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Life Sciences - Biochemistry & Biophysics | Transcriptional Regulation - Methods and Protocols

Transcriptional Regulation

Methods and Protocols

Series: Methods in Molecular Biology, Vol. 809

Vancura, Ales (Ed.)

2012, XVI, 627p. 104 illus..

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  • Presents detailed protocols developed using mammalian, yeast, and Drosophila cells and tissues, which can easily be applied across species and cell types
  • Features reliable methods tested in laboratories around the world
  • Includes expert tips and implementation advice to ensure successful results

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.

Content Level » Professional/practitioner

Keywords » ChIP assays - Chromatin immunoprecipitation - Chromatin structure - Drosophila cells - Gene expression - Histone modifications - Mass spectrometry - Pre-initiation complex (PIC) - Promoter elements - RNA synthesis - Transcription factors - Yeast and mammalian cells

Related subjects » Biochemistry & Biophysics - Human Genetics

Table of contents 

Part I: Promoter Elements, Transcription Factors, and Pre-Initiation Complex (PIC) Assembly


1. Genome Wide In Vivo Crosslinking of Sequence Specific Transcription Factors

            Xiao-Yong Li and Mark D. Biggin


2. Characterization of Complex Regulatory Networks and Identification of Promoter Regulatory Elements in Yeast: “In Silico” and “Wet-Lab” Approaches

            Nuno P. Mira, Miguel C. Teixeira, and Isabel Sá-Correia


3. Electrophoretic Mobility Shift Assay Analysis of NFkB Transcriptional Regulation by Nuclear IkBa 

            Ashish Juvekar, Sitharam Ramaswami, Subrata Manna, Tzu-Pei Chang, Adeel Zubair, and Ivana Vancurova


4. Probing Endogenous RNA Polymerase II Pre-Initiation Complexes by Electrophoretic Mobility Shift Assay

            Emmanuelle Wilhelm, Christopher Takacs, and Brendan Bell


5. Elucidating Protein: DNA Complex by Oligonucleotide DNA Affinity Purification

            Teddy T.C. Yang and Chi-Wing Chow


6. Chromatin Immunoprecipitation (ChIP) Assay as Tool for Analyzing Transcription Factor Activity

            Padmaja Gade and Dhan V. Kalvakolanu


7. Two-Step Crosslinking for Analysis of Protein-Chromatin Interactions

            Bing Tian, Jun Yang, and Allan R. Brasier


8. Chromatin Immunoprecipitation Analysis of NFkB Transcriptional Regulation by Nuclear IkBa in Human Macrophages

            Sitharam Ramaswami, Subrata Manna, Ashish Juvekar, Steven Kennedy, Ales Vancura, and Ivana Vancurova


9. In Vivo ChIP for the Analysis of Microdissected Tissue Samples

            Chris Murgatroyd, Anke Hoffmann, and Dietmar Spengler


10. Quantification of Protein-DNA Interactions by In Vivo Chromatin Immunoprecipitation in Yeast

            Amparo Pascual-Ahuir and Markus Proft


11. Mapping Protein-DNA Interactions Using ChIP-Sequencing

            Charles E. Massie and Ian G. Mills


12. ChIP and ReChIP Assays:  Investigating Interactions Between Regulatory Proteins, Histone Modifications, and the DNA Sequences to which They Bind

            Agnieszka D. Truax and Susanna F. Greer


13. Transcriptional Regulation of Genes Via Hypoxia-Inducible Factor

            Olga Roche and Michael Ohh


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

            William A. Sands, Hayley D. Woolson, Stephen J. Yarwood, and Timothy M. Palmer


Part II: Chromatin Structure


15. Computational Analysis of Promoter Elements and Chromatin Features in Yeast

            John J. Wyrick


16. Chromatin Affinity Purification (ChAP)

            Ryoko Harada and Alain Nepveu


17. Determination of Histone Acetylation Status by Chromatin Immunoprecipitation

            Luciano Galdieri, John Moon, and Ales Vancura


18. Immunostaining of Drosophila Polytene Chromosomes to Investigate Recruitment of Chromatin Binding Proteins

            Magdalena Murawska and Alexander Brehm


19. Detection of Transcriptional Activators, Co-Activators, and Chromatin Remodeling by Chromatin Immunoprecipitation Coupled with Real-Time PCR

            Tamara Y. Erkina and Alexandre M. Erkine


20. Chromatin Endogenous Cleavage and Psoralen Crosslinking Assays to Analyze rRNA Gene Chromatin In Vivo

            Joachim Griesenbeck, Manuel Wittner, Romain Charton, and Antonio Conconi


21. UV-Induced DNA Damage and DNA Repair in Ribosomal Genes Chromatin

            Julie Pelloux, Maxime Tremblay, Raymund J. Wellinger, and Antonio Conconi


22. Analysis of SUC2 Promoter Structure by Nucleosome Scanning

            Jennifer Chang and Ales Vancura


23. Chromatin Immunoprecipitation of Mouse Embryos

            Anne K. Voss, Mathew P. Dixon, Tamara McLennan, Andrew J. Kueh, and Tim Thomas


24. Chromatin Immunoprecipitation (ChIP) in Mouse Hippocampal Cells and Tissues

            Badi Sri Sailaja, Takumi Takizawa, and Eran Meshorer


Part III: Chromatin Modifying Complexes


25. Approaches for Studying Nucleosome Movement by ATP-Dependent Chromatin Remodeling Complexes

            Swetansu K. Hota and Blaine Bartholomew


26. Mapping Protein-DNA and Protein-Protein Interactions of ATP-Dependent Chromatin Remodelers

            Swetansu K. Hota, Mekonnen Lemma Dechassa, Punit Prasad, and Blaine Bartholomew


27. Evaluation of Histone Modifying Enzymes in Stem Cell Populations

            Leanne Stalker and Christopher Wynder


28. Purification of Multiprotein Histone Acetyltransferase Complexes

            Yuan-Liang Wang, Francesco Faiola, and Ernest Martinez


29. Reconstitution of Active and Stoichiometric Multisubunit Lysine Acetyltransferase Complexes in Insect Cells

            Kezhi Yan, Chao-Jung Wu, Nadine Pelletier, and Xiang-Jiao Yang


30. Affinity Purification of MLL3/MLL4 Histone H3K4 Methyltransferase Complex

            Young-Wook Cho, SunHwa Hong, and Kai Ge


31. Methods for Analyzing Histone Citrullination in Chromatin Structure and Gene Regulation

            Pingxin Li, Jing Hu, and Yanming Wang


Part IV: RNA Synthesis and Regulation


32. Analysis of mRNA Abundance and Stability by Ribonuclease Protection Assay

            Cristina Romero-López, Alicia Barroso-delJesus, Pablo Menendez, and Alfredo Berzal-Herranz


33. Array-Based Nuclear Run-On (ANRO) Analysis

            Jinshui Fan, Yu-Chi Chen, Tonya Watkins, Chi V. Dang, Myriam Gorospe, and Chris Cheadle


34. In Vivo Run on Assays to Monitor Nascent Precursor RNA Transcripts

            Piergiorgio Percipalle and Emilie Louvet


35. Genome Wide Full-Length Transcript Analysis Using 5’ and 3’ Paired-End-Tag Next Generation Sequencing (RNA-PET)

            Xiaoan Ruan and Yijun Ruan


36. Analysis of Cotranscriptionnal RNA Processing by RNA-ChIP Assay

            Danielle Bittencourt and Didier Auboeuf


37. Quantitative Analysis of Transcription Elongation by RNA Polymerase I In Vitro

            David Alan Schneider


38. Detection and Characterization of Transcription Termination

            Ghada Ghazal, Jules Gagnon, and Sherif Abou Elela


39. Promoter-Associated Noncoding RNA from the CCND1 Promoter

            Xiaoyuan Song, Xiangting Wang, Shigeki Arai, and Riki Kurokawa

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