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] Supplemental material is available at http://www.genesdev.org.

The Rpd3 histone deacetylase (HDAC) functions in a large complex containing many proteins including Sin3 and Sap30. Previous evidence indicates that the pho23, rpd3, sin3, and sap30 mutants exhibit similar defects in PHO5 regulation. We report that pho23 mutants like rpd3, sin3, and sap30 are hypersensitive to cycloheximide and heat shock and exhibit enhanced silencing of rDNA, telomeric, and HMR loci, suggesting that these genes are functionally related. Based on these observations, we explored whether Pho23 is a component of the Rpd3 HDAC complex. Our results demonstrate that MycPho23 co-immunoprecipitates with HA-Rpd3 and HASap30. Furthermore, similar levels of HDAC activity were detected in immunoprecipitates of HA-Pho23, HARpd3, or HA-Sap30. In contrast, HDAC activity was not detected in immunoprecipitates of HA-Pho23 or HASap30 from strains lacking Rpd3, suggesting that Rpd3 is the HDAC associated with these proteins. However, HDAC activity was detected in immunoprecipitates of HA-Sap30 or HA-Rpd3 from cells lacking Pho23, although levels were significantly lower than those detected in wild-type cells, indicating that Rpd3 activity is compromised in the absence of Pho23. Together, our genetic and biochemical studies provide strong evidence that Pho23 is a component of the Rpd3 HDAC complex, and is required for the normal function of this complex. Modifications of chromatin by histone acetyltransferases (HATs)1 and histone deacetylases (HDACs) play important roles in transcriptional regulation (1-4). Many proteins possessing intrinsic HAT activity have been identified from various organisms, and many of these proteins have been shown to be transcriptional coactivators or have other transcription-related functions. Similarly, several HDACs have been identified in different organisms as multiprotein complexes that are associated with transcriptional repressors and co-repressors (5-7). In many cases, HATs and HDACs are targeted to specific promoters through their interaction with DNA-binding transcription factors, suggesting that they regulate transcriptional activity by modifying the local chromatin structure at target promoters (8 -10). However, recent reports suggest that HATs also function in an untargeted manner to acetylate histones on a genome-wide scale (11,12).Packaging of DNA into chromatin is thought to affect transcription by impeding the access of transcription factors to DNA regulatory sequences. HATs acetylate lysine residues on core histones, thereby neutralizing the positive charge of the histone tails and decreasing their affinity for DNA and/or adjacent nucleosomes in higher order chromosomal structures (7, 13). Such a modification of chromatin is thought to increase the accessibility of DNA to transcription regulatory complexes (14,15). Thus, in general, hyperacetylation of histones correlates with activation of gene expression, whereas deacetylation represses transcription (16,17). Consistent with this model, the targeted recruitment of the Gcn5 HAT to specific promoters correlates with bo...

Retrotransposons are RNA elements that reverse transcribe their RNA genomes and make a cDNA copy that is inserted back into a new genomic location by the element-encoded integrase protein. Ty1 is a long terminal repeat (LTR) retrotransposon in Saccharomyces cerevisiae that inserts into an ∼700-bp integration window upstream of tRNA genes with a periodicity of ∼80 bp. ATP-dependent chromatin remodeling by Isw2 upstream of tRNA genes leads to changes in chromatin structure and Ty1 integration site selection. We show that the N terminus of Bdp1p, a component of the RNA polymerase III transcription factor TFIIIB, is required for periodic integration of Ty1 into the integration window. Deletion of the Bdp1p N terminus and mutation of ISW2 result in similar disruption of nucleosome positioning upstream of some tRNA genes, and the N-terminal domain of Bdp1p is required for targeting of Isw2 complex to tRNA genes. This study provides the first example for recruitment of an ATP-dependent chromatin-remodeling factor by a general transcription factor in vivo.[Keywords: RNA polymerase III; chromatin; integrase; retrotransposon; transcription factor; tRNA gene] Supplemental material is available at http://www.genesdev.org.

LTR-containing retrotransposons reverse transcribe their RNA genomes, and the resulting cDNAs are integrated into the genome by the element-encoded integrase protein. The yeast LTR retrotransposon Ty1 preferentially integrates into a target window upstream of tDNAs (tRNA genes) in the yeast genome. We investigated the nature of these insertions and the target window on a genomic scale by analyzing several hundred de novo insertions upstream of tDNAs in two different multicopy gene families. The pattern of insertion upstream of tDNAs was nonrandom and periodic, with peaks separated by ∼80 bp. Insertions were not distributed equally throughout the genome, as certain tDNAs within a given family received higher frequencies of upstream Ty1 insertions than others. We showed that the presence and relative position of additional tDNAs and LTRs surrounding the target tDNA dramatically influenced the frequency of insertion events upstream of that target.