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© 2002 Nature Publishing Group http://structbio.nature.com letters The essential histone variant H2A.Z regulates the equilibrium between different chromatin conformational states Jun Y. Fan1, Faye Gordon2, Karolin Luger3, Jeffrey C. Hansen2 and David John Tremethick1 1 The John Curtin School of Medical Research, The Australian National University, P.O. Box 334, Canberra, Australian Capital Territory 2601, Australia. 2Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229, USA. 3Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA Published online: 19 February 2002, DOI: 10.1038/nsb767. Explaining the determinants involved in regulating the equilibrium between different chromatin structural states is fundamental to understanding differential gene expression. Histone variant H2A.Z is essential to chromatin architecture in higher eukaryotes but its role has not yet been explained. We show here that H2A.Z facilitates the intramolecular folding of nucleosomal arrays while simultaneously inhibiting the formation of highly condensed structures that result from intermolecular association. This makes a case for H2A.Z playing a fundamental role in creating unique chromatin domains poised for transcriptional activation. These results provide new insights into understanding how chromatin fiber dynamics can be altered by core histone variants to potentially regulate genomic function. Within the eukaryotic nucleus, DNA and histones are assembled into chromatin to form a highly dynamic nucleoprotein fiber. Chromatin a must be stable enough for efficient packaging of DNA into the nucleus while at the same time labile enough to allow access of DNA to DNAbinding proteins like transcription factors. The nucleosome is the fundamental unit of chromatin, consisting of DNA wrapped around an octamer of core histones. Cells exploit alterations in histone–DNA and histone–histone Fig. 1 Characterization of H2A- (control) and H2A.Zcontaining nucleosomal arrays. a, Structure of the 12 repeats of 208-12 DNA template used in this investigation. b, Micrococcal nuclease digestion of assembled arrays. 208-12 DNA (300 µg) was assembled into an array using either control or H2A.Z-containing octamers (300 µg). Arrays containing an equivalent of 3 µg of DNA were cleaved with micrococcal nuclease in the presence of 1.0 mM CaCl2. c, EcoRI digestion of nucleosomal arrays. The same reconstitutes were digested with EcoRI. The digested array (0.5 µg and 1.5 µg) was then loaded and electrophoresed on a 1% agarose gel. The percentage of octamer-free DNA was quantitated using densitometry. d, Sedimentation velocity analysis. The same reconstitutes, in low salt buffer, were analyzed by sedimentation velocity in the XL-A analytical ultracentrifuge, and the data were analyzed to yield the integral distribution of sedimentation coefficients. 172 c interactions within and between nucleosomes to regulate gene expression, partly through modulation of the equilibrium between open and highly condensed conformational states. One extensively studied mechanism for altering the higher order conformation of chromatin is posttranslational modification of histones1. However, there are probably other equally important ways to modify chromatin structure, such as altering the biochemical composition of nucleosomes by the replacement of major histone types with specific histone variants. The structural consequences of having the variant histone H2A.Z incorporated into chromatin is not known, but its presence and high level of conservation from yeast to humans (>90% sequence identity among different organisms) indicate that H2A.Z has an important, specific role in chromosome function2. Consistent with this proposal, H2A.Z is essential for Drosophila3, Tetrahymena4 and mouse survival5. Recently, we defined the region of Drosophila H2A.Z required for its essential function6. This region is part of the docking domain, which is involved in maintaining the interactions between the histone H3/H4 tetramer and the histone H2A/H2B dimer within the nucleosome. The docking domain also contributes to surface features of the nucleosome. Most recently, the crystal structure of a nucleosome containing H2A.Z was solved7. Amino acid changes in the docking domain of H2A.Z result in an altered nucleosomal surface that could potentially modulate interactions between nucleosomes. Here, we addressed the issue of whether H2A.Z can modulate nucleosome–nucleosome interactions involved in higher-order chromatin condensation. H2A.Z facilitates intranucleosomal interactions To determine the role of H2A.Z in this nucleosome–nucleosome interactions, we employed a highly defined in vitro chromatin model system. This model system uses the 208-12 DNA template, which consists of 12 repeats of a 208-base pair (bp) nucleosome positioning sequence from the sea urchin 5S RNA gene (Fig. 1a). One major advantage of using this system to investigate the influ- b d nature structural biology • volume 9 number 3 • march 2002 © 2002 Nature Publishing Group http://structbio.nature.com letters a b c d Fig. 2 H2A.Z facilitates the intrinsic 29S–55S folding pathway. a, Sedimentation coefficient distribution plot of control and H2A.Zcontaining arrays in 0.25 mM MgCl2. b, Sedimentation coefficient distribution plot of control and H2A.Z-containing arrays in 1.5 mM MgCl2. c, Effect of MgCl2 on the average S of H2A (control) and H2A.Z nucleosomal arrays. The average Save (Smidpoint) is defined as the boundary fraction = 0.5 of the sedimentation coefficient distribution plot. d, Effect of ZnCl2 on the average S of control H2A and H2A.Z nucleosomal arrays. The same reconstitutes, in different ZnCl2 concentrations, were analyzed by sedimentation velocity. Save was determined for each sedimentation coefficient distribution plot. Plasmid linear DNA 2.74 kb in length (pGEM digested with EcoRI) was assembled into a nucleosomal template using control or H2A.Z-containing octamers. e, Sedimentation coefficient distribution plots of control and H2A.Z-containing nucleosomal templates in low salt buffer. f, Sedimentation coefficient distribution plots of control and H2A.Z nucleosomal templates in 1.0 mM MgCl2. repeats released by EcoRI digestion gives a precise determination of the extent of nucleosome assembly9–11. This analysis revealed that the nucleosomal arrays (Fig. 1b) have 93.0 and 93.5% of 208 DNA repeats occupied with H2A and H2A.Z histone octamers, respectively (Fig. 1c; Table 1). H2A-containing mononucleosomes migrate at two distinct positions (Fig. 1c). The faster migrating band may be due to some partial dissociation during e f electrophoresis under the gel-shift conditions used here. H2AZ mononucleosomes run at single position, perhaps indicating that they are more stable (see discussion). Next, we performed sedimentation velocity studies of H2Aand H2A.Z-containing nucleosome arrays under low salt conditions using the analytical ultracentrifuge. This analysis provides an extremely sensitive measure of the sedimentation coefficients and degree of saturation of all species in a sample. Previously, 208-12 DNA was shown to be assembled with 12 histone octamers adopting an extended 29S conformation in low salt ence of H2A.Z on chromatin dynamics is that regularly spaced buffer8–10. Because the arrays do not fold under these conditions, nucleosomal arrays can be assembled using only purified DNA any differences in the sedimentation coefficients of arrays and histones, which yield a highly homogenous population of assembled with H2A or H2A.Z would indicate a difference in nucleosomal arrays that can be characterized rigorously by ana- nucleosome density8–11. The nucleosomal arrays assembled with lytical methods8–11. In addition, this model system has been used either H2A or H2A.Z display identical sedimentation distributo extensively analyze the macromolecular determinants involved tion profiles (Fig. 1d). Because the degree of DNA wrapping is in chromatin fiber condensation8–11. Here, we compare the solu- identical for both types of nucleosomes, as shown by the crystal tion dynamics of chromatin model systems assembled from structure of an H2A.Z-containing nucleosome7, both templates either control Xenopus histone octamers or Xenopus histone have been assembled to the same nucleosome density (Fig. 1d). Because the average sedimentation coefficient for both temoctamers containing H2A.Z7 (Fig. 1a). A major concern in this analysis was to ensure that the 208-12 plates is 28.5S, the majority of both the control and H2A.Z DNA templates contained an identical degree of saturating H2A arrays are fully saturated (≥50% loaded with 12 nucleosomes per or H2A.Z histone octamers. To accomplish this, three approach- DNA template) (Table 1). es were used. First, we cleaved the H2A or H2A.Z nucleosomal Finally, to confirm these results, quantitative agarose gel arrays with EcoRI and subjected the digestion products to non- electrophoresis was carried out to determine the effective radius denaturing gel-shift analysis. A pair of EcoRI digestion sites flank (Re) and gel-free mobility (µo) of these arrays. Both of these each 208-bp repeat (Fig. 1a). Consequently, digestion of a parameters are sensitive measures of the level of nucleosome satnucleosomal array with EcoRI releases 208-bp fragments of DNA uration8,9. Both the Re and µo values are indistinguishable for that are either histone-free or contain a histone octamer (and control and H2A.Z-containing arrays and are similar to reported partially digested di-nucleosomes). Following gel-shift analysis, values obtained for nucleosomal arrays containing 12 histone careful quantitation of the amount of histone-free 208 DNA octamers per 208-12 DNA (Table 1)8,9,11. Having established that control and H2A.Z nucleosomal arrays are assembled Table 1 Properties of purified 208-12 control and H2A.Z-containing nucleosomal with histone octamers to the same extent, arrays in low salt buffers we used sedimentation velocity analysis to Save (S)1 Re (nm)2 –µo (× 10–4 cm2 V–1 s–1) % free DNA3 address whether H2A.Z influences H2A 28.5 28.33 ± 0.4 1.84 ± 0.33 7.0 ± 0.3 intramolecular folding of nucleosomal H2A.Z 28.5 28.55 ± 0.9 1.81 ± 0.05 6.5 ± 0.3 arrays. The addition of increasing concen1Values represent the S trations of Mg2+ ions to an unfolded array in 20,W obtained at the boundary fraction midpoint in TE buffer (Fig. 1d). 2Quantitative agarose gels were carried out as described9. Values represent the mean ± standard low salt was previously shown to cause a deviation of three experiments at Pe ≥ 200 nm. series of conformational changes that result 3The mean ± standard deviation of the percentage of unoccupied 5S rDNA sites (Fig. 1c). in the formation of a highly folded state9,10. nature structural biology • volume 9 number 3 • march 2002 173 © 2002 Nature Publishing Group http://structbio.nature.com letters Specifically, with increasing salt, the extended 29S array first compacts into a moderately folded 40S intermediate and finally to a highly folded 55S conformation12. Linker histones stabilize the array in the 55S conformation, which is as condensed as the canonical 30 nm fiber9–11. Under physiological conditions, a fully saturated nucleosomal array lacking linker histones is in equilibrium between the 29S, 40S and 55S conformational states9,10,12. The sedimentation coefficient distribution profiles of control and H2A.Z arrays in the presence of 0.25 mM (Fig. 2a) and 1.5 mM MgCl2 (Fig. 2b) indicate that H2A.Z facilitates the intrinsic 29S–55S folding pathway. In both MgCl2 concentrations (Fig. 2c), the H2A.Z-containing arrays adopted a significantly more folded conformation than the control H2A arrays, as indicated by the right-shifted sedimentation coefficients at all points in the distribution plot. H2A.Z facilitates the formation of both the 40S intermediate and the highly folded 55S structure. In 1.5 mM MgCl2, the sedimentation coefficient distribution of the H2A.Z arrays, but not the control arrays, reached 55S (Fig. 2b). To determine whether the effect of H2A.Z on higher order folding is ion-specific, the sedimentation velocity experiments were repeated using ZnCl2. At all salt concentrations tested, H2A.Z increased the average degree of folding (Fig. 2d), consistent with the results obtained in MgCl2. Given that the role of cations in the folding process is neutralization of DNA charge, ZnCl2 was more efficient than MgCl2 in promoting the folding of both types of arrays (Fig. 2c,d), because Zn2+ binds more tightly to DNA than Mg2+. Taken together, these data demonstrate unequivocally that the conformational equilibrium of H2A.Z arrays is shifted toward the folded states relative to control H2A arrays. Next we determined whether H2A.Z facilitates folding of a nucleosomal array when a different DNA sequence is used. To address this, we assembled linear plasmid DNA of 2.74 kb in length (pGEM digested with EcoRI) with control or H2A.Zcontaining histone octamers to the same nucleosome density (≥50% loaded with 13 nucleosomes per DNA template, equivalent to one nucleosome per 210 bp of DNA) (Fig. 2e). In the presence of 1.0 mM MgCl2, plasmid DNA containing H2A.Z adopted a more folded conformation (Fig. 2f). Therefore, we conclude that H2A.Z can promote intramolecular folding independent of DNA sequence and the positioning of nucleosomes in a regular array. H2A.Z inhibits internucleosomal interactions We noted another feature during the sedimentation velocity experiments. At 0.6 mM ZnCl2 and 2.0 mM MgCl2, control arrays pelleted within seconds in the analytical ultracentrifuge, whereas H2A.Z arrays sedimented much more slowly. Above the range where folding occurs, divalent cation concentrations induce a cooperative, reversible oligomerization reaction in which 208-12 nucleosomal arrays self-associate to form large, soluble nucleoprotein complexes. These complexes share many features of interphase chromosomal fibers12. The N-terminal domains of the core histones are required for oligomerization10, and acetylation impedes the oligomerization process in vitro13,14. To investigate whether H2A.Z also impedes oligomerization, we used a microfuge-based assay15 to determine the extent of oligomerization as a function of Zn2+ concentration for both control and H2A.Z-containing nucleosomal arrays. Using the same control and H2A.Z nucleosomal arrays as the sedimentation velocity experiments (Fig. 2a–d), H2A.Z arrays were shown to oligomerize less effectively than control arrays, both in the absence (Fig. 3a) and presence of 50 mM NaCl (Fig. 3b), as revealed by the requirement for greater amounts of divalent cations to achieve equivalent levels of oligomerization. We conclude that H2A.Z, like histone acetylation, impedes the formation of highly condensed chromatin that results from intermolecular associations in vitro. This finding may explain why H2A.Z-containing arrays are more sensitive than control arrays to micrococcal nuclease digestion (carried out in the presence of CaCl2, Fig. 1b). H2A.Z can alter nucleosome positioning To investigate the possibility that H2A.Z may alter nucleosome positioning, we determined whether H2A or H2A.Z histone octamers occupy different positions on the 208-12 DNA template. Studies have shown that ∼50% of chicken erythrocyte histone octamers occupy a single translational position, with the remaining octamers distributed in alternate translational positions16. To measure nucleosomal positioning we exploited a unique AluI restriction site located 48 bp from the 5′ boundary of the 5S positioning sequence (Fig. 1a). Control and H2A.Z arrays were digested with micrococcal nuclease to produce a homogeneous population of nucleosome core particles. The core particle DNA was purified and digested with AluI, and the DNA fragments were run on a polyacrylamide gel (Fig. 4). Consistent with previous studies16, control histone octamers are located primarily at two major locations, with position 1 being the favored position (44% of the total positions). Although H2A.Z-containing octamers occupy the same positions, a a b Fig. 3 H2A.Z inhibits the formation of highly condensed chromatin fibers. The percentage of control and H2A.Z-containing nucleosomal arrays that remained in the supernatant after a centrifugation step (using the same reconstitutes as in Fig. 2) are shown. Each data point represents the average and standard deviation of two independent experiments. a, Control and H2A.Z-containing nucleosomal templates in low salt buffer. b, Control and H2A.Z-containing nucleosomal templates in 50 mM NaCl. 174 nature structural biology • volume 9 number 3 • march 2002 letters © 2002 Nature Publishing Group http://structbio.nature.com Fig. 4 H2A.Z alters nucleosome positioning. Control and H2A.Z nucleosomal arrays were digested with micrococcal nuclease (in 1.0 mM CaCl2) into trimmed mononucleosomes. The DNA was dissociated from histones and purified on a 6% polyacrylamide gel. A single DNA fragment, with an apparent length of 155 bp, was excised, purified and digested with AluI (because the 5S DNA is structured, core particle DNA runs at ∼150–155 bp). Following digestion, the products were run on a 12% polyacrylamide gel, and the digestion products were quantitated using densitometry. Shown is a representative densitometer trace. P1 and P2 refer to the AluI digestion products, which define the two major nucleosomal positions as described16. U is undigested nucleosomal DNA. Shown below the traces are the position of the two major nucleosomal positions and the percentage of nucleosomes located at these positions. significantly greater fraction of the H2A.Z histone octamers are found in the major positional frame (position 1 = 70%). Thus, H2A.Z can influence nucleosome positioning in vitro, which may also be important to the biological functions of this variant histone in vivo. Conclusions Modulating the equilibrium from a compacted to a decondensed chromatin state is a key step in gene expression17. Removal of linker histones destabilizes folded chromatin fibers9–11. However, the intrinsic folding of nucleosomal arrays is sufficient to repress transcription13. Therefore, identifying the molecular determinants that regulate nucleosomal array condensation is of fundamental importance to understanding gene regulation. Here we show that the histone variant H2A.Z is critical to the chromatin condensation process. We show that H2A.Z promotes intramolecular folding of nucleosomal arrays (Fig. 2). In contrast, H2A.Z also significantly impedes in vitro oligomerization (Fig. 3). No other known effector of chromatin condensation shows these characteristics. Changes in intramolecular folding involve local alterations that influence fiber conformation, whereas oligomerization is correlated with global chromosomal fiber condensation. This later stage of compaction involves interactions between chromatin fibers, yielding a highly condensed state. Our data suggest that H2A.Z may create a transcriptionally poised higher order nature structural biology • volume 9 number 3 • march 2002 chromatin domain. The effect of H2A.Z to promote global decondensation mimics the effect of hyperacetylation in vitro13. As such, regions of decondensed genomic chromatin enriched in H2A.Z will be, in principle, marked as a functionally primed chromatin domains18. Assuming that the histone acetyltransferase Gcn5 participates in global acetylation of the yeast genome19, this could explain why disruption of the histone acetyltransferase GCN5 gene in yeast made cells highly dependent upon H2A.Z for viability20. At the same time, the globally decondensed fiber containing H2A.Z will be more stably folded. This stable folding may explain the need for more localized structural changes caused by SWI/SNF and histone-modifying enzymes. Alternatively, higher order folding induced by H2A.Z may be required for establishing a specific ‘active’ chromatin architecture at the promoter regions of certain genes20. This ability of H2A.Z to inhibit the formation of highly condensed chromatin while facilitating intramolecular folding can explain the role of H2A.Z in both gene activation21 and the formation of specialized structures at the HMR locus22. H2A.Z probably affects higher order folding by altering core histone tail domain interactions with nearby nucleosomes. The tail domains are required for both higher order folding and oligomerization8,10,11. H2A.Z-containing nucleosomes have a more extensive acidic patch on the nucleosome surface compared to nonvariant nucleosomes7. This may promote tail domain-dependent nucleosome–nucleosome interactions, thereby facilitating the close approach of neighboring nucleosomes and concomitant intramolecular folding. The basis for the inhibitory effect of H2A.Z on oligomerization remains to be explained, but may be related to competition between folding and oligomerization for the requisite tail domains12. During the preparation of this manuscript, a conflicting study was published, which reported that H2A.Z-containing nucleosomes were less stable compared to nucleosomes containing major H2A, and, because of this, H2A.Z arrays were less folded23. This study differs from ours in several ways. First, we used a histone expression and purification system shown to produce nucleosomes that yield high-resolution crystallographic structures. Second, their study used a monovalent cation instead of divalent cations to study array folding, and monovalent cations have been clearly established to be unable to induce the formation of a stable 55S structure9. Third, their study did not demonstrate that the arrays were fully in complex with nucleosomes. In addition, H2A.Z was shown to stabilize the association of H2A.Z/H2B dimers in nucleosomes isolated from chicken erythrocyte chromatin24. Consistent with this enhanced nucleosome stability, we have also found that H2A.Z strengthens the interaction between the H2A.Z/H2B dimer and the H3/H4 tetramer using our in vitro assembled nucleosomal arrays in salt dissociation 175 © 2002 Nature Publishing Group http://structbio.nature.com letters experiments (data not shown). Potentially, enhanced nucleosome stability can explain why H2A.Z-containing octamers are found in one major position on the 5S RNA gene (Fig. 4); however, we cannot rule out that intranucleosomal interactions during the folding process may facilitate positioning of H2A.Z nucleosomes. In summary, we report the first biochemical analysis of a homogenous preparation of H2A.Z-containing nucleosomal arrays. H2A.Z uniquely effects chromatin condensation; higher order folding is accentuated, whereas oligomerization is inhibited. These data suggest that a major function of H2A.Z is to regulate the conformational equilibria of the chromatin fiber to promote specialized functional chromosomal domains. Methods Nucleosome array reconstitution and sedimentation velocity analysis. H2A.Z and control histone octamers were assembled from recombinant histones25. These different octamers were used to assemble the 208-12 DNA template into nucleosomal arrays26. Sedimentation velocity studies using a Beckman XL-A and analysis of boundaries were carried out as described15. These data yielded the integration distribution of sedimentation coefficients plotted as the boundary fraction versus S20,W (sedimentation coefficient corrected for water at 20 °C). Average sedimentation coefficients (Save) were determined from the rate of sedimentation at the boundary midpoint (boundary fraction = 0.5). Determination of nucleosome array saturation. Nucleosomal arrays (∼2 µg) were digested with EcoRI and subjected to native agarose gel electrophoresis as described15. Densitometry was used to quantify the extent of nucleosome assembly, accounting for the population of partially digested di-nucleosomes. To correct for histone quenching of fluorescence, the ethidium signal from nucleosomal bands was multiplied 2.5-fold15. Quantitative gel electrophoresis was performed as described9. Microcentrifuge assay for oligomerization self-association. The ability of H2A.Z and control arrays to oligomerize at different ZnCl2 concentrations was determined using a differential centrifugation assay15. 176 Nucleosome positioning. Nucleosomal arrays were extensively digested with micrococcal nuclease. The monomer was DNA purified and digested with EcoRI and AluI. The DNA digestion products were analyzed by polyacrylamide gel electrophoresis26. Acknowledgments This work was supported by a Human Frontier Science Program grant to K.L. and D.J.T., and an NIH grant to J.C.H. Competing interests statement The authors declare that they have no competing financial interests. Correspondence should be addressed to D.J.T. email: [email protected] Received 12 November, 2001; accepted 24 January, 2002. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. Strahl, B.D. & Allis, C.D. Nature 403, 41–45 (2000). Jackson, J.D. & Gorovsky, M.A. Nucleic Acids Res. 28, 3811–3816 (2000). van Daal, A. & Elgin, S.C. Mol. 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