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DNase I Digestion of Isolated Nulcei for Genome-Wide Mapping of DNase Hypersensitivity Sites in Chromatin
DNase I hypersensitivity (DHS) analysis is a powerful method to analyze chromatin structure and identify genomic regulatory elements. Integration of a high-throughput detection method into DHS analysis makes genome-wide mapping of DHS sites possible at a reasonable cost. Here we describe methods for DHS analysis carried out with mouse liver nuclei, involving DNase I digestion followed by isolation of DNase I-released DNA fragments suitable for high-throughput, next generation DNA sequencing (DNase-seq). A real-time PCR-based assay used to optimize DNase I digestion conditions is also described. (Source: Springer protocols ...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Methods for Studies of Protein Interactions with Different DNA Methyltransferases
There are now many methods available for studying protein interactions between DNA methltransferases (DNMTs) and their binding partners. Here we describe a step-by-step procedure to identify whether proteins of interest interact with DNMTs by co-immunoprecipitation (co-IP) assay in transiently transfected cells. Though one mammalian cell is described, investigators can use the same method with other cell lines or primary cells for in vivo protein interaction studies. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Fluorescence Anisotropy Microplate Assay to Investigate the Interaction of Full-Length Steroid Receptor Coactivator-1a with Steroid Receptors
Estrogens, acting via estrogen receptor (ER) play key roles in growth, differentiation, and gene regulation in the reproductive, central nervous, and skeletal systems. ER-mediated gene transcription contributes to the development and spread of breast, uterine, and liver cancer. Steroid receptor coactivator-1a (SRC1a) belongs to the P160 family of coactivators, which is the best known of the many coactivators implicated in ER-mediated transactivation. Binding of full-length P160 coactivators to steroid receptors has been difficult to investigate in vitro. This chapter details how to investigate the interaction of SRC1a with...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Mammalian Two-Hybrid Assays for Studies of Interaction of p3 with Transcription Factors
The two-hybrid system is a powerful genetic assay that allows the interaction between two proteins to be detected in vivo. It was originally described in 1989 and since then it has been one of the main techniques used to identify interactions between proteins from different cellular organisms. Here we describe the methods to study the interaction of p300 with other transcription factors, specifically between p300 and two transcription factors related to hypoxia and inflammation, HIF-1α and NF-κB-p65, respectively. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Sedimentation and Immunoprecipitation Assays for Analyzing Complexes that Repress Transcription
Co-repressor proteins function as platforms for the assembly of multi-subunit complexes that mediate transcriptional repression. Common components of such complexes are histone deacetylases, which catalyze the removal of acetyl groups from the tails of histones within nucleosomes, resulting in chromatin compaction and gene repression. In addition, co-repressor complexes generally interact with sequence-specific DNA-binding proteins that direct association with regulatory elements in the genome. Thus, identifying proteins that stably associate with co-repressors can provide insights regarding the biochemical function and ta...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Isolation of Nuclei for Use in Genome-Wide DNase Hypersensitivity Assays to Probe Chromatin Structure
DNase hypersensitivity (DHS) analysis coupled with high-throughput DNA sequencing (DNase-seq) has emerged as a powerful tool to analyze chromatin accessibility and identify regulatory sequences in genomic DNA on a global scale. In this method, intact nuclei are isolated from fresh tissue or cultured cells and then subjected to limited digestion using DNase I. The resulting short DNA fragments released by DNase digestion, which correspond to regions of open chromatin structure, are subsequently purified and identified by high throughput next generation DNA sequencing. This chapter describes methods used to isolate intact nu...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Histone Deacetylase Inhibitor Valproic Acid as a Small Molecule Inducer to Direct the Differentiation of Pluripotent Stem Cells
Pluripotent stem cells can be directed into myogenic differentiation by small molecular inducers, which preferentially activate muscle-specific transcription networks. Here we describe how to efficiently direct the differentiation of pluripotent P19 cells into skeletal muscle lineage by using histone deacetylase inhibitor, valproic acid and the ligand of retinoic acid receptor, all-trans retinoic acid. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Use of Histone Deacetylase Inhibitors to Examine the Roles of Bromodomain and Histone Acetylation in p3-Dependent Gene Expression
The bromodomain is an evolutionarily conserved motif harbored by many transcription regulators and nearly all nuclear histone acetyltransferases including the transcriptional coactivator p300. The function of p300 is required for the expression of an array of genes, in part through histone acetylation. Here, we describe an experimental approach to examine the role of either the wild-type or a bromo-deficient p300 in the expression of p300-dependant genes. The role of histone acetylation in the expression of p300-dependent genes can also be assessed by targeting histone deacetylase activities using an inhibitor approach. (S...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Analysis of p3 Occupancy at the Early Stage of Stem Cell Differentiation by Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) is an invaluable method to study the specific interaction of regulatory proteins with genomic DNA. Since its first development, it has been modified extensively to make it applicable to many different cell types and experimental systems. The cross-linking of regulatory proteins to genomic DNA requires monolayer cells or single cell suspensions. Here, we describe a ChIP protocol using embryoid bodies formed at the early stage differentiation of pluripotent stem cells, which we have used to determine long-range p300-dependent regulatory elements of myogenic-specific genes. (Source: Spring...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Heavy Methyl-SILAC Labeling Coupled with Liquid Chromatography and High-Resolution Mass Spectrometry to Study the Dynamics of Site-Specific Histone Methylation
Histone lysine and arginine methylation involved in gene activation and silencing is dynamically regulated. However, partly limited to the research technologies previously available, the dynamics of global histone methylation on a site-specific basis have not been fully pursued. Heavy methyl-SILAC (Stable Isotope Labeling of Amino Acids in Cell Culture) labeling provides a remarkable signpost to distinguish the preexisting and newly generated methyl marks on histones. Using this technology coupled with quantitative LC-MS analysis make it possible to monitor changes in the dynamics of histone site-specific methylation. In t...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Simple and Efficient Identification of Chromatin Modifying Complexes and Characterization of Complex Composition
We describe here simple and efficient approaches for the identification of chromatin modifying complexes and subsequent characterization of complex composition. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Immunoaffinity Purification of Protein Complexes from Mammalian Cells
In this chapter, we describe a purification scheme designed to isolate multisubunit protein complexes gently and quickly from crude extracts of mammalian cells using immunoaffinity purification of epitope tagged proteins and the multisubunit complexes with which they associate. As an example we describe isolation of the mammalian Mediator complex from HeLa S3 cells. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Peptide Microarrays for Profiling of Serine/Threonine Kinase Activity of Recombinant Kinases and Lysates of Cells and Tissue Samples
Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be investigated as well. The method for performing experiments on dynamic peptide microarrays with real-time readout is described, as well as the influence of assay parameters and suggestions for optimization of experiments. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Preparation of Cell Lines for Single-Cell Analysis of Transcriptional Activation Dynamics
Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Gene Regulation
This review concisely highlights the complexity of regulatory events. It provides examples of how interconnectivity of regulatory hubs could maintain transcriptional synergy and orchestrate the proper spatial and temporal patterns of gene expression. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news