Conditional Gene Expression 3.5 Mouse Necropsy and Sample Collection 3.6 FACS Analysis of Primary Tumor to Identify the Phenotype of Cells Comprising the Tumor 3.7 Generating Cell Lines from Primary Tumor Tissue 3.8 Evaluating Conditional Expression of Tet System In Vivo 3.9 In Vivo Tumor Transplantation Studies 4 Notes References Chapter 17: Chromogenic In Situ Hybridization (CISH) as a Method for Detection of C-Myc Amplification in Formalin-Fixed Paraff... 1 Introduction 2 Materials 3 Methods 3.1 CISH Procedure (See Also Note 1) 3.2 Evaluation of Results 4 Notes References Chapter 18: MYC Copy Number Detection in Clinical Samples Using a Digital DNA-Hybridization and Detection Method 1 Introduction 1.1 Hybridization 1.2 Purification 1.3 Data Collection 1.4 Data Analysis 2 Materials 2.1 Fragmentation 2.2 Precipitation 2.3 Hybridization 2.4 Purification (Prep Station Step) 2.5 Data Collection (Digital Analyzer Step) 2.6 Data Analysis 3 Methods 3.1 Hybridization 3.1.1 Alu1 Restriction Enzyme Digestion 3.1.2 Covaris AFA-Based Fragmentation 3.1.3 Hybridization 3.2 Purification (Prep Station Step) 3.3 Data Collection (Digital Analyzer Step) 3.4 Data Analysis 3.4.1 Raw Data 3.4.2 Normalization 3.4.3 Quality Control 3.4.4 LogRatio Calculation 4 Notes References Chapter 19: Cell-Based Methods for the Identification of Myc-Inhibitory Small Molecules 1 Introduction 2 Materials 2.1 Cell Lines 2.2 Lentiviral and Retroviral Vectors 2.3 Other Reagents 2.4 Equipment 3 Methods 3.1 Overview 3.2 Procedure for Screening of Small Molecules for Myc Inhibition 3.3 Filtering for False Positives 3.3.1 Luciferase Inhibition/Quenchers 3.3.2 General Transcription Inhibition 3.4 Cell-Based Assays for Inhibition of Endogenous Myc-Dependent Phenotypes 3.4.1 Proliferation Assay 3.4.2 The Cell Cycle Distribution Assay 4 Notes References Index