Crystal structures of heterotypic nucleosomes containing histones H2A.Z and H2A

H2A.Z is incorporated into nucleosomes located around transcription start sites and functions as an epigenetic regulator for the transcription of certain genes. During transcriptional regulation, the heterotypic H2A.Z/H2A nucleosome containing one each of H2A.Z and H2A is formed. However, previous homotypic H2A.Z nucleosome structures suggested that the L1 loop region of H2A.Z would sterically clash with the corresponding region of canonical H2A in the heterotypic nucleosome. To resolve this issue, we determined the crystal structures of heterotypic H2A.Z/H2A nucleosomes. In the H2A.Z/H2A nucleosome structure, the H2A.Z L1 loop structure was drastically altered without any structural changes of the canonical H2A L1 loop, thus avoiding the steric clash. Unexpectedly, the heterotypic H2A.Z/H2A nucleosome is more stable than the homotypic H2A.Z nucleosome. These data suggested that the flexible character of the H2A.Z L1 loop plays an essential role in forming the stable heterotypic H2A.Z/H2A nucleosome.


Introduction
The nucleosome is a basic unit of eukaryotic chromatin, in which genomic DNA is compacted and accommodated within the nucleus. In the nucleosome, two copies each of histones H2A, H2B, H3 and H4 form the histone octamer, which wraps about 150 base-pairs of DNA on its surface [1]. Nucleosomes are connected with linker DNAs and form poly-nucleosomes. The local higher-order configurations of poly-nucleosomes are considered as determinants for the expression or repression of genes in certain loci [2,3].
The local higher-order chromatin configuration may be amended by various nucleosomes containing histone modifications and histone variants. Posttranslational modifications of histones occur on specific chromosome loci and epigenetically regulate the gene expression of these loci through the higherorder chromatin configuration and dynamics [4][5][6][7][8][9]. Histone variants are also important epigenetic markers, which may dictate the functional regions of chromosomes or the specific loci of the genomic DNA [10 -12]. Histone variants are encoded as non-allelic histone genes and have different amino acid sequences from those of the canonical histones [13].
Among the histone variants, H2A.Z is known as a universal nucleosome component and has been suggested to function as a regulator of transcription [14 -16]. The contributions of H2A.Z in chromosome stability and DNA repair have also been reported [17][18][19][20][21][22]. H2A.Z is an essential factor for early development and stem cell differentiation in metazoans, but its role in these developmental stages remains poorly understood [23][24][25][26][27]. H2A.Z is known to accumulate around transcription start sites (TSSs), which frequently contain nucleosome-depleted regions (NDRs), especially in transcriptionally active genes [15,16,[28][29][30][31][32][33]. Importantly, the depletion of H2A.Z reportedly enhances the nucleosomal barrier to RNA polymerase, suggesting that the H2A.Z nucleosome just downstream of the TSS (þ1 nucleosome) may function to relieve the RNA polymerase pausing by the nucleosomal barrier at the TSS [34]. Therefore, the H2A.Z nucleosomes around TSSs may be required for transcriptional activation, due to their unstable character. Consistent with this idea, previous experiments designed to detect whole nucleosome disruption suggested that the H2A.Z nucleosome is less stable than the canonical nucleosome [35][36][37]. The instability of the H2A.Z nucleosome may be more significant, when the histone H3.3 variant is incorporated into the H2A.Z nucleosome [38]. However, a fluorescence resonance energy transfer assay, which specifically detects the H2A-H2B dissociation from the nucleosome, suggested that the dissociation of H2A.Z-H2B is more salt-resistant than that of H2A-H2B under high salt conditions (around 550 mM NaCl) [39].
The H2A.Z nucleosome (þ1) of active genes reportedly shifts upstream and occupies the TSS regions during mitosis, when transcription is generally suppressed [40]. Intriguingly, in mouse trophoblast stem cells, the H2A.Z nucleosomes around TSS regions convert from homotypic (H2A.Z/ H2A.Z) to heterotypic (H2A.Z/H2A) after DNA replication [41,42]. The heterotypic H2A.Z/H2A nucleosomes may occupy the TSS to regulate the transcription status of the related genes [41,42]. However, the previous crystal structures of the homotypic H2A.Z nucleosomes indicated that the L1 loop structure of H2A.Z is very different from that of canonical H2A and may cause steric hindrance when it forms the heterotypic nucleosome with canonical H2A [36,42,43].
To understand this intriguing discrepancy, in this study, we reconstituted the heterotypic H2A.Z/H2A nucleosomes and determined their crystal structures. The structures revealed that the H2A.Z L1 loop configuration drastically changes upon heterotypic nucleosome formation, without any structural change of the canonical H2A structure. To our surprise, we found that the heterotypic H2A.Z/H2A nucleosome is more stable than the homotypic H2A.Z nucleosome. These structural and biochemical properties of nucleosomes containing H2A.Z, homotypically and heterotypically, are important to understand the mechanism by which the H2A.Z-dependent transcriptional regulation is epigenetically maintained and promoted in cells.

Results and discussion
2.1. Preparation of the heterotypic H2A.Z/H2A nucleosome To understand the mechanism by which the H2A.Z and H2A molecules are heterotypically accommodated within a nucleosome, we reconstituted the heterotypic H2A.Z/H2A nucleosome, with H3.1 as the histone H3 subunit, by a method based on previous studies [44,45] (figure 1a). In mammals, H2A.Z.1 and H2A.Z.2 are found as two non-allelic isoforms [46]. The H2A.Z.1 knockout in mice is lethal, indicating its essential role in development [24]. Therefore, in this study, we used H2A.Z.1 as the representative H2A.Z. Human histone H2A was prepared as a fusion protein containing an additional 144 amino acid peptide (Tag) at its N-terminus, just before the thrombin recognition sequence, Leu-Val-Pro-Arg-Gly-Ser (H2A Tag ; electronic supplementary material, table S1). The nucleosomes were reconstituted by the salt-dialysis method with H2A.Z and H2A Tag , in the presence of H2B, H3.1 and H4. In this step, the homotypic H2A Tag nucleosome, the homotypic H2A.Z nucleosome and the heterotypic H2A.Z/H2A Tag nucleosome were reconstituted (figure 1b, lane 2). These three nucleosomes were separated very well by native polyacrylamide gel electrophoresis (PAGE) (figure 1b, lane 2). We then purified the heterotypic nucleosome by preparative native PAGE (

Stability of the heterotypic H2A.Z/H2A nucleosome
We then performed a thermal stability assay to evaluate the stabilities of the reconstituted nucleosomes [45,47,48]. In this method, the histones thermally dissociated from the nucleosome are detected by the fluorescent signal of SYPRO Orange bound to free histones. Consistent with the previous results [48], the canonical H2A nucleosome showed a bi-phasic thermal denaturation curve (figure 1d, upper panel) with two dissociation temperature peaks at 74-758C and 81-828C, corresponding to the H2A-H2B and H3-H4 dissociations, respectively, under the conditions without NaCl (figure 1d, lower panel). In the homotypic H2A.Z nucleosome, H2A.Z-H2B dissociated from the nucleosome at a lower temperature than H2A-H2B in the canonical H2A nucleosome (figure 1d). These results indicated that the homotypic H2A.Z nucleosome is less stable than the canonical H2A nucleosome. To our surprise, the heterotypic H2A.Z/H2A nucleosome was clearly more stable than the homotypic H2A.Z nucleosome, but slightly less stable than the canonical H2A nucleosome (figure 1d). This moderate stability of the heterotypic H2A.Z/H2A nucleosome was also confirmed under the conditions with 250 mM NaCl (figure 1e). Therefore, the presence of canonical H2A may facilitate the H2A.Z-H2B association with the H3-H4 tetramer and/or DNA in the nucleosome.
2.3. The H2A.Z L1 loop structure is drastically altered in the heterotypic H2A.Z/H2A nucleosome To reveal the structural basis for the heterotypic H2A.Z/H2A nucleosome formation and stability, we determined the crystal structure of the heterotypic H2A.Z/H2A nucleosome at 2.2 Å resolution (figure 2a and table 1). In the crystal structure, the electron densities for the H2A.Z-specific residues, such as Thr49, Gly92 and Gly106, are clearly distinguishable from the corresponding H2A-specific residues, Gly46, Asn89 and Gln104 (figure 2b  ... (IV) ... (III) ... (II) 0 mM NaCl 250 mM NaCl  We then compared the L1 loop structures of H2A.Z and H2A in the heterotypic nucleosome with those in the homotypic nucleosomes. We found that the H2A.Z L1 loop structure is drastically altered in the heterotypic nucleosome, when compared with that in the homotypic H2A.Z nucleosome (figure 3a). In this H2A.Z Ll loop structure, the H2A.Z.1-specific Ser38 residue, which is replaced by Thr in H2A.Z.2, does not contact the other residues, and thus it may not affect the L1 loop structure in the heterotypic H2A.Z/H2A nucleosome if it is replaced by Thr. Surprisingly, in the heterotypic H2A.Z/H2A nucleosome, no obvious difference was observed in the H2A L1 loop structures between the heterotypic and homotypic H2A nucleosomes (figure 3b). These results indicated that the conformation of the H2A.Z L1 loop flexibly changes to fit the H2A L1 loop structure in the heterotypic H2A.Z/H2A nucleosome.

Preparation of recombinant human histones and histone complexes
The DNA fragment encoding the 144 amino acid (144aa) peptide tag sequence was inserted just upstream of the His 6 -tag sequence in the pET15b-H2A plasmid vector (electronic supplementary material, table S1). The 144aa tagged H2A peptide containing a His 6 -tag peptide was produced in E. coli BL21 (DE3) cells and was purified by Ni-NTA agarose (Qiagen) column chromatography. The 144aa and His 6 tagged H2A peptide was dialysed against water four times and then lyophilized. H2A.Z.1, H2B, H3.1 (or H3.3) and H4 were purified as described previously [36,55,56]. The 144aa and After this dialysis step, the samples were incubated at 558C for 2 h to prevent improper histone -DNA binding. The heterotypic H2A.Z/tagged H2A nucleosome was purified by preparative native PAGE [45]. The tag peptide was proteolytically removed by thrombin protease (GE Healthcare) and the heterotypic H2A.Z/H2A nucleosome was further purified by another round of preparative native PAGE. For crystallization, the heterotypic H2A.Z/H2A nucleosome was dialysed against 20 mM potassium cacodylate buffer (pH 6.0), containing 1 mM EDTA.

Thermal stability assay of nucleosomes
The nucleosome stability was monitored by a thermal stability assay, as described previously [45,47,48]. Purified nucleosomes were mixed with SYPRO Orange dye (Sigma-Aldrich) in 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM dithiothreitol, in the presence or absence of 250 mM NaCl. The SYPRO Orange fluorescence was detected with a StepOnePlus Real-Time PCR system (Applied Biosystems), using a temperature gradient from 258C to 958C, in steps of 18C min 21 . or 28% PEG400, and 2% trehalose, and were flash-cooled in a stream of N 2 gas (21808C). The diffraction datasets of the heterotypic H2A.Z/H2A nucleosomes with H3.1 or H3.3 were collected with an X-ray wavelength of 1.1 Å at 21738C on the BL-1A beamline at the Photon Factory (Tsukuba, Japan). The diffraction dataset of the homotypic H2A.Z nucleosomes with H3.3 was collected with an X-ray wavelength of 1.0 Å at 21738C on the BL41XU beamline at SPring-8. The datasets were processed using the HKL2000 and CCP4 programs [57,58]. The structures of the heterotypic H2A.Z/H2A nucleosomes with H3.1 or H3.3 were determined by molecular replacement with the PHASER program, using the crystal structure of the canonical nucleosome (PDB ID: 3AFA) as the search model [56,59]. The structure of the homotypic H2A.Z nucleosome with H3.3 was determined by molecular replacement with the PHASER program, using the crystal structure of the nucleosome containing H3.3 (PDB ID: 3AV2) as the search model [59,60]. The refinements and model building of the atomic coordinates were performed using the PHENIX and COOT programs [61,62]. The Ramachandran plot of the heterotypic H2A.Z/H2A nucleosome with H3.1 showed 98.1% of the residues in the favoured region, 1.9% of the residues in the  rsob.royalsocietypublishing.org Open Biol. 6: 160127 allowed region and no outlying residues. The Ramachandran plot of the heterotypic H2A.Z/H2A nucleosome with H3.3 showed 98.1% of the residues in the favoured region, 1.9% of the residues in the allowed region and no outlying residues. The Ramachandran plot of the homotypic H2A.Z nucleosome with H3.3 showed 95.8% of the residues in the favoured region, 4.2% of the residues in the allowed region and no outlying residues. A summary of the data collection and refinement statistics is provided in table 1. Structural graphics were displayed using the PYMOL program (http://pymol.org).

B-factor calculation
The B-factors for the Ca atoms of the H2A.Z.1 molecules in the heterotypic H2A.Z/H2A nucleosome and the homotypic H2A.Z nucleosome (PDB ID: 3WA9) were calculated using the PHENIX program. rsob.royalsocietypublishing.org Open Biol. 6: 160127