Chromatin remodeling by imitation switch (ISWI) class ATP-dependent remodelers is stimulated by histone variant H2A.Z

Abstract

ATP-dependent chromatin remodeling complexes rearrange nucleosomes by altering the position of DNA around the histone octamer. Although chromatin remodelers and the histone variant H2A.Z colocalize on transcriptional control regions, whether H2A.Z directly affects remodeler association or activity is unclear. We determined the relative association of remodelers with H2A.Z chromatin and tested whether replacement of H2A.Z in a nucleosome altered the activity of remodeling enzymes. Many families of remodelers showed increased association with H2A.Z chromatin, but only the ISWI family of chromatin remodelers showed stimulated activity in vitro. An acidic patch on the nucleosome surface, extended by inclusion of H2A.Z in nucleosomes and essential for viability, is required for ISWI stimulation. We conclude that H2A.Z incorporation increases nucleosome remodeling activity of the largest class of mammalian remodelers (ISWI) and that it correlates with increased association of other remodelers to chromatin. This reveals two possible modes for regulation of a remodeler by a histone variant.

FIGURE 1.

All major families of ATP-dependent chromatin remodelers are bound to H2A.Z-containing chromatin. Western blots on purified H2A and H2A.Z chromatin were performed for ATP-dependent core motor proteins (A) and other remodeling complex subunits (B). The primary antibody used in each blot is indicated on the left. A Western blot against the affinity tag on the histone (FLAG) was used as a loading control. Changes in amount of protein present in the input of the purifications were not evident between HeLa, H2A-FLAG, or H2A.Z-FLAG nuclear extracts as determined by Western blot (not shown). The two lower bands observed for Mi-2α migrate at the expected molecular weight for the protein and likely reflect alternatively modified forms of the protein. The top slower migrating species observed only with H2A.Z is >100 kDa larger than expected and is of unknown identity.

FIGURE 2.

Human ACF complex remodels H2A.Z-containing nucleosomes more rapidly than H2A nucleosomes. Cy5 end-labeled recombinant canonical nucleosomes (red) were mixed in equal proportion (30 nm) to Cy3 end-labeled recombinant H2A.Z nucleosomes (green) and remodeled in the presence of 2 mm ATP by the enzyme complex indicated on the left. Remodeling is inferred by digestion with Pst1. The reactions were stopped at the indicated time points, proteinase-digested, and resolved on a 6% acrylamide gel before scanning on a Typhoon imager. Time points from independent reactions with H2A-Cy5 and H2A.Z-Cy3 nucleosomes alone are shown first and indicate that both preparations of nucleosomes are competent for remodeling. The bottom panel is a replicate of ACF remodeling shown in the top panel but done with half the total concentration of each nucleosome (15 nm).

FIGURE 3.

Increased efficiency of remodeling H2A.Z-containing nucleosomes is a general feature of ISWI family remodeling enzymes. A, colloidal blue staining of purified remodeling enzymes. B, sample time courses of restriction enzyme accessibility remodeling reactions for core motor proteins on P³² end-labeled nucleosome substrates. C, graphs of phosphorimaging quantitation of uncut substrate versus time from reactions shown in B. k_obs for remodeling reactions is determined for the enzyme indicated by fitting the reaction to the exponential decay curve [S] = [S]_ie^−kt where [S]_i is the initial substrate concentration and k is k_obs. The ratios of k_obs are shown on the right. D, summary of ratio k_obs for reactions performed with recombinant H2A.Z-containing nucleosomes to recombinant canonical nucleosomes. The error bars represent standard deviations from the average of 7–26 different remodeling reactions from multiple enzyme preparations and nucleosome assemblies. E, k_obs was measured by the rate of appearance of P³² labeled phosphate and is displayed as the ratio between H2A.Z nucleosomes to canonical nucleosomes. Free phosphate was resolved from hydrolyzed ATP by thin layer chromatography in 1 m formic acid, 0.5 m LiCl. F, rates were calculated and graphed as the percentage of the maximal rate within each titration to compare between replicates. The error bars represent the standard deviation between three replicates.

FIGURE 4.

ISWI family stimulation is independent of nucleosome position and dependent on the extended acidic patch on H2A.Z-containing nucleosomes. A, native PAGE of eluted glycerol gradient fractions of mononucleosomes recombinantly assembled on the TPT positioning sequence with a 45-bp overhang. Fractions of similar mobility within the native gel were compared as similarly positioned nucleosomes in REA assays with SNF2H. A summary of ratio of k_obs between the indicated fractions is shown in the graph on the right. B, k_obs of remodeling determined by REA assay are compared between the mutant H2A.Z mononucleosomes (NK) and canonical mononucleosomes for the enzyme indicated. The calculations were done as in Fig. 3D. The dashed line is from the data shown in Fig. 3D and is displayed as a reference point to compare H2A.Z-NK remodeling to wild type H2A.Z remodeling by SNF2H. C, REA assay with Cy5 end-labeled recombinant mononucleosomes (red) mixed with either Cy3 end-labeled H2A.Z nucleosomes or mutant H2A.Z (NK) mononucleosomes (green). Separate SNF2H and ACF remodeling reactions are indicated. The Cy3 label on the H2A.Z nucleosomes appears relatively less bright than the Cy3 label on the NK nucleosomes most likely because of decay of the Cy3 on the H2A.Z nucleosomes that were assembled earlier.

FIGURE 5.

H2A.Z stimulation of remodeling is shared with multiple ISWI complexes regardless of degree of in vivo enrichment. A, colloidal blue staining of recombinant ISWI complexes. B, summary of REA remodeling reactions for H2A.Z enriched SNF2H complex (NoRC) and both H2A.Z depleted complexes containing SNF2H (WICH) and SNF2L (RSF/L). The ratio of k_obs of either H2A.Z or H2A.Z-NK nucleosomes to k_obs for canonical nucleosomes is graphed for each enzyme complex. The error bars reflect standard deviation from the average of at least seven reactions on multiple nucleosome assemblies. C, REA assay with Cy5 end-labeled recombinant canonical nucleosomes (red) and Cy3 end-labeled recombinant H2A.Z nucleosomes (green) were performed as in Fig. 2.