Two distinct mechanisms of chromatin interaction by the Isw2 chromatin remodeling complex in vivo

Abstract

We have previously shown that Saccharomyces cerevisiae Isw2 complex slides nucleosomes to remodel chromatin in vivo. Our data suggested a model in which Isw2 complex binds the histone octamer and DNA separately to generate the force necessary for nucleosome movement. Here we find that the histone H4 "basic patch" is the only portion of any amino-terminal histone tail required for both target-specific association of Isw2 complex with chromatin and chromatin remodeling in vivo, whereas it is dispensable for basal levels of chromatin binding. Similarly, we find that nonremodeled chromatin structure and integrity of Isw2 complex are required only for target-specific association of Isw2 with chromatin. These data demonstrate fundamental differences between the target-specific and basal modes of chromatin binding by Isw2 complex in vivo and suggest that only the former involves contributions from DNA, histone H4, and sequence-specific DNA binding proteins. We propose a model for target recognition and chromatin remodeling by Isw2 complex in vivo.

FIG. 1.

A portion of the histone H4 basic patch is required for Isw2-dependent chromatin remodeling. (A) Diagram of the amino-terminal tails of the four core histones. The H4 basic patch is underlined. (B to D) Nucleosome positions at the POT1 gene were analyzed in wild type (WT) and the indicated histone tail mutants using DNase I as a probe of chromatin structure. Triangles and circles indicate DNase I-cut sites that resemble the wild-type and isw2 mutant patterns, respectively. Brackets indicate DNase I-cut sites that resemble intermediate states between the wild-type and isw2 mutant patterns. Size standards, numbered with respect to the POT1 initiation codon, are indicated to the left.

FIG. 2.

The basic patch is the only portion of any N-terminal histone tail required for specific association of Isw2p-K215R with its chromatin targets in vivo. Chromatin immunoprecipitation was performed as described in Materials and Methods. Radioactive duplex PCR was carried out for the POT1 or INO1 and ACT1 promoters, using a serial dilution of input chromatin (to ensure linearity of the PCR) and precipitated chromatin. The fraction of the input that immunoprecipitated was quantitated for each sample using a phosphorimager, and the ratios of the signals from Isw2 targets (POT1 or INO1) to those from a control locus (ACT1) were calculated. The averages and the data points from two experiments are shown by bars and vertical lines, respectively. The signals at ACT1 were similar in all strains used (data not shown). bp, R17A R19A basic patch mutant.

FIG. 3.

The H4 basic patch is required for binding of Isw2 complex to NCPs. (A) Electromobility shift assay measuring binding of Isw2 complex to wild-type or mutant NCPs. NCPs bound by Isw2 complex and unbound NCPs are indicated by white and black arrows, respectively. In this experiment we used purified catalytically inactive Isw2 complex, but essentially identical results were obtained with wild-type Isw2 complex (data not shown). There were no detectable effects of Mg-ATP on nucleosome binding by Isw2 complex (data not shown). (B) Wild-type NCPs were incubated with Isw2 complex and the indicated antibody. The supershifted species is marked with an arrowhead. M, anti-Myc; F, anti-FLAG. (C) Wild-type (WT) and mutant histone octamers containing the H4 R17A R19A basic patch double mutation (b.p.) were reconstituted into nucleosome arrays on immobilized templates and digested with micrococcal nuclease (MNase). Purified DNA was subjected to Southern blotting to analyze nucleosome density and verify chromatin assembly; “lo” and “hi” refer to lower and higher nucleosome densities in the assembled chromatin. (D) ATPase assays measuring the fraction of ATP hydrolyzed by Isw2 complex were carried out on samples identical to those described in panel C. The averages and standard deviations of three ATPase assays per sample are shown.

FIG. 4.

The H4 basic patch mutant is broadly required for the function of Isw2 complex in vivo. The top 3% of genes upregulated in the H4 R17A R19A double mutant were compared to the top 3% of Isw2-regulated genes (170 genes each) as described in Materials and Methods.

FIG. 5.

Itc1p is required for specific association of Isw2p-K215R with Isw2 targets in vivo. Chromatin immunoprecipitations of Isw2p and Isw2p-K215R were carried out in cells with wild type (ITC1) or null mutations (itc1) of the ITC1 gene. Each immunoprecipitation was carried out a total of three times using two independent preparations of chromatin for each strain. Shown are the average ratios and standard deviations of experimental loci relative to PPA1, calculated as follows: enrichment (n-fold) = experimental locus (immunoprecipitation as percentage of input)/PPA1 (immunoprecipitation as percentage of input).

FIG. 6.

Isw2 complex requires nonremodeled chromatin structure for robust localization to target promoters in vivo. WT (wild type) and K215R refer to the genotype of each copy of the ISW2 gene. One allele of ISW2 was fused to three copies of the FLAG (FL) epitope. (A and B) The indicated diploid yeast strains were digested with DNase I, and their chromatin structure at Isw2 target genes POT1 (A), STE6 (B), and the nearby tRNA tT(CGU)K (B) was analyzed by indirect end-labeling analysis as above. (C) Chromatin immunoprecipitation of the diploid strains indicated in panel A. The relative enrichment (n-fold) for each ChIP was calculated as described in the legend of Fig. 5, except that ACT1 was used as the negative control in this experiment. We have found that neither ACT1 nor PPA1 is targeted by Isw2 complex, and therefore both can be used interchangeably as negative controls for Isw2 ChIP experiments (data not shown).

FIG. 7.

A model for Isw2-dependent chromatin remodeling in vivo. (Top) Isw2 is targeted to specific loci via interactions with sequence-specific DNA binding proteins (DBP), linker DNA (bulge), and the basic patch (small circle) of the histone H4 tail (thick curved line). These interactions allow Isw2 to generate the necessary force to push DNA into nucleosomes (arrow). (Bottom) After nucleosome sliding, Isw2 is released from its targets as shown by an arrow. This may be due to the lack of sufficiently long linker DNA, reduced availability of histone H4 tail (dashed line), or a combination of both after remodeling.