Chromatin allows the eukaryotic cell to package its DNA efficiently. To understand how chromatin structure is controlled across the Saccharomyces cerevisiae genome, we have investigated the role of the ATP-dependent chromatin remodelling complex Isw2 in positioning nucleosomes. We find that Isw2 functions adjacent to promoter regions where it repositions nucleosomes at the interface between genic and intergenic sequences. Nucleosome repositioning by Isw2 is directional and results in increased nucleosome occupancy of the intergenic region. Loss of Isw2 activity leads to inappropriate transcription, resulting in the generation of both coding and noncoding transcripts. Here we show that Isw2 repositions nucleosomes to enforce directionality on transcription by preventing transcription initiation from cryptic sites. Our analyses reveal how chromatin is organized on a global scale and advance our understanding of how transcription is regulated.

Genetic control elements are usually situated in local regions of chromatin that are hypersensitive to structural probes such as DNase I. We have reconstructed the chromatin structure of the hsp70 promoter using an in vitro nucleosome assembly system. Binding of the GAGA transcription factor on existing nucleosomes leads to nucleosome disruption, DNase I hypersensitivity at the TATA box and heat-shock elements, and rearrangement of adjacent nucleosomes. ATP hydrolysis facilitates this process, suggesting that an energy-dependent pathway is involved in chromatin remodelling.

We report the purification of an ATP-dependent nucleosome remodeling factor (NURF) from Drosophila embryo extracts. NURF is composed of at least four polypeptides that act in concert with the GAGA transcription factor to alter chromatin structure at the hsp70 promoter. The energy requirement is attributed to an ATPase activity that is stimulated by nucleosomes but not by free DNA or histones, suggesting that NURF acts directly on a nucleosome to perturb its structure. This finding and the physical properties of NURF contrast sharply with the multisubunit SWI2/SNF2 complex, which has also been shown to alter nucleosomes in an ATP-dependent manner. The results suggest that two distinct systems may be involved in remodeling chromatin for transcription.

We have identified and characterized two Imitation Switch genes in