Genome-wide identification of Isw2 chromatin-remodeling targets by localization of a catalytically inactive mutant.
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.
Gelbart
Marnie E
ME
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Bachman
Nurjana
N
Delrow
Jeffrey
J
Boeke
Jef D
JD
Tsukiyama
Toshio
T
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D017931:DNA Primers; D016681:Genome, Fungal; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; C086468:ISWI protein; D018626:Retroelements; D014157:Transcription Factors; D012343:RNA, Transfer; D000251:Adenosine Triphosphatases