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Studies in vivo and in vitro have shown that the packaging of DNA into chromatin can affect gene expression.Here, binding of the yeast transcriptional activator GAL4 to DNA in chromatin has been investigated in vivo with a yeast episome. A positioned nucleosome that is present in cells grown in glucose and contains a single GAL4 binding site is disrupted by GAL4 binding in galactose. GAL4 can also bind to DNA in chromatin when the carboxylterminal activation domain of GAL4 is either masked by GAL80 or is absent. These results show that a transcription factor can bind to its site in vivo in what would appear to be a repressive chromatin structure.