Blockage to exonuclease III digestion in the chromatin of Saccharomyces cerevisiae maps to the in vitro ? determined binding site of a trans-acting regulatory factor

Abstract

A series of transposable element-induced mutations at the HIS4 locus in Saccharomyces cerevisiae have been attributed to the transposition of a Ty element into the 5' regulatory region of this gene. Various Ty-containing His+ revertants have been isolated and the HIS4/Ty junction region sequenced. The only difference found in this region between a His- and a weak His+ strain was a single point mutation, an A----G transition. The position of Ty remained unaltered. Examination of lacZ fusion plasmids further implicated this A----G transition as being responsible for the altered phenotype, the bp transition representing an allele of a cis-acting regulatory element. Subsequent gel retardation and methylation interference experiments revealed that this A----G mutation enabled the binding of a trans-acting factor (TyBf) in vitro. In this paper we show that the TyBf binding site is in a region of chromatin hypersensitive to digestion by DNase I. The binding site is protected in vivo from digestion with exonuclease III, suggesting the presence of a bound protein in His+ ("on") but not His- ("off") Ty-containing strains. We propose that a trans-acting factor binding in vivo, presumably TyBf, is responsible for the activation of HIS4 expression in these insertion mutants.