Summary How is chromatin architecture established and what role does it play in activation of transcription? We show that a regulatory locus in yeast (the UASg) bears, in addition to binding sites for the activator Gal4, sites bound by the protein RSC. RSC tightly positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that suffices to impose characteristic features of chromatin architecture. Removal of RSC allows ordinary nucleosomes to form more broadly over the UASg, and these nucleosomes compete with (but do not exclude) Gal4 binding to its sites. Taken with our previous work, the results show that both prior to and following induction specific DNA binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are found scattered throughout the yeast genome. We surmise, also, that Gal4 works in higher eukaryotes despite whatever obstacle broadly positioned nucleosomes present.

The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose) and repression (by glucose) of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome “remodeler” rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4′s action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription—one that requires the action of SWI/SNF recruited by the activator, and a slower one that does not—clarifies our understanding of the early events of gene activation, and in particular corrects earlier reports that SWI/SNF plays no role in GAL gene induction. Our finding that chromatin structure is irrelevant for repression as studied here—that is, repression sets in as efficiently whether or not promoter nucleosomes are allowed to reform—contradicts the widely held, but little tested, idea that nucleosomes are required for repression. These findings were made possible by our nucleosome occupancy assay. The assay, we believe, will prove useful in studying other outstanding issues in the field.

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It has been shown in vitro that Saccharomyces cerevisiae strand exchange protein 1 (Sep1) promotes the transfer of one strand of a linear duplex DNA to a homologous single-stranded DNA circle. Sep1 also has an exonuclease active on DNA and RNA. By using exonuclease III-treated linear duplex DNA with various lengths of single-stranded tail as well as Ca 2؉ to inhibit the exonuclease activity of Sep1, we show that the processivity of exonuclease activity of Sep1 is greater than previously reported. The results in this work also demonstrate that the joint molecule between the linear duplex and single-stranded circle observed from the Sep1-promoted strand transfer reaction is just the pairing between the long single-stranded tail of the linear duplex DNA (generated by the exonuclease activity of Sep1) and the single-stranded circular DNA. When a synthetic Holliday junction was used as substrate, branch migration facilitated by Sep1 could not be detected. Finally, using electron microscopy no ␣-structure, a joint molecule with displaced single-stranded DNA tail that indicates branch migration could be observed. The results imply that Sep1 cannot promote branch migration in vitro. Further investigation is needed to determine the role of Sep1 in recombination in vivo.