Assays for transcription factors access to nucleosomal DNA.
Several promoters have been shown to have sequence specifically positioned nucleosomes that determine the architecture of the promoter. DNA binding proteins that regulate gene expression are in many cases known to bind to their cognate DNA segments organized within such positioned nucleosomes. It has become increasingly evident that the cooperation of chromatin and transcription factors results in an efficient and fine-tuned regulation of transcription. The first step in a gene induction event must be the access of transcription factors for the regulatory promoter/enhancer target sites. In this perspective it becomes interesting to evaluate the affinity of DNA binding proteins for their cognate binding site in a nucleosome context. Here we describe the preparation of nucleosome probe, a method for in vitro nucleosome reconstitution by salt dilution, purification of the reconstituted mononucleosomes, and characterization of the translational and rotational positions of the nucleosomal DNA. In addition, methods for affinity determination and characterization of protein-nucleosomal DNA interaction, such as methylation protection and methylation interference by dimethyl sulfate, quantitative DNase I footprinting, and electrophoretic mobility shift assay, are described.
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Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.
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D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D009707:Nucleosomes; D013463:Sulfuric Acid Esters; D014157:Transcription Factors; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D003850:Deoxyribonuclease I; C007482:dimethyl sulfate