Chapter 1: A Review of Post-translational Modifications and Subcellular Localization of Ets Transcription Factors: Possible Con
1. Introduction
1.1. The Ets Factors
2. Regulation of Ets Transcriptional Factors by the Post-translational Modifications
2.1. Cooperation of Phosphorylation and Sumoylation as Mechanisms of Downregulation of Tel Repressor Function
2.2. Antagonism Between Phosphorylation and Sumoylation Regulates Elk-1 Activity
2.3. Diverse Post-translational Modifications Regulate the Dual Role of Net as a Repressor and/or Activator
2.4. Multiple Phosphorylations and Acetylation Regulates Er81 Activity
2.5. Synergism Between Phosphorylation and Glycosylation Regulates Elf-1 Activity, Subcellular Localization, and Degradation
2.6. Phosphorylation Tightly Regulates Fli-1 and Mef Half-Life and Activity
2.7. Sumoylation and Ubiquitination Negatively Regulate the Activity of Ets-1, Pea3, and Erm
3. Ets Post-translational Modifications in Cancer
3.1. Ets Post-translational Modifications in Human Diseases
3.2. Ets Factor Deregulation and Post-translational Modification in Cancer
3.2.1. Ets Deregulation
3.2.2. Ets Post-translational Modifications
3.2.3. Phosphorylation
3.2.4. Acetylation
3.2.5. Sumoylation and Subcellular Transport
3.3. Ets Factors as Targets for Cancer Diagnosis and Therapy
4. Involvement of Ets Factors in the Hypoxic Response
4.1. Transcriptional Regulation of Ets Factors in Hypoxia
4.2. Cooperation of Ets Factors and HIF in Hypoxic Induction of Target Genes
4.3. Involvement of Ets Factors in the Hypoxic Induction of Target Genes
4.4. The Ets Factor Net is a Regulator of HIF-1a Protein Stability
5. Studying Ets Factors Post-translational Modifications in Living Animals
5.1. Strategies for Studies of Ets Factors Phosphorylation in Mouse Cancer Models
5.1.1. Ets-2 Phosphorylation: Mutation of the Modification Site
5.1.2. Elk-1 Phosphorylation: Imaging with Bioluminescence
5.1.3. Net Phosphorylation: Antibodies Designed to Target Modification
5.2. Ets-2 Phosphorylation in Macrophages in Mouse
5.3. Elk-1 Phosphorylation in Neurons in the Rat Model
5.4. Ets Post-translational Modifications in Invertebrate Animal Models
5.4.1. Drosophila melanogaster
5.4.2. Caenorhabditis elegans
6. Perspectives
References
Chapter 2: Regulation of Transcription Factor Function by Targeted Protein Degradation: An Overview Focusing on p53, c-Myc, an
1. Introduction
2. Regulation of Transcription Factor Expression by Targeted Protein Degradation
2.1. p53
2.2. c-Myc
2.3. c-Jun
3. Conclusions
References
Chapter 3: Review of Molecular Mechanisms Involved in the Activation of the Nrf2-ARE Signaling Pathway by Chemopreventive Age
1. Introduction
2. The Importance of the Nrf2 Transcription Factor in the Induction of Genes Encoding Antioxidant and Phase II Detoxification
3. Structure and Functions of the Members of the Cytoplasmic Oxidative Stress System (Nrf2–Keap1)
4. The Role of the Cytoplasmic Oxidative Stress System (Nrf2–Keap1) Under Basal (Reducing) Conditions
5. Effects of ARE-Inducers-Dependent Post-translational Modifications (Modification of Keap1 Cysteines, Phosphorylation) on th
5.1. Modification of Keap1 Cysteines (C257, C273, C258, C297 and in Particular C151) by ARE Inducers and Disruption of the Ke
5.2. Modification of Keap1 Cysteines by ARE Inducers and Disruption of the Keap1–Cul3 Interaction
5.3. Role of Ubiquitination of Keap1 in Downregulation of Nrf2 Ubiquitination by Cul3 Keap1–Nrf2 Complex and Increased Nr
5.4. Nrf2/Keap1 Phosphorylation by Protein Kinases and Activation of the Nrf2–Keap1 Complex
6. Alteration of Import and Export of Nrf2 by ARE Inducers
6.1. Nrf2 Phosphorylation Events by Chemopreventive Agents and Nrf2 Localization
6.2. Modification of Nrf2 Cysteines (C183, C506), Nrf2 Nuclear Accumulation, and Transactivation of Target Genes
7. Conclusions
References
Chapter 4: Subnuclear Localization and Intranuclear Trafficking of Transcription Factors
1. Introduction
2. Materials
2.1. Preparation of Metaphase Chromosome Spreads from Suspension and Adherent Cell Cultures
2.2. Nuclear Matrix Intermediate Filament Preparation
2.3. Microscopy
3. Methods
3.1. Preparation of Metaphase Chromosome Spreads from Suspension and Adherent Cell Cultures
3.1.1. Suspension Cells
3.1.2. Adherent Cells
3.1.3. Slide Preparation
3.2. Nuclear Matrix Intermediate Filament Preparation
3.2.1. Whole Cell (WC) Preparation
3.2.2. Cytoskeleton (CSK) Preparation
3.2.3. Nuclear Matrix Intermediate Filament (NMIF) Preparation
3.2.4. Immunostaining of the Samples
3.3. Microscopy
3.3.1. Fluorescence Microscopy
3.3.2. Viewing Live Cells Using the Confocal Microscope
3.3.3. Preparation of Live Cell Stage, a Closed System Chamber
3.3.4. Microscope Preparation
3.4. Fluorescence Recovery after Photobleaching (FRAP)
3.5. Intranuclear Informatics
4. Notes
References
Chapter 5: Analysis of Ligand-Dependent Nuclear Accumulation of Smads in TGF-b Signaling
1. Introduction
2. Materials
2.1. Cell Culture
2.2. Live Cell Treatment
2.3. Cellular Fractionation
2.4. Immuno-fluorescence
2.5. Live Cell Imaging
3. Methods
3.1. Cellular Fractionation of Cytoplasm and Nuclear Proteins
3.2. Determining Protein Concentration and Performing Western Blot Analysis
3.3. Immuno-fluorescence Detection of Smad2 and Smad4
3.4. Image Analysis for Immunofluorescence and Live Cell Imaging
3.5. Live Cell Imaging of GFP-Smad4 and YFP-Smad2
4. Notes
References
Chapter 6: Raf/MEK/MAPK Signaling Stimulates the Nuclear Translocation and Transactivating Activity of FOXM1
1. Introduction
2. Materials
2.1. Cell Culture, Synchronization, and Flow Analysis
2.2. Immunostaining
2.3. Western Blotting to Monitor MAPK Activity
2.4. Transient Reporter Assays
3. Methods
3.1. Synchronization of BJ1 Cells
3.2. Study of FOXM1 Nuclear Translocation by Immunostaining
3.3. Study of FOXM1 Nuclear Translocation Following Pharmacological Modulation of MAPK Signaling
3.4. Transient Reporter Assays to Test the Effect of Raf/MEK/MAPK Signaling on FOXM1 Transactivating Activity
4. Notes
References
Chapter 7: Coupling of Dephosphorylation and Nuclear Export of Smads in TGF-b Signaling
1. Introduction
2. Materials
2.1. Cell Culture Reagents (Hyclone)
2.2. Chemicals and Buffers
2.3. Plasmids and Reporter Reagents
2.4. Antibodies
3. Methods
3.1. Mammalian Cell Culture
3.2. Cell Fractionation Assay
3.3. In Vitro Export Assay
3.4. Quantitative Smad2 Export Assay
3.5. In Vitro Phosphatase Assay
4. Notes
References
Chapter 8: Assessing Sequence-Specific DNA Binding and Transcriptional Activity of STAT1 Transcription Factor
1. Introduction
2. Materials
3. Methods
3.1. Cell Culture
3.2. Plasmid Construction
3.3. Pharmacological Suppression of STAT Signaling
3.4. Western Blotting
3.5. Fluorescence Microscopy
3.6. Immunocyto-chemistry
3.7. Antibody Microinjection Assay
3.8. Purification of Recombinant STAT1
3.9. Import Assays with Permeabilized Cells
3.10. Digitonin Assay for Assessing Increased DNA-Binding Activity
3.11. Depho-sphorylation Assays
3.12. Fluorescence Recovery After Photobleaching
3.13. Pull-Down Assay with Biotinylated Oligos
3.14. Electrophoretic Mobility Shift Assay
3.15. Reporter Gene Assay
4. Notes
References
Chapter 9: Analysis of Nuclear Export Using Photoactivatable GFP Fusion Proteins and Interspecies Heterokaryons
1. Introduction
2. Materials
2.1. Keratinocyte Culture
2.2. PEI Transfection Mix
2.3. Microscopy Equipment
2.4. Expression Vectors and Pharmacological Inhibitors
2.5. Microscopy Experiments and Heterokaryon Formation Assays
3. Methods
3.1. Preparation of Collagen-Coated Culture Substrates
3.2. Preparation of Primary Murine Keratinocyte Cultures
3.3. Transient Transfection and Inhibitor Treatments
3.4. Optimization of PA-GFP Fusion Protein Photoactivation
3.5. Photoactivation and Confocal Microscopy
3.6. Interspecies Heterokaryon Assays
3.7. Indirect Immunofluorescence Microscopy Analysis of Heterokaryons
4. Notes
References
Chapter 10: Determination of Nuclear Localization Signal Sequences for Krüppel-Like Factor 8
1. Introduction
2. Materials
2.1. PCR and DNA Agarose Gel Electrophoresis (DAGE)
2.2. Cell Culture and Transfection
2.3. SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting
2.4. Immuno-fluorescent Staining and Microscopy
3. Methods
3.1. Overall Research Flow (Fig. 1)
3.2. NLS Sequence Search (Fig. 2)
3.3. PCR-Based Mutagenesis and DAGE to Disrupt the Predicted NLS Sequences (Fig. 3)
3.3.1. Mutant Primer Design
3.3.2. Overlapping PCR
3.4. DAGE and DNA Purification
3.4.1. Making and Running Agarose Gel
3.4.2. Purification of DNA Fragments from DNA Agarose Gels
3.4.3. Restriction Digestion
3.4.4. Ligation
3.4.5. Transformation
3.4.6. Plasmid DNA Preparation
3.5. Transfection and Western Blotting to Confirm the Expression of the Mutant Proteins
3.5.1. Transfection
3.5.2. SDS-PAGE
3.5.3. Western Blotting
3.6. IF Staining and Microscopy to Determine the Requirement of the Predicted NLS Sequences for the Nuclear Localization (Fig.
3.6.1. Set up Cells for Transfection
3.6.2. Immunostaining for Fluorescent Microscopy
3.6.3. GFP-based Fluorescent Microscopy to Determine the Sufficiency of the Predicted NLS Sequences for the Nuclear Localizati
4. Notes
References
Chapter 11: Methods to Measure Nuclear Export of b-Catenin Using Fixed and Live Cell Assays
1. Introduction
2. Materials
2.1. Cell Culture
2.2. Immuno-fluorescence
2.3. Nuclear Export Assays
2.4. Nuclear Export Using Fluorescent Recovery After Photobleaching
3. Methods
3.1. Cell Culture, Transfection, and Drug Treatment
3.2. Localization of b-Catenin by Immunofluorescence Microscopy
3.3. Nuclear Export of Endogenous b-Catenin in Permeabilized Cells
3.4. Identification of Sequences with Nuclear Export Activity Using a Transfection-Based Assay
3.5. Nuclear Export of b-Catenin in Live Cells Using Fluorescence Recovery After Photobleaching
4. Data Analysis
5. Notes
References
Chapter 12: Imaging of Transcription Factor Trafficking in Living Cells: Lessons from Corticosteroid Receptor Dynamics
1. Introduction
2. Materials
2.1. Plasmid Construct
2.2. Transfection
2.3. Cell Culture
3. Live Cell Imaging
4. Methods
4.1. Plasmid Construction
4.2. Cell Culture and Transfection
4.3. Examination of Transcriptional Activity
4.4. Live Cell Imaging
4.4.1. Time-Lapse Imaging
4.4.2. FRAP Analysis
4.4.3. FRET Analysis
4.4.4. Ratio Imaging
4.4.5. Emission Spectra
4.4.6. Acceptor Photobleaching
5. Notes
References
Chapter 13: Hypoxia-Inducible Factors: Post-translational Crosstalk of Signaling Pathways
1. Introduction
1.1. HIFs Family: Basic Biology
1.2. HIF Regulation
1.2.1. Hydroxylation
1.2.2. Ubiquitination
1.2.3. Acetylation
1.2.4. Phosphorylation
1.2.5. S-Nitrosylation
1.2.6. SUMOylation
1.3. Conclusion
2. Materials
2.1. Cell Culture Techniques
2.2. Recombinant Proteins
2.2.1. GST-Recombinant Protein Expression and Purification in Bacteria
2.2.2. 35S-Labeled Protein In Vitro Translation
2.3. Protein–Protein Interaction Techniques
2.3.1. CoIP
2.3.2. GST Pull-Down
2.4. Hydroxylation
2.4.1. Hydroxylation Activity Assay
2.4.1.1. HIF Hydroxylation
2.5. Acetylation
2.5.1. Acetyltransferase Assay
2.5.2. In Vitro Acetylation Assay
2.6. Phosphorylation
2.6.1. Growth Factor Treatment/Inhibitory Studies
2.6.2. Phosphorylation of GST-Fusion Proteins by Recombinant GSK3b In Vitro
3. Methods
3.1. Cell Culture Techniques
3.2. Recombinant Proteins
3.2.1. GST-Recombinant Protein Expression in Bacteria and Purification
3.2.2. 35S-Labeled Protein In Vitro Translation
3.3. Protein–Protein Interaction Techniques
3.3.1. CoimmunoprecipiŁtation
3.3.2. GST Pull-Down Assay
3.4. Hydroxylation
3.4.1. HIF Hydroxylation
3.5. Acetylation
3.5.1. Acetyltransferase Assay
3.5.2. In Vitro Acetylation Assay
3.6. Phosphorylation
3.6.1. Growth Factor Treatment/Inhibitor Studies
3.6.2. Phosphorylation of GST-Fusion Proteins by Recombinant GSK3b In Vitro
4. Notes
References
Chapter 14: The Basic-Helix-Loop-Helix-Leucine Zipper Gene Mitf : Analysis of Alternative Promoter Choice and Splicing
1. Introduction
2. Materials
2.1. Embryo Harvesting and Tissue Grinding
2.2. Cell Culture and Transfection
2.3. RNA Extraction and cDNA Preparation
2.4. Polymerase Chain Reaction
2.5. Agarose Gel
2.6. Real-Time PCR Analysis
3. Methods
3.1. Sample Preparation for Isoform Analysis
3.1.1. Harvesting Embryonic Tissue for Detection of Alternate Promoter Isoforms of Mitf at Different Developmental Stages. Exa
3.1.2. Harvesting Transfected Cells for Assaying Alternative Splicing of Different Mitf Exons
3.2. Preparation of RNA and cDNA From Embryonic Tissue or Transfected Cells
3.2.1. RNA Extraction Using RNeasy Mini RNA Extraction Kit From Qiagen
3.2.2. cDNA Preparation Using Superscript First-Strand Synthesis System for RT-PCR
3.2.3. Setting Up a PCR
3.2.4. Agarose Gel Electrophoresis
3.3. Quantitative Real-Time PCR
3.3.1. Primer Design for Alternative Promoter Isoforms
3.3.2. Primer Design for Alternatively Spliced Exons
3.3.3. Generation of Standard Curve and Real-Time Analysis of Alternative Promoter Isoforms
3.3.4. Generation of Standard Curve and Real-Time Analysis of Alternatively Spliced Exons
4. Notes
References
Chapter 15: Phosphorylation Control of Nuclear Receptors
1. Introduction
2. Identification of Nuclear Receptors Phosphorylation Sites
3. Identification of Protein Kinases Involved in NRs Phosphorylation
4. Dynamics of NRs Phosphorylation
5. Phosphorylation and NRs Subcellular Localization
6. NRs Phosphorylation and Transcription Regulation
7. How Phosphorylation Regulates NR Transcriptional Activity
7.1. Phosphorylation and NRs Structure
7.2. NR Phosphorylation and Ligand Binding
7.3. Phosphorylation and Coregulators Interactions
7.4. Phosphorylation and NRs DNA Binding
8. Phosphorylation and Other NR Modifications
9. Conclusion
References
Chapter 16: Regulation of Krüpple-Like Factor 5 by Targeted Protein Degradation
1. Introduction
2. Materials
2.1. Cell Culture and Transfection
3. Methods
3.1. Chase Assays for the KLF5 Degradation by WWP1 In Vivo
3.1.1. The CHX Chase Assay
3.1.2. The Pulse Chase Assay
3.2. The Ubiquitination Assay In Vitro for KLF5
3.3. Immuno-precipitation (IP) Under Denaturing Conditions for the KLF5 Protein Ubiquitination by WWP1 In Vivo
4. Notes
References
Chapter 17: Post-translational Control of ETS Transcription Factors: Detection of Modified Factors at Target Gene Promoters
1 Introduction
2 Materials
2.1 Cell Culture, Cross-Linking, Lysis and Sonication
2.2 Chromatin Immunoprecipitation and Washing Immune Complexes
2.3 Elution, Cross-Link Reversal and DNA Purification
2.4 PCR Analysis
3 Methods
3.1 Mitogen Stimulation, Cross-Linking, Cell Lysis and Sonication
3.2 ChIP and Washing Immune Complexes
3.3 Elution, Cross-Link Reversal and DNA Purification
3.4 PCR Analysis
4 Notes
References
Chapter 18: Integration of Protein Kinases into Transcription Complexes: Identifying Components of Immobilised In Vitro Pre-i
1. Introduction
2. Materials
2.1. Cell Culture, Mitogen Stimulation and Nuclear Extract Preparation
2.2. Preparation of Immobilised Templates
2.3. Assembly of Pre-initiation Complexes and Elution
2.4. SDS-PAGE and Immunoblotting
3. Methods
3.1. Mitogen Stimulation and Nuclear Extract Preparation
3.2. Preparation of Immobilised Templates
3.3. Pre-initiation Complex Assembly and Elution
3.4. SDS-PAGE and Immunoblotting
4. Notes
References
Chapter 19: Post-translational Modification of p53 by Ubiquitin
1. Introduction
2. Materials
2.1. Cell Culture, Transfection, and Lysis
2.2. SDS-PolyaŁcrylamide Gel Electrophoresis (SDS-PAGE)
2.3. Western Blotting for Ubiquitinated p53
2.4. In Vitro Reconstituted Ubiquitination Assay
2.5. In Vitro Transcription/Translation
3. Methods
3.1. Transfections and Prepration of Samples for Immunoprecipitation
3.2. ImmunopreciŁpitation
3.3. SDS-PAGE and Western Transfer of Proteins
3.4. Immunoblotting for Detection of Modified p53 Protein
3.5. Preparation of p53 Protein Through In Vitro Translation
3.6. In Vitro Ubiquitination Assay
4. Notes
References
Chapter 20: Phosphorylation-Dependent Regulation of SATB1, the Higher-Order Chromatin Organizer and Global Gene Regulator
1. Introduction
2. Materials
2.1. Purification of SATB1
2.2. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue Staining
2.3. In Vitro Phosphorylation
2.4. Drying of the Gel and Autoradiography
2.5. Western Blotting of Phosphorylated SATB1
2.6. Cells, Cell Culture, and Treatments
2.7. Immunoprecipi-tation (IP) of SATB1
2.8. Reporter Assay
3. Methods
3.1. Purification of SATB1
3.2. SDS-PAGE and Coomassie Staining
3.3. In Vitro Phosphorylation of SATB1
3.4. Western Blotting
3.5. Immunoprecipi-tation of SATB1
3.6. Treatments to the Cells and Reporter Assays
4. Notes
References
Chapter 21: In Vivo and In Vitro Tools to Identify and Study Transcriptional Regulation of USF-1 Target Genes
1. Introduction
2. Materials
2.1. Cell Culture Reagents
2.2. RNA Purification, Quality Control, and Reverse Transcription (RT) Reaction
2.3. Real Time PCR
2.4. Transient Tranfections Assays of Recombinant Proteins and Promoter-Genes
2.5. Luciferase Assays
3. Methods
3.1. Cell Culture and RT-qPCR
3.1.1. Day 1 Procedures
3.1.2. Day 2 Procedures
3.1.3. Day 3 Procedures
3.1.4. Day 4 Procedures
3.1.5. Data Analysis
3.2. Luciferase Assay
3.2.1. Day 1 Procedures
3.2.2. Day 2 Procedures
3.2.3. Day 4 Procedures
4. Notes
References
Chapter 22: Measuring the Absolute Abundance of the Smad Transcription Factors Using Quantitative Immunoblotting
1. Introduction
1.1. Relevant Background Information on Immunoblotting
1.2. Outline of a Quantitative Immunoblotting Experiment
2. Materials
2.1. Cell Culture and TGF-b Treatment
2.2. Cell Lysis and Sample Preparation
2.3. SDS-PAGE
2.4. Coomassie Staining
2.5. Immunoblotting
3. Methods
3.1. Cell Culture and TGF-b Treatment
3.2. Cell Lysis and Sample Preparation
3.3. SDS-PAGE
3.4. Coomassie Staining
3.5. Immunoblotting
3.6. Densitometry Using ImageJ
3.7. Data Analysis and Calculations for Coomassie-Stained Gels Using Microsoft Excel
3.8. Data Analysis and Calculations for Immunoblot Data Using Microsoft Excel
4. Notes
References
Chapter 23: Flow Cytometry Analysis of Transcription Factors in T Lymphocytes
1. Introduction
2. Materials
2.1. Mice
2.2. Instruments and Disposables
2.3. Antibodies and Reagents
2.4. Media and Buffers
3. Methods
3.1. Overview
3.2. Tissue and Cells Suspension Preparation
3.3. Staining of Lymph Node or Spleen Cellular Suspensions for Flow Cytometry Analysis
3.3.1. Surface Staining for CD4 and CD8 Markers
3.3.2. Fixation for Flow Cytometry
3.3.3. Permeabilization for Flow Cytometry
3.3.4. Addition of Anti-BCL11B and Secondary Antibodies
3.3.5. Flow Cytometry Data Acquisition and Analysis
3.4. Purification of CD4+ T Lymphocytes
3.5. ImmunocytoŁchemistry
3.6. Cytoplasmic/Nuclear Fractionation
4. Notes
References
Chapter 24: Identification of Specific Protein/E-Box-Containing DNA Complexes: Lessons from the Ubiquitously Expressed USF Tran
1. Introduction
2. Materials
2.1. Cell Nuclear Extract Preparation
2.2. Probe Preparation
2.3. EMSA–ŁCompetition–Supershift
2.4. Probe Binding Reaction Reagents
2.5. Competition and Supershift Assays
2.6. ChIP
2.6.1. Crosslinking Step
2.6.2. Chromatin Preparation
2.6.3. Immunoprecipitation
2.7. DNA Preparation
2.8. Real Time PCR
3. Methods
3.1. Cell Nuclear Extract Preparation
3.2. Probe Selection, Annealing, and Labeling
3.3. EMSA
3.4. Competition Assay
3.5. Supershift
3.6. ChIP
3.6.1. Crosslinking Step (Day 1)
3.6.2. Chromatin Preparation (Day 1)
3.6.3. Immunoprecipitation (Day 1)
3.6.4. Collection of Complexes (Day 2)
3.6.5. DNA Preparation and PCR (Day 3)
4. Notes
References