In nucleo enzymatic assays for the identification and characterization of histone modifying activities.
The impact of histone phosphorylation, acetylation, and methylation onto transcription and various other cellular DNA-mediated processes is now well established. Numerous histone modification marks, specific sites carrying particular post-translational modifications, have been described and analyzed in detail. Whereas, many methods for the study of histone post-translational modifications involve sophisticated equipment and techniques, we describe the revival of a very simple procedure for the analysis of histone modifications and histone modifying enzymes, which is derived from experiments first carried out several decades ago. This method is based on the isolation of cell nuclei containing intact chromatin structures that are then incubated with defined enzymatic substrates of histone modifying enzymes. We provide quick protocols for the isolation of nuclei from yeast and mammalian cells and give basic procedures for the phosphorylation, acetylation, and methylation of histones (and other proteins) using these subcellular sources that can be carried out in any laboratory. Simple methods for the analysis of histone modifications using these assays are discussed.
Fischle
Wolfgang
W
Laboratory of Chromatin Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. fischlw@rockefeller.edu
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D019411:Clinical Laboratory Techniques; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011868:Radioisotopes / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism
D004798:Enzymes; D006657:Histones; D011868:Radioisotopes; D000105:Acetyl Coenzyme A; D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate