Precise regulation of the levels and timing of gene expression is fundamental to all biological processes and is largely determined by the activity of cis-regulatory modules, containing the binding sites for transcription factors, within promoters and enhancers. The global identi ...
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 de ...
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) a ...
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. St ...
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. ...
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 nucle ...
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 t ...
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 skelet ...
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 thro ...
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 cro ...
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 pursu ...
Affinity purification and mass spectrometry analysis have been used to identify and characterize protein complexes. Wdr82-associated chromatin modifying complexes were purified by single-step FLAG affinity purification from human cells induced to express FLAG-tagged W ...
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 associ ...
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 inve ...
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, w ...
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.
Identification and verification of novel transcription factor interactions is an inherent step in the discovery of molecular mechanisms driving gene transcription and regulation. Co-immunoprecipitation and GST-pull down are often key techniques in the verification proc ...
Fluorescence cross-correlation spectroscopy (FCCS) is an established spectroscopic method to observe the interaction between the different fluorescent molecules. Using FCCS, researchers can assess the interaction of target molecules in the aqueous condition, and can app ...
TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiati ...
Proteins that bind to DNA can elicit changes in DNA conformation, such as bending and looping, which are important signals for later events such as transcription. TATA-binding protein (TBP) is one example of a protein that elicits a conformational change in DNA; TBP binds and sharply bends its reco ...
