Overall Structure of the Globular Domain of H5 Bound to the Nucleosome

Since MNase digestion experiments show that the tails of H5 do not affect the interactions between the globular domain and the nucleosome (Allan et al., 1980;Puigdomenech et al., 1983) and the globular domain in complex with the nucleosome has been used as a model for the chromatosome in earlier studies (Allan et al., 1980;Zhou et al., 1998), we reconstituted the globular domain of H5 (H5_22-98) in complex with the nucleosome for structural determination. The nucleosome contains 167 bp DNA centered with the 147 bp ‘601’ positioning sequence (Dorigo et al., 2004;Thastrom et al., 2004). We crystallized the complex and determined its structure at 3.5 Å resolution using X-ray diffraction and the molecular replacement method (Figure 1A,Figure S1 andTable 1). The structure is well defined with observable electron densities for all backbones and most of the side chains (Figure 1B-E). In the structure (pdbID: 4QLC), the globular domain sits on the nucleosome dyad and interacts with both DNA linkers. The L3 loop and the N-terminal region of the α2 helix interact with the dyad. The L1 loop interacts with one DNA linker and the α3 helix interacts with the other. Accordingly, we termed the two DNA linkers linker-L1 and linker-α3, respectively. The C-terminus of the globular domain is close to the ends of the linker DNA, consistent with its role in the stem structure formation of longer linker DNAin vitro (Bednar et al., 1998;Hamiche et al., 1996;Syed et al., 2010). The structure of the nucleosome core particle region in the complex is similar to that of the free form (Figure 1F).

We next used¹³C-labeled methyl groups in residues Ile, Leu, and Val of the globular domain (Tugarinov et al., 2004) and paramagnetic spin labeling (Clore et al., 2007) to investigate the location and orientation of the globular domain on the nucleosome in solution. In these experiments, we first assigned chemical shifts of the methyl groups of the globular domain in complex with the nucleosome by comparing them with those of free globular domain, by measuring the NOEs among the methyl groups and by mutating specific residues (Figure 2A andFigure S2). We mutated residues H2A Thr119 or H3 Lys37 in the nucleosome to Cys and linked them to the paramagnetic spin label (MTSL) through a disulfide bond. Residues H2A Thr119 and H3 Lys37 in the nucleosome are largely disordered but close to the folded region. Spin labels at these positions should cause little perturbation to the structure of the nucleosome but have relatively defined location (Zhou et al., 2013). The effects of the spin labels on the NMR peak intensities of the methyl groups depend on their distances to the corresponding methyl groups of the globular domain in an inversely related manner (Battiste and Wagner, 2000). We found that the measured intensities of the methyl NMR peaks are in agreement with the calculated distances between the methyl groups and the spin label sites in the crystal structure, showing the expected profile and a 50% decrease in peak intensity at ∼17 Å distance (Figure 2B-D) (Battiste and Wagner, 2000). For example, spin labels at H3 Lys37 in the nucleosome do not have substantial effects (< 20%) on any of the peak intensities of the methyl groups (Figure 2B) and the methyl groups of the globular domain are distant (> 23 Å) from the spin labels at both H3 K37 sites (Figure 2E). Spin labels of H2A Thr119 have large effects (> 50%) only on the peak intensities of the methyl groups of residues Leu66, Leu70 and Val87 of the globular domain (Figure 2C) and these methyl groups are closest to the spin labels at the two H2A Thr119 sites (< 16 Å) (Figure 2F).

Interactions Between the Globular Domain and DNA

In the structure of the complex, the globular domain has broad contacts with DNA (Figure 3A, B andFigure S3). Residues Lys40, Arg42, Arg94 and Lys97 are close to linker-L1. Residues Arg47, Lys69, Arg73 and Lys85 interact with the DNA at the dyad. Residue Arg74 interacts with linker-α3. Electron densities are clearly observable for the side chains of Arg47, Arg73, Arg74 and Arg94 (Figure S3B, D, F), indicating that the globular domain has stable interactions with the dyad and both DNA linkers. The electron densities for the side chains of Lys40, Arg42, Lys69, Lys85, and Lys97 are either weak or invisible (Figure S3B, C, F), suggesting that these long side chains have substantial local dynamic motions. In addition to the electrostatic interactions, the charge-neutral residues Gln48, Val87 and Ser90 insert into the minor groove of the DNA at the dyad (Figure 3B). Residues His25, Tyr28 and Gln67 are in close contact with linker-α3 (Figure 3B). The side chains of these residues have observable electron densities (Figure S3C, D, E).

To investigate the energetic roles of globular domain residues in binding of the nucleosome, we mutated residues on the surface of the globular domain in H5_22-142 (with high expression yield) to Ala and measured the effect of each mutation on the equilibrium disassociation constant (K_D) (Figure 3C, D,Figure S4A, B, andTable 2). NMR experiments show that the additional 40 residues in the C-terminal tail of H5_22-142 are largely disordered in the H5_22-142-nucleosome complex (Figure S1F) and H5_22-102 and H5_22-142 show similar K_D values (0.35 and 0.23 μM, respectively) (Figure 3D andTable 2). The globular domain residues whose Ala mutations increase K_D by a factor of two or more are situated close to the linker-L1, dyad and linker-α3 (Figure 3A, B, C andTable 2), which include both positively charged and charge-neutral residues. The residues whose mutations decreased binding affinity strongly are largely associated with the dyad and linker-α3. Thus, the mutation results are fully consistent with the crystal structure in which the globular domain interacts more closely with the dyad and linker-α3 than with linker-L1 (Figure 3A, B).

Previous mutation studies have revealed two nucleosome-binding sites of the H5 globular domain: site I includes residues Lys69, Lys85, and Arg73 and site II includes residues Lys40, Arg42, Lys52 and Arg94 (Duggan and Thomas, 2000;Goytisolo et al., 1996). Additionally, the globular domain with simultaneous mutation of site II residues to Ala protects ∼157 bp DNA in the nucleosome against MNase digestion (Duggan and Thomas, 2000). These results are consistent with our structure in which site II residues (Lys40, Arg42 and Arg94) interact with linker–L1 and site I residues (should include Lys52) (Figure 3A) are close to the dyad. In contrast, based on the structural similarity between the globular domain and two DNA-binding proteins (CAP and HNF-3γ), the α3 helix of the globular domain was predicted to bind to the major groove of DNA (Ramakrishnan et al., 1993), whereas in our structure the a3 helix binds to the minor groove of DNA, in a way similar to core histones.

Structural Analysis of Previous Models for Nucleosome Recognition by Linker Histones

Three major structural models have been proposed for the nucleosome bound to the globular domain of H5 or mouse H1°, which belong to the same subtype of linker histones and have been renamed H1.0 based on phylogeny analysis (Talbert et al., 2012). In the symmetric DNA extension model, the globular domain of H5 interacts with dyad and 10 bp DNA from each of the two linkers (Allan et al., 1980). In the two asymmetric models, the globular domain interacts with the nucleosomal DNA near the dyad and 20 bp DNA (An et al., 1998a;Brown et al., 2006) or 10 bp DNA (Zhou et al., 1998) from one linker. The symmetric model is consistent with our structure and a computational docking model (Fan and Roberts, 2006), which has identified three DNA binding sites (site I, site II and site III containing Arg74) on the globular domain.

A detailed asymmetric model with 20 bp DNA from one linker is built based on the results from mutation and fluorescence recovery after photobleach (mutation-FRAP) experiments (Brown et al., 2006). The globular domain residues whose mutations have large effects on the half-lives (t_50%) of chromatin association of mouse linker histone H1° are mapped on the structure of the H5 globular domain (Figure 4A). Two clusters of positively charged residues are identified and modeled to bind at two DNA sites: nucleosomal DNA at the vicinity of the dyad and an extended 20 bp DNA linker from one side of the nucleosomal DNA. We found that one cluster (Arg42, Arg94 and Lys97) (site II) is close to linker-L1 and the other (Arg47, Lys69, Lys73, Arg74 and Lys85) binds to two DNA sites: the dyad and linker-α3, instead of one nucleosomal DNA site as suggested (Brown et al., 2006) (Figure 4B). Since the FRAP t_50% was measured using the full-length protein while the crystal structure includes the globular domain only, the agreement between these results strongly supports that the globular domains of the linker histones bind to the nucleosomein vitro or to the native chromatinin vivo in a similar manner and that the tails of the linker histones of H5/H1° do not alter the binding mode of the globular domains (Figure S4C, D).

The asymmetric model with one 10 bp linker DNA (Zhou et al., 1998) is based on a study using a site-specific protein-DNA photo-crosslinking method with a crosslinking distance of ∼11 Å (Pendergrast et al., 1992). The H5 globular domain residue Ser41 is crosslinked to the DNA site at the vicinity of the dyad of chicken nucleosomes with 168 bp heterogeneous DNA sequences (Zhou et al., 1998). The globular domain residues Ser29 and Ser71 are cross-linked to the DNA position that is 3-4 bp nucleotides away from the end of one DNA linker. In our structure, the Cα atom of residue Ser41 is ∼11 Å away from an atom in a nucleotide at the vicinity of the dyad and the Cα atoms of residues Ser29 and Ser71 are ∼11 Å away from an atom in the nucleotide that is three base pair nucleotides away from the end of linker-α3 (Figure 4D). Thus, the crosslinking results are not sufficiently definitive to determine the location and orientation of the globular domain on the nucleosome, but they are fully consistent with our structure.

We note that a previous model with interactions of 5 bp DNA from one linker and 15 bp DNA from the other with core histones in the nucleosome through allosteric effects of linker histone (Hayes et al., 1994;Pruss et al., 1996) has been refuted by later studies, which reveal that results interpreted as “protection” by linker histones in the initial study can also be obtained from naked DNA or nucleosome core particle (An et al., 1998b) and that both site I and site II of the globular domain of H5 are required for protection of 20 bp linker DNA against MNase digestion (Duggan and Thomas, 2000) instead of site I alone (Hayes et al., 1996). However, an asymmetric model with 20 bp DNA from one linker, which is based on the results fromLytechinus variegatus 5S DNA nucleosome and MNase digestion, is unexplained by our structure (An et al., 1998a). It is possible that the linker histone might still bind to the dyad but have changed the positioning of the nucleosome.

Different Binding Modes Can Lead to Distinct Higher-Order Structures of Chromatin

The L1 and L3 loops of the globular domain in complex with the nucleosome interact with DNA and show induced conformational changes when compared with the corresponding regions in the free globular domain structure (Figure 5A), whereas the L2 loop is exposed to the solvent and largely retains its conformation (Figure 5A). NMR measurement of backbone dynamics of the free globular domain indicates that the L3 loop region is highly flexible (Figure S1D). Notably, in all modeling studies of the globular domain in complex with the nucleosome, the free globular domain of H5 has been used and kept rigid. In comparison with the linker DNAs in the structural models of the tetra-nucleosome in the absence of linker histones (Schalch et al., 2005) and the 30 nm nucleosome array condensed by H1.4 (Song et al., 2014), we also found that the globular domain of H5 alters the trajectories of the two DNA linkers and brings them closer to the dyad by ∼10 Å at their shortest distance (Figure 5B, C). The agreement between the mutation data and the crystal structure (Figure 3A-C) suggests that crystal packing has no major effect on the conformation of the two DNA linkers.

It has been suggested that linker histones might control the trajectory of linker DNA and subsequent packing of nucleosomes in the higher-order chromatin structures (Robinson et al., 2006;Sivolob and Prunell, 2003;Song et al., 2014). Different trajectories could be conferred to linker DNA by the globular domains with different binding modes (Figure 5B, C), which might lead to distinct higher-order structures of chromatin. To test this hypothesis, we performed sedimentation experiments on a nucleosome array (12 nucleosome × 177 bp nucleosome repeat length) in the presence of 0.3 mM Mg²⁺ and the globular domain of linker histone H5 orDrosophila H1. The sedimentation coefficient of the nucleosome array containing the globular domain of H5 was substantially higher (by ∼6S) than the one containing the globular domain ofDrosophila H1 when the ratio of the globular domain over the nucleosome is at one (Figure 6). This result indicates that the binding mode of linker histones indeed can play an important role in defining the higher-order structure of chromatin. In line with the above results, previous cryo-EM studies have shown that instead of forming the twisted double helix as in the nucleosome array condensed by H1.4 (Song et al., 2014), the same nucleosome array condensed by linker histone H5 might have an interdigitated structure (Robinson et al., 2006). In addition, the results from mutation-FRAP studies on mouse H1c (George et al., 2010), which is an ortholog of human H1.4, are largely consistent with the off-dyad binding model of H1.4 (Figure S6A, B) (George et al., 2010), in contrast to the on-dyad binding mode of mouse H1° (Figure 4B) (Brown et al., 2006).

To better understand the relationship between the binding mode of linker histones and the structure of condensed chromatin, we compared the structures of the free mono-nucleosome core particle and the two available condensed chromatin arrays: the 9 Å resolution tetra-nucleosome (Schalch et al., 2005) and the 11 Å resolution H1.4-condensed nucleosome array (Song et al., 2014). We found that the DNA regions near the nucleosome entry/exit sites in the two condensed nucleosome arrays move away from the corresponding regions in the mono-nucleosome by ∼4 Å at the points closest to the histone cores (Figure S5A, B). In contrast, the corresponding DNA conformations in the nucleosome in complex with the globular domain of H5 and in the mono-nucleosome structure remain largely the same (Figure S5C). These observations suggest that the two condensed nucleosome arrays could impose restraints on the conformation of the linker DNA. In addition, one linker DNA in the tetra-nucleosome structure would clash with the globular domain of H5 bound to the dyad but could largely accommodate the globular domain ofDrosophila H1 bound to the nucleosome off the dyad (Figure S5D, E). Thus, the globular domain on the nucleosome dyad in the two condensed nucleosome arrays would either make poor interactions with the DNA linkers (Figure 5B, C andFigure S5A, B) or clash with them (Figure S5D, E). To form close contacts between the globular domain and linker DNA, either the linker DNA needs to move closer to the dyad or the globular domain needs to be at the off-dyad location, which would affect subsequent nucleosome packing differently and lead to distinct higher-order structures of chromatin.

Linker Histone Subtypes and Their Binding Modes

We observed differences at nine key positions between the amino acid sequences of the globular domains of H5 andDrosophila H1 (Figure S6A). Four positively charged residues (Arg47, Lys50, Arg74 and Lys97) in the H5 globular domain correspond to neutral residues (Leu68, Thr76, Ser96 and Ala119) in the globular domain ofDrosophila H1, whereas two neutral residues (Gln50 and Val87) in the globular domain of H5 correspond to positively chargedDrosophila H1 globular domain residues (Lys72 and Lys109). These differences are likely responsible for their on- and off-dyad binding modes (Figure S6C). Human linker histones have 11 subtypes (Figure S6A). The globular domains of human H1.0 and chicken H5 share more than 80% sequence identity and all of the residues important for nucleosome binding in H5 are conserved in H1.0. Thus, H1.0 should also bind to the nucleosome on the dyad.

Human H1.1 to H1.5 share more than 89% sequence identity and all of the positively charged residues are conserved (Figure S6A). They should bind to the nucleosome with the same binding mode. Intriguingly, in contrast to the off-dyad binding model of the H1.4-condensed nucleosome array (Figure S6B) (Song et al., 2014), hydroxyl radical foot-printing experiments suggest that the globular domain of H1.5 binds to the nucleosome on the dyad in the di- and tri-nucleosomes in a way similar to the globular domain of H5 (Syed et al., 2010). Mapping the positively charged residues of the globular domain of H1.4 to the corresponding positions in the structure of the globular domain of H5 in our crystal structure suggests that the globular domain of H1.4 on the nucleosome dyad could make broad interactions with the dyad and the two DNA linkers (Figure S6D). Although the observed electron densities for the H1.4-condensed nucleosome array also suggest that the globular domain of H1.4 has close contacts with both DNA linkers (Song et al., 2014), the L1 loop of the globular domain facing the linker DNA in the structural model of the H1.4-condensed nucleosome array seems too far away from the linker DNA (Figure S6B). It is possible that the observed electron density between the L1 loop of the globular domain of H1.4 and the linker DNA might represent the cross-linking reagent. Indeed, mutation-FRAP results suggest that the L1 loop of the mouse H1c (corresponding to human H1.4) globular domain does not show interactions with DNA (George et al., 2010). In addition, the binding of the globular domain of mouse H1c to the chromatin is substantially weaker compared to mouse H1° (George et al., 2010). Therefore, the globular domains of H1.1 to H1.5 could bind to the dyad of the nucleosome in open chromatin but might be forced to bind off the dyad of the nucleosome in the condensed nucleosome array, assuming chromatin has the intrinsic tendency to form the zigzag two-start helical structure when condensed (Schalch et al., 2005).

It should be noted that the tails of linker histone H1.4 to H1.5 are unlikely to play important roles in determining the binding modes of the globular domains. Only about five residues immediately before and after the globular domain of H1.4 are structured in the H1.4-condensed nucleosome array; the C-terminal tail residues have no interactions with DNA and the N-terminal tail residues interact with one linker DNA (Song et al., 2014). In addition, the seven residues immediately before the globular domain are conserved in H1.4 and H1.5 and deletion of the tails of H1.5 did not affect the pattern of hydroxyl radical footprinting for the 10 bp of linker DNA on each side of the nucleosome (Syed et al., 2010). In general, linker histone tails do not have binding preferences for specific DNA conformations, whereas the globular domain of linker histones preferentially binds to the nucleosomal and one or both linker DNAs. Therefore, it is unlikely that the tails could substantially alter the binding position of the globular domain on the nucleosome. These results are consistent with the view that linker histone tails are intrinsically disordered regions and likely to interact non-specifically with the linker DNA, which allows chromatin to fold by reducing electrostatic repulsion (Vuzman and Levy, 2012).

The globular domains of other linker histone subtypes (H1x, H1t, H1t2, H1oo, HILS1) have much lower sequence similarity to H1.0 or H1.4/H1.5 (Figure S6A). Using solution NMR, we assigned the backbone chemical shifts of H1x (unpublished results). Based on the Cα chemical shift values, we found that the α1 helix of the globular domain of H1x is two helical turns longer than that of the globular domain H5. Human HILS1 andDrosophila BigH1 show deletions of residues corresponding to the β1-strand and loop L3 regions of the globular domain of H5, respectively. Thus, human HILS1 andDrosophila BigH1 would also fold to structures that are distinct from that of the globular domain of H5. These results suggest that the chromatosomes containing H1x, HILS1 and BigH1 may have alternative new binding modes.

Linker Histone Function Beyond the Formation of 30 nm Chromatin Fiber

For some cell types, recent studies show that chromatin may exist predominantly as 10 nm fibers (Maeshima et al., 2014) or heterogeneous groups of nucleosomes that are associated with linker histones (Ricci et al., 2015). The more open chromatin with one or few nucleosomes in a cluster could be maintained through posttranslational modifications of the tails of core histones (Shogren-Knaak et al., 2006), where the globular domain of linker histones could function by binding to the nucleosome as it does in the chromatosome. It has been observed that the globular domain of H1c alone is sufficient for directing gene-specific transcriptional repression inXenopus embryos (Vermaak et al., 1998).

Recent studies also show that the nucleosome unravels asymmetrically under tension and that DNA sequence and modifications regulate its flexibility on the histone core and the direction of transcription (Ngo et al., 2015). If so, it is likely that linker histones also play a role in controlling the DNA dynamics on the nucleosome, and the on- and off-dyad binding of linker histones to the nucleosome may have different effects on the direction of transcription and gene expression. For example, globular domains that occlude ∼10 bp linker DNA from both sides of the nucleosome should be capable of suppressing gene expression more than those that occlude ∼10 bp linker DNA from one side of the nucleosome. The off-dyad binding of linker histones could affect the direction of transcription by sealing of the DNA at one end of the nucleosome through preferential interaction with one linker DNA.

It has long been known that linker histones bind to the nucleosome and play an essential role in the formation of 30 nm chromatin fibers. However, it has not been possible to obtain higher-resolution structural information to answer how linker histones interact with the nucleosome. Our 3.5 Å resolution structure of the globular domain of chicken H5 bound to the nucleosome provides first insights into the detailed structural mechanisms of nucleosome recognition by linker histones and helps resolve the long debate on this issue. It has also long been implied that all linker histones condense the chromatin to conform the same 30 nm chromatin fibers. We have found that different linker histones could bind to the nucleosomevia distinct structural modes, which subsequently lead to distinct higher-order structures of chromatin. Thus, various subtypes of linker histones might use diverse binding modes to regulate higher-order structures and functions of chromatin.

Sample Preparations

Histone proteins were expressed inEscherichia coli and purified using Ni-NTA beads and reverse phase HPLC. The DNA fragments were released from the plasmid vector by restriction enzymes. The globular domain of H5 was either¹⁵N/¹³C or¹⁵N/(Ile, Leu, Val) methyl-labeled by growing the cells in M9 medium containing the corresponding isotopes. H2A Thr119 or H3 Lys37 was mutated to Cys and linked to MTSL (S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanethiosulfonate). The nucleosome in complex with globular domain of linker histone H5 was reconstituted by adding the globular domain to the nucleosome directly. The nucleosome arrays in complex with the globular domains or the full-length linker histones were reconstituted by mixing the linker histones and the nucleosome array at higher salt concentration followed with dialysis in low salt buffers.

Crystal Structure Determination

Crystals were screened with the H5_22-98-nucleosome complex at 1.1:1 ratio using the crystallization robot. The X-ray diffraction data was collected at the Advanced Photon Source (APS). The diffraction data were processed with XDS or HKL2000. The structures were solved using the molecular replacement method.

NMR, ITC and Sedimentation Experiments

NMR spectra were collected on Bruker 700 and 900 MHz NMR instruments equipped with cyro-probes and processed with NMRPipe and NMRView. ITC experiments were performed on a VP-ITC microcalorimeter at 25°C by titrating linker histones to the nucleosome solution. The titration thermograph was fitted with the model containing a single equilibrium disassociation constant for binding. Sedimentation velocity data were collected at 15,000 rpm and 20 °C using a Beckman XL-A analytical ultracentrifuge. The data was analyzed in terms of a continuous c(s) distribution and sedimentation coefficients were corrected to standard conditionss_20,w.

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