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RESEARCH PAPER

Genome-wide identification of Isw2 chromatin-remodeling targets by localization of a catalytically inactive mutant

¹ Division of Basic Sciences and ² Genomics Resource, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA; ³ Molecular and Cellular Biology Program, University of Washington and Fred Hutchinson Cancer Research Center, Seattle, Washington 98195, USA; ⁴ Department of Molecular Biology and Genetics and High Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA

Isw2 ATP-dependent chromatin-remodeling activity is targeted to early meiotic and MATa-specific gene promoters in Saccharomyces cerevisiae. Unexpectedly, preferential cross-linking of wild-type Isw2p was not detected at these loci. Instead, the catalytically inactive Isw2p-K215R mutant is enriched at Isw2 targets, suggesting that Isw2p-K215R, but not wild-type Isw2p, is a sensitive chromatin immunoprecipitation (ChIP) reagent for marking sites of Isw2 activity in vivo. Genome-wide ChIP analyses confirmed this conclusion and identified tRNA genes (tDNAs) as a new class of Isw2 targets. Loss of Isw2p disrupted the periodic pattern of Ty1 integration upstream of tDNAs, but did not affect transcription of tDNAs or the associated Ty1 retrotransposons. In addition to identifying new Isw2 targets, our localization studies have important implications for the mechanism of Isw2 association with chromatin in vivo. Target-specific enrichment of Isw2p-K215R, not wild-type Isw2p, suggests that Isw2 is recruited transiently to remodel chromatin structure at these sites. In contrast, we found no evidence for Isw2 function at sites preferentially enriched by wild-type Isw2p, leading to our proposal that wild-type Isw2p cross-linking reveals a scanning mode of the complex as it surveys the genome for its targets.

[Keywords: Chromatin; ATP-dependent chromatin remodeling; ISWI; genome-wide localization; retrotransposon; tRNA]

Received January 18, 2005; revised version accepted March 3, 2005.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1298905.

⁵ Corresponding author.
E-MAIL ttsukiya{at}fhcrc.org; FAX (206) 667-6497.

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