Two DNA-binding sites on the globular domain of histone H5 are required for binding to both bulk and 5 S reconstituted nucleosomes

We have previously shown the existence of two DNA-binding sites on the globular domain of H5 (termed GH5), both of which are required for nucleosome organisation, as judged by the protection of a 166 bp chromatosome intermediate during micrococcal nuclease digestion of chromatin. This supports a model in which GH5 contacts two duplexes on the nucleosome. However, studies of a nucleosome assembled on the 5 S rRNA gene have argued against the requirement for two DNA-binding sites for chromatosome protection, which has implications for the role of linker histones. We have used this proposed difference in the requirement for a second site on the globular domain in the two models as a means of investigating whether bulk and reconstituted 5 S nucleosomes are indeed fundamentally different. GH5 protects a 166 bp chromatosome in both "bulk" and 5 S systems, and in both cases protection is abolished when all four basic residues in site II are replaced by alanine. Binding to four-way DNA junctions, which present a pair of juxtaposed duplexes, is also abolished. Single mutations of the basic residues did not abolish chromatosome protection in either system, or binding to four-way junctions, suggesting that the residues function as a cluster. Both bulk and 5 S nucleosomes thus require a functional second DNA-binding site on GH5 in order to bind properly to the nucleosome. This is likely to reflect a similar mode of binding in each case, in which two DNA duplexes are contacted in the nucleosome. There is no indication from these experiments that linker histones bind fundamentally differently to 5 S and bulk nucleosomes.     

Cooperative binding of the globular domains of histones H1 and H5 to DNA.

In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.     

Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H1(0) linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.     

Nucleosome Interaction Surface of Linker Histone H1c Is Distinct from That of H10

The fully organized structure of the eukaryotic nucleosome remains unsolved, in part due to limited information regarding the binding site of the H1 or linker histone. The central globular domain of H1 is believed to interact with the nucleosome core at or near the dyad and to bind at least two strands of DNA. We utilized site-directed mutagenesis and in vivo photobleaching to identify residues that contribute to the binding of the globular domain of the somatic H1 subtype H1c to the nucleosome. As was previously observed for the H1⁰ subtype, the binding residues for H1c are clustered on the surface of one face of the domain. Despite considerable structural conservation between the globular domains of these two subtypes, the locations of the binding sites identified for H1c are distinct from those of H1⁰. We suggest that the globular domains of these two linker histone subtypes will bind to the nucleosome with distinct orientations that may contribute to higher order chromatin structure heterogeneity or to differences in dynamic interactions with other DNA or chromatin-binding proteins.     

Distinct properties of the two putative "globular domains" of the yeast linker histone, Hho1p

The putative linker histone in Saccharomyces cerevisiae, Hho1p, has two regions of sequence (GI and GII) that are homologous to the single globular domains of linker histones H1 and H5 in higher eukaryotes. However, the two Hho1p "domains" differ with respect to the conservation of basic residues corresponding to the two putative DNA-binding sites (sites I and II) on opposite faces of the H5 globular domain. We find that GI can protect chromatosome-length DNA, like the globular domains of H1 and H5 (GH1 and GH5), but GII does not protect. However, GII, like GH1 and GH5, binds preferentially (and with higher affinity than GI) to four-way DNA junctions in the presence of excess linear DNA competitor, and binds more tightly than GI to linker-histone-depleted chromatin. Surprisingly, in 10 mM sodium phosphate (pH 7.0), GII is largely unfolded, whereas GI, like GH1 and GH5, is structured, with a high alpha-helical content. However, in the presence of high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate) GII is also folded; the anions presumably mimic DNA in screening the positive charge. This raises the possibility that chromatin-bound Hho1p may be bifunctional, with two folded nucleosome-binding domains.     

The Saccharomyces cerevisiae linker histone Hho1p, with two globular domains, can simultaneously bind to two four-way junction DNA molecules

Saccharomyces cerevisiae encodes a single linker histone, Hho1p, with two globular domains. This raised the possibility that Hho1p could bind to two nucleosome cores simultaneously. To evaluate this idea, we studied the ability of a four-way junction, immobilized on the surface of a magnetic bead, to pull down a radiolabeled four-way junction in the presence of different Hho1 proteins. Four-way junctions are known to bind to H1, presumably due to structure similarities to the DNA at the nucleosomal entry/exit point. We found a significant increase in the ability of full-length Hho1p to pull down radiolabeled four-way junction DNA under ionic conditions where both globular domains could bind. The binding was structure specific, since the use of double-stranded DNA, or a mutant Hho1p in which the second DNA binding site of globular domain 1 was abolished, resulted in a significant decrease in bridged binding. Additionally, bridged binding required a covalent attachment between the two globular domains, since factor Xa protease treatment of the complex formed by a modified Hho1p that contained a factor Xa cleavage site between the two globular domains resulted in a significant release of radiolabeled four-way junction. These findings demonstrated that the two globular domains independently associated with two different four-way junction molecules in a manner that required amino acid residues implicated in structure-specific binding in the nucleosome. We discuss the implication of these findings on the chromatin structure of yeast and propose a model where a single Hho1 protein binds to two serially adjacent nucleosomes.     

The interaction of histone H5 and its globular domain with core particles, depleted chromatosomes, polynucleosomes, and a DNA decamer

Certain features of linker histone behavior were analyzed using a precipitation and a nitrocellulose filter binding assay. Chromatosomes, depleted of the linker histones, present one unique binding site to the globular domain of histone H5 (GH5) which involves the two 10-base pair DNA ends of the chromatosome. Additional binding to lower affinity sites is intrinsically different and results in aggregation as does all binding to core particles. These findings, as well as the binding study on a synthetic DNA decamer, lend support to earlier hypotheses of more than one DNA binding site on the globular domain. Our studies provide a deeper insight into the long standing question of H5/nucleosome stoichiometry. A salt dependence analysis of GH5 binding to H5-depleted chromatosomes indicates that GH5 displaces a number of ions similar to the total H1 linker histone, suggesting a delocalized binding of the carboxyl- and amino-terminal tails.     

Linker histone-dependent organization and dynamics of nucleosome entry/exit DNAs

A DNA sequence-dependent nucleosome structural and dynamic polymorphism was recently uncovered through topoisomerase I relaxation of mononucleosomes on two homologous approximately 350-370 bp DNA minicircle series, one originating from pBR322, the other from the 5S nucleosome positioning sequence. Whereas both pBR and 5S nucleosomes had access to the closed, negatively crossed conformation, only the pBR nucleosome had access to the positively crossed conformation. Simulation suggested this discrepancy was the result of a reorientation of entry/exit DNAs, itself proposed to be the consequence of specific DNA untwistings occurring in pBR nucleosome where H2B N-terminal tails pass between the two gyres. The present work investigates the behavior of the same two nucleosomes after binding of linker histone H5, its globular domain, GH5, and engineered H5 C-tail deletion mutants. Nucleosome access to the open uncrossed conformation was suppressed and, more surprisingly, the ability of 5S nucleosome to positively cross was largely restored. This, together with the paradoxical observation of a less extensive crossing in the negative conformation with GH5 than without, favored an asymmetrical location of the globular domain in interaction with the central gyre and only entry (or exit) DNA, and raised the possibility of the domain physical rotation as a mechanism assisting nucleosome fluctuation from one conformation to the other. Moreover, both negative and positive conformations showed a high degree of loop conformational flexibility in the presence of the full-length H5 C-tail, which the simulation suggested to reflect the unique feature of the resulting stem to bring entry/exit DNAs in contact and parallel. The results point to the stem being a fundamental structural motif directing chromatin higher order folding, as well as a major player in its dynamics.     

A K52Q substitution in the globular domain of histone H1t modulates its nucleosome binding properties

A comparison of the globular domain sequences of the somatic H1d and testis-specific H1t revealed a single substitution of lysine 52 in H1d to glutamine 54 in H1t, which is one of the three crucial residues within the second DNA binding site. The globular domains of both histones were modeled using the crystal structure of chicken GH5 as a template and was also docked onto the nucleosome structure. The glutamine residue in histone H1t forms a hydrogen bond with main chain carbonyl of methionine-52 (in H1t) and is spatially oriented away from the nucleosome dyad axis. A consequence of this change was a lower affinity of recombinant histone H1t towards Four-way junction DNA and reconstituted 5S mononucleosomes. When Gln-54 in Histone H1t was mutated to lysine, its binding affinity towards DNA substrates was comparable to that of histone H1d. The differential binding of histones H1d and H1t towards reconstituted mononucleosomes was also reflected in the chromatosome-stop assay.     

Roles of H1 domains in determining higher order chromatin structure and H1 location

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.     

On the structure and dynamics of the complex of the nucleosome and the linker histone

Several different models of the linker histone (LH)–nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.     

Binding Dynamics of Disordered Linker Histone H1 with a Nucleosomal Particle

Linker histone H1 is an essential regulatory protein for many critical biological processes, such as eukaryotic chromatin packaging and gene expression. Mis-regulation of H1s is commonly observed in tumor cells, where the balance between different H1 subtypes has been shown to alter the cancer phenotype. Consisting of a rigid globular domain and two highly charged terminal domains, H1 can bind to multiple sites on a nucleosomal particle to alter chromatin hierarchical condensation levels. In particular, the disordered H1 amino- and carboxyl-terminal domains (NTD/CTD) are believed to enhance this binding affinity, but their detailed dynamics and functions remain unclear. In this work, we used a coarse-grained computational model, AWSEM-DNA, to simulate the H1.0b-nucleosome complex, namely chromatosome. Our results demonstrate that H1 disordered domains restrict the dynamics and conformation of both globular H1 and linker DNA arms, resulting in a more compact and rigid chromatosome particle. Furthermore, we identified regions of H1 disordered domains that are tightly tethered to DNA near the entry-exit site. Overall, our study elucidates at near-atomic resolution the way the disordered linker histone H1 modulates nucleosome’s structural preferences and conformational dynamics.     

Extranucleosomal DNA binding directs nucleosome sliding by Chd1

Chd1- and ISWI-type chromatin remodelers can sense extranucleosomal DNA and preferentially shift nucleosomes toward longer stretches of available DNA. The DNA-binding domains of these chromatin remodelers are believed to be responsible for sensing extranucleosomal DNA and are needed for robust sliding, but it is unclear how these domains contribute to directional movement of nucleosomes. Here, we show that the DNA-binding domain of Chd1 is not essential for nucleosome sliding but is critical for centering mononucleosomes on short DNA fragments. Remarkably, nucleosome centering was achieved by replacing the native DNA-binding domain of Chd1 with foreign DNA-binding domains of Escherichia coli AraC or Drosophila melanogaster engrailed. Introducing target DNA sequences recognized by the foreign domains enabled the remodelers to rapidly shift nucleosomes toward these binding sites, demonstrating that these foreign DNA-binding domains dictated the direction of sliding. Sequence-directed sliding occluded the target DNA sequences on the nucleosome enough to promote release of the remodeler. Target DNA sequences were highly stimulatory at multiple positions flanking the nucleosome and had the strongest influence when separated from the nucleosome by 23 or fewer base pairs. These results suggest that the DNA-binding domain's affinity for extranucleosomal DNA is the key determinant for the direction that Chd1 shifts the nucleosome.     

Co-operative binding of the globular domain of histone H5 to DNA.

The globular domain of histone H5 (GH5) was prepared by trypsin digestion of H5 that was extracted from chicken erythrocyte nuclei with NaCl. Electron microscopy, sucrose gradient centrifugation, native agarose gel electrophoresis and equilibrium density gradient ultracentrifugation show that GH5 binds co-operatively to double-stranded DNA. The electron microscopic images suggest that the GH5-DNA complexes are very similar in structure to co-operative complexes of intact histone H1 (or its variants) with double-stranded DNA, studied previously, which have been proposed to consist of two parallel DNA double helices sandwiching a polymer of the protein. For complexes with GH5 or with intact H1, naked DNA co-sediments with the protein-DNA complexes through sucrose gradients, and DNA also appears to protrude from the ends and sides of the complexes; measurements of the protein-DNA stoichiometry in fractionated samples may not reflect the stoichiometry in the complexes. An estimate of the stoichiometry obtained from the buoyant density of fixed GH5-DNA complexes in CsCl suggests that sufficient GH5 is present in the complexes for the GH5s to be in direct contact, as required by a simple molecular mechanism for the co-operative binding. Chemical crosslinking demonstrates that GH5s are in close proximity in the complexes. In the absence of DNA, GH5-GH5 interactions are weak or non-existent.     

A nucleosome interaction module is required for normal function of Arabidopsis thaliana BRAHMA

The BRAHMA (BRM) gene encodes the SNF2-type ATPase of the putative Arabidopsis thaliana SWI/SNF chromatin remodelling complex. This family of ATPases is characterized by the presence of a conserved catalytic domain and an arrangement of auxiliary domains, whose functions in the remodelling activity remains unclear. Here, we characterize, at the molecular and functional level, the carboxy-terminal part of Arabidopsis BRM. We have found three DNA-binding regions that bind various free DNA and nucleosomal probes with different specificity. One of these regions contains an AT-hook motif. The carboxy terminus also contains a bromodomain able to bind histones H3 and H4. We propose that this array of domains constitute a nucleosome interaction module that helps BRM to interact with its substrate. We also characterize an Arabidopsis mutant that expresses a BRM protein lacking the last 454 amino acid residues (BRM-DeltaC), encompassing the bromodomain and two of the three DNA-binding activities identified. This mutant displays an intermediate phenotype between those of the wild-type and a null allele mutant, suggesting that the nucleosome interaction module is required for the normal function of BRM but it is not essential for the remodelling activity of BRM-containing SWI/SNF complexes.     

Unraveling linker histone interactions in nucleosomes

Considerable progress has been made recently in defining the interactions of linker histones (H1s) within nucleosomes. Major advancements include atomic resolution structures of the globular domain of full-length H1s in the context of nucleosomes containing full-length linker DNA. Although these studies have led to a detailed understanding of the interactions and dynamics of H1 globular domains in the canonical on-dyad nucleosome binding pocket, more information regarding the intrinsically disordered N-terminal and C-terminal domains is needed. In this review, we highlight studies supporting our current understanding of the structures and interactions of the N-terminal, globular, and C-terminal domains of linker histones within the nucleosome.     

Molecular modeling of the chromatosome particle

In an effort to understand the role of the linker histone in chromatin folding, its structure and location in the nucleosome has been studied by molecular modeling methods. The structure of the globular domain of the rat histone H1d, a highly conserved part of the linker histone, built by homology modeling methods, revealed a three-helical bundle fold that could be described as a helix–turn–helix variant with its characteristic properties of binding to DNA at the major groove. Using the information of its preferential binding to four-way Holliday junction (HJ) DNA, a model of the domain complexed to HJ was built, which was subsequently used to position the globular domain onto the nucleosome. The model revealed that the primary binding site of the domain interacts with the extra 20 bp of DNA of the entering duplex at the major groove while the secondary binding site interacts with the minor groove of the central gyre of the DNA superhelix of the nucleosomal core. The positioning of the globular domain served as an anchor to locate the C-terminal domain onto the nucleosome to obtain the structure of the chromatosome particle. The resulting structure had a stem-like appearance, resembling that observed by electron microscopic studies. The C-terminal domain which adopts a high mobility group (HMG)-box-like fold, has the ability to bend DNA, causing DNA condensation or compaction. It was observed that the three S/TPKK motifs in the C-terminal domain interact with the exiting duplex, thus defining the path of linker DNA in the chromatin fiber. This study has provided an insight into the probable individual roles of globular and the C-terminal domains of histone H1 in chromatin organization.     

Two homologous domains of similar structure but different stability in the yeast linker histone, Hho1p

The Saccharomyces cerevisiae homologue of the linker histone H1, Hho1p, has two domains that are similar in sequence to the globular domain of H1 (and variants such as H5). It is an open question whether both domains are functional and whether they play similar structural roles. Preliminary structural studies showed that the two isolated domains, GI and GII, differ significantly in stability. In 10 mM sodium phosphate (pH 7), the GI domain, like the globular domains of H1 and H5, GH1 and GH5, was stably folded, whereas GII was largely unstructured. However, at high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate), which might mimic the charge-screening effects of DNA phosphate groups, GII was folded. In view of the potential significance of these observations in relation to the role of Hho1p, we have now determined the structures of its GI and GII domains by NMR spectroscopy under conditions in which GII (like GI) is folded. The backbone r.m.s.d. over the ordered residues is 0.43 A for GI and 0.97 A for GII. Both structures show the "winged-helix" fold typical of GH1 and GH5 and are very similar to each other, with an r.m.s.d. over the structured regions of 1.3 A, although there are distinct differences. The potential for GII to adopt a structure similar to that of GI when Hho1p is bound to chromatin in vivo suggests that both globular domains might be functional. Whether Hho1p performs a structural role by bridging two nucleosomes remains to be determined.