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Through many recent remarkable developments, perhaps the most significant advancements in the study of transcriptional regulation are the development of genome-wide approaches for measuring gene expression, exemplified by gene chips (chip), and chromatin immunoprecipitation assays (ChIP) for measuring in vivo protein-DNA interactions at any genomic loci. "Transcriptional Regulation: Methods and Protocols" takes this progress and builds upon it with a collection of key protocols used in expert laboratories around the world. Divided into four convenient sections, this detailed volume explores promoter elements, transcription factors, and preinitiation complex (PIC) assembly, chromatin structure, chromatin modifying complexes, and RNA synthesis and regulation.
Written in the highly successful "Methods in Molecular Biology" series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and vital tips on troubleshooting and avoiding known pitfalls. Comprehensive and accessible, Transcriptional Regulation: Methods and Protocols equally serves senior researchers and scientists experienced in transcriptional regulation as well as graduate students and scientists who wish to study transcriptional regulation for the first time.
Part I: Promoter Elements, Transcription Factors, and Pre-Initiation Complex (PIC) Assembly
1. Genome Wide In Vivo Crosslinking of Sequence Specific Transcription Factors
2. Characterization of Complex Regulatory Networks and Identification of Promoter Regulatory Elements in Yeast: "In Silico" and "Wet-Lab" Approaches
3. Electrophoretic Mobility Shift Assay Analysis of NFkB Transcriptional Regulation by Nuclear IkBa
4. Probing Endogenous RNA Polymerase II Pre-Initiation Complexes by Electrophoretic Mobility Shift Assay
5. Elucidating Protein: DNA Complex by Oligonucleotide DNA Affinity Purification
6. Chromatin Immunoprecipitation (ChIP) Assay as Tool for Analyzing Transcription Factor Activity
7. Two-Step Crosslinking for Analysis of Protein-Chromatin Interactions
8. Chromatin Immunoprecipitation Analysis of NFkB Transcriptional Regulation by Nuclear IkBa in Human Macrophages
9. In Vivo ChIP for the Analysis of Microdissected Tissue Samples
10. Quantification of Protein-DNA Interactions by In Vivo Chromatin Immunoprecipitation in Yeast
11. Mapping Protein-DNA Interactions Using ChIP-Sequencing
12. ChIP and ReChIP Assays: Investigating Interactions Between Regulatory Proteins, Histone Modifications, and the DNA Sequences to which They Bind
13. Transcriptional Regulation of Genes Via Hypoxia-Inducible Factor
14. Exchange Protein Directly Activated By Cyclic AMP 1 (Epac1)-Regulated Recruitment of CCAAT/Enhancer Binding Proteins (C/EBPs) to the Suppressor of Cytokine Signalling-3 (SOCS-3) Promoter
Part II: Chromatin Structure
15. Computational Analysis of Promoter Elements and Chromatin Features in Yeast
16. Chromatin Affinity Purification (ChAP)
17. Determination of Histone Acetylation Status by Chromatin Immunoprecipitation
18. Immunostaining of Drosophila Polytene Chromosomes to Investigate Recruitment of Chromatin Binding Proteins
19. Detection of Transcriptional Activators, Co-Activators, and Chromatin Remodeling by Chromatin Immunoprecipitation Coupled with Real-Time PCR
20. Chromatin Endogenous Cleavage and Psoralen Crosslinking Assays to Analyze rRNA Gene Chromatin In Vivo
21. UV-Induced DNA Damage and DNA Repair in Ribosomal Genes Chromatin
22. Analysis of SUC2 Promoter Structure by Nucleosome Scanning
23. Chromatin Immunoprecipitation of Mouse Embryos
24. Chromatin Immunoprecipitation (ChIP) in Mouse Hippocampal Cells and Tissues
Part III: Chromatin Modifying Complexes
25. Approaches for Studying Nucleosome Movement by ATP-Dependent Chromatin Remodeling Complexes
26. Mapping Protein-DNA and Protein-Protein Interactions of ATP-Dependent Chromatin Remodelers
27. Evaluation of Histone Modifying Enzymes in Stem Cell Populations
28. Purification of Multiprotein Histone Acetyltransferase Complexes
29. Reconstitution of Active and Stoichiometric Multisubunit Lysine Acetyltransferase Complexes in Insect Cells
30. Affinity Purification of MLL3/MLL4 Histone H3K4 Methyltransferase Complex
31. Methods for Analyzing Histone Citrullination in Chromatin Structure and Gene Regulation
Part IV: RNA Synthesis and Regulation
32. Analysis of mRNA Abundance and Stability by Ribonuclease Protection Assay
33. Array-Based Nuclear Run-On (ANRO) Analysis
34. In Vivo Run on Assays to Monitor Nascent Precursor RNA Transcripts
35. Genome Wide Full-Length Transcript Analysis Using 5' and 3' Paired-End-Tag Next Generation Sequencing (RNA-PET)
36. Analysis of Cotranscriptionnal RNA Processing by RNA-ChIP Assay
37. Quantitative Analysis of Transcription Elongation by RNA Polymerase I In Vitro
38. Detection and Characterization of Transcription Termination
39. Promoter-Associated Noncoding RNA from the CCND1 Promoter
