Antagonistic forces that position nucleosomes in vivo Iestyn Whitehouse & Toshio Tsukiyama ATP-dependent chromatin remodeling complexes are implicated in many areas of chromosome biology. However, the physiological role of many of these enzymes is still unclear. In budding yeast, the Isw2 complex slides nucleosomes along DNA. By analyzing the native chromatin structure of Isw2 targets, we have found that nucleosomes adopt default, DNA-directed positions when ISW2 is deleted. We provide evidence that Isw2 targets contain DNA sequences that are inhibitory to nucleosome formation and that these sequences facilitate the formation of nuclease-accessible open chromatin in the absence of Isw2. Our data show that the biological function of Isw2 is to position nucleosomes onto unfavorable DNA. These results reveal that antagonistic forces of Isw2 and the DNA sequence can control nucleosome positioning and genomic access in vivo. Chromatin allows the cell to efficiently package its genome. The fundamental repeating unit of chromatin is the nucleosome 1 . Nucleo- somal DNA is tightly bound by the core histone proteins, which render the DNA less accessible. An implication of this is that basic cellular processes such as transcription, replication and recombination, which rely on access to the genome, can be regulated by chromatin. Indeed, chromatin remodeling factors that alter the position, composition or integrity of chromatin have important roles in these processes 2 . We have a limited understanding of what determines a particular nucleosome arrangement in vivo. Along with a number of histone and nonhistone proteins, the underlying DNA sequence can influence nucleosome positioning. There have been cases in which the favored position of nucleosomes assembled in vitro is the same as the dominant position found in vivo 3,4 . This arises because certain DNA sequences are more readily bound by the histone octamer than others. Studies that map global nucleosome occupancy in vivo have found that intergenic regions frequently contain fewer nucleosomes than coding regions 5,6 . This may be because these regions are overrepre- sented in dA-dT–rich elements 7,8 , which wrap poorly into nucleo- somes 9,10 . These and other investigations support a hypothesis that DNA elements that are unfavorable for nucleosome formation may influence chromatin structure by altering nucleosome positioning or occupancy 8 . There are, however, examples where unfavorable DNA is found within nucleosomes, and nucleosomes can form on various dA- dT–rich sequences both in vitro 11 and in vivo 12,13 . Nucleosome positioning can also be controlled by a family of enzymes that use the energy from ATP hydrolysis to alter histone- DNA contacts. These ATP-dependent chromatin remodeling enzymes may be subdivided into a number of classes, including SWI/SNF, ISWI, CHD, Rad54 and INO80 (ref. 14). In vitro, ISWI complexes have been shown to reposition nucleosomes along DNA 15,16 , assemble chromatin 17 and form regularly spaced nucleosome arrays 18 . In vivo, ISWI complexes function in the regulation of transcription 19–23 , global chromosome structure 24 , DNA replication 25,26 , cell cycle 27 , ribosomal DNA silencing 28 and cohesin loading 29 . The Saccharomyces cerevisiae Isw2 complex belongs to the ISWI class of chromatin remodeling enzymes 30 . Isw2 is required for the repression of distinct subsets of genes 19–21 . Nuclease digestion studies in vivo have determined how Isw2 alters the positions of nucleosomes around promoter elements transcribed by both RNA polymerase II 19–21,31,32 and RNA polymerase III 33 . A common feature of Isw2 action at these loci is the repositioning of nucleosomes along DNA toward sites of recruitment 34 . This process limits promoter DNA accessibility and generates a more tightly packed nucleosome array that leads to repression of transcription by RNA polymerase II or changes in integration site selection by Ty1 retrotransposons 33 . To understand the determinants of nucleosome positioning in vivo, we have investigated how chromatin is organized at Isw2 target loci in budding yeast. Our analysis shows that Isw2 repositions nucleo- somes onto unfavorable DNA sequences to generate tightly packed, nuclease-inaccessible arrays. We also show that when Isw2 is deleted, or the target becomes active, the chromatin adopts a DNA-directed positioning that facilitates genomic access. Our results show that interplay between Isw2 and the underlying DNA can control nucleo- some positioning. RESULTS Nucleosome sliding by Isw2 at POT1 Isw2 alters chromatin structure at a diverse set of loci across the S. cerevisiae genome 33 . Although these sites contain no obvious similarity, Isw2 activity results in highly similar nucleosome reorga- nization 19,21,31–34 . This led us to investigate whether these sites share common features that specify chromatin structure and direct Isw2-dependent chromatin remodeling. We first investigated how Isw2 alters nucleosome positions at the POT1 locus. The POT1 gene encodes a peroxisomal oxoacyl thiolase Received 21 February; accepted 17 May; published online 2 July 2006; doi:10.1038/nsmb1111 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. Correspondence should be addressed to T.T. (ttsukiya@fhcrc.org). NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 7 JULY 2006 633 ARTICLES © 2006 Nature Publishing Group http://www.nature.com/nsmb