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Methods for accurately quantitating changes in histone post-translational modifications are necessary for developing an understanding of how their dynamic nature influences nuclear events involving access to genomic DNA. This article describes methods for the use of in vivo stable isotope label incorporation for quantitating the levels of modification at specific residues in histone proteins. These...
We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how to measure the degree of chromatin assembly in the...
Acetylation of histone tails by histone acetyltransferase (HAT) enzymes is a key post-translational modification of histones associated with transcriptionally active genes. Acetylation of the physiological nucleosome substrate is performed in cells by megadalton complexes such as SAGA and NuA4. To understand how HAT enzymes specifically recognize their nucleosome and not just histone tail substrates,...
Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein–DNA interactions. Here, we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite...
Regulation of many biological processes often occurs by DNA sequences positioned over a large distance from the site of action. Such sequences, capable of activating transcription over a distance, are termed enhancers. Several experimental approaches for analysis of the mechanisms of communication over a distance between DNA regions positioned on the same molecule and, in particular, for analysis...
The eukaryotic genome is packaged into chromatin, and chromatin modification and remodeling play an important role in transcriptional regulation, DNA replication, recombination and repair. Recent findings have shown that various post-translational histone modifications cooperate to recruit different effector proteins that bring about mobilization of the nucleosomes and cause distinct downstream consequences...
The core histone tail domains play important roles in different stages of chromatin condensation. The tails are required for folding nucleosome arrays into secondary chromatin structures such as the ∼30nm diameter chromatin fiber and for mediating fiber–fiber interactions important for formation of tertiary chromatin structures. Crosslinking studies have demonstrated that inter-nucleosomal tail–DNA...
DNA packaging into chromatin imposes several levels of regulation on the central nuclear processes of DNA replication, recombination, repair and transcription. ATP-dependent chromatin-remodeling enzymes play a critical role in this regulation by altering the accessibility of nucleosomal DNA. Remodeling can result in large-scale changes in chromatin, such as the formation of heterochromatin, or smaller...
Atomic force microscopy (AFM) is a technique that can directly image single molecules in solution and it therefore provides a powerful tool for obtaining unique insights into the basic properties of biological materials and the functional processes in which they are involved. We have used AFM to analyze basic features of nucleosomes in arrays, such as DNA–histone binding strength, cooperativity in...
Macromolecular assemblies and machines undergo large-scale conformational changes as essential features of their normal function. Modern stopped-flow instrumentation and biotechnology combine to provide a powerful tool for characterizing the rates and natures of these conformational changes. Standard commercially available instruments provide extraordinary sensitivity and speed, allowing analysis...
Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing...
Elucidating how the metazoan genome is organised into distinct functional domains is fundamental to understanding all aspects of normal cellular growth and development. The “histone code” hypothesis predicts that post-translational modifications of specific histone residues regulate genomic function by selectively recruiting nuclear factors that modify chromatin structure. A paradigm supporting this...
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