Localization of linker histone in chromatosomes by cryo-atomic force microscopy

Linker histones play a fundamental role in determining higher order chromatin structure as a consequence of their association with nucelosomal DNA. Yet the locations and structural consequences of linker histone binding are still enigmatic. Here, using cryo-atomic force microscopy, we show that the linker histone H5 in native chromatin and in chromatosomes reconstituted on the 5S rDNA template is located at the dyad of the nucleosome core particle, within the "stem" structure. Direct measurement also indicates that the length of free linker DNA between chromatosomes in native chromatin is approximately 30 bp, slightly shorter than that estimated from nuclease digestion assays.     

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.     

Neutron scattering studies of chromatosomes

Neutron scattering data establish that the radius of gyration of the DNA in chicken erythrocyte chromatosome particles is significantly higher, by about 0.3 nm, than the radius of gyration of the DNA in the core particle. Corresponding information of the radius of gyration of the protein component in the chromatosomes (3.75 nm) indicated an enlargement, compared to the radius of gyration of the octamer of histone proteins both in core particles and in the histone octamer stabilised in 2 M NaCl (3.25 nm). From the latter data, we could calculate the distance in the chromatosome between the centre of mass of the linker histone and the histone octamer as 5.5 nm. These results impose severe limitations for the organisation of the 22 bp extra DNA and the possible location of H1/H5 in the chromatosome, implying that the H1/H5 is close to the centre turn of the core particle DNA.     

Quantitative determination of linking number differences between circular polynucleosomes and histone H1-bound circular polynucleosomes

With the aim of discovering contribution of histone H1 to linking number changes of DNA, determination of linking number differences between histone H1-free circular polynucleosomes and histone H1-bound circular polynucleosomes was carried out during our investigations. Our results showed that on average, binding of ～11.5 histone H1 molecules causes one linking number change in circular polynucleosomes in the presence of 1.5?mM spermidine. When concentrations of spermidine decreases or increases, these linking number differences decrease significantly. It is therefore evident that linking number changes caused by histone H1 are spermidine concentration-dependent.     

Comparative filter binding study of H5 to nucleosome core particles,H1, H5 depleted chromatosomes and DNA fragments

The filter-binding technique with PEI treated glass fiber is used to study the interaction of histone H5 to core particles, chromatosomes and DNA derived from it. By working at very low concentrations of interacting particles we are able to study the effective binding process independent of interfering insoluble complexes. The interactions are characterized by a very high affinity. An intrinsically higher affinity of H5 for cores and chromatosomes versus chromatosome derived DNA is demonstrated. Both chromatosomes and DNA derived from these bind about twice the amount as compared to core particles, which saturate at about one H5 per core particle.Abbreviations GH5 globular domain of histone H5 - PEI polyethyleneimine     

Structural appearance of linker histone H1/siRNA complexes

Efficient non-viral vectors for the in vivo siRNA transfer are still being searched for. Comparing the differences of the structural appearance of siRNA and pDNA one would assume differences in the assembling behaviour between these polyanions when using polycationic vectors such as nuclear proteins. The spontaneous assembly of nuclear proteins such as histone H1 (H1) with pDNA as polyanion which has intensively been investigated over the last decade, showed a particulate structure of the resulting complexes. For an efficient in vivo use small almost monomolecular structures are searched for. Using siRNA as the polyanion might enforce this structural prerequisite lacking unwanted aggregation processes, exploiting the molecular size of siRNA. We therefore investigated the structure of H1/siRNA complexes. Five commonly used methods characterizing the resulting assemblies such as retardation gels, static and dynamic light scattering, reduction of ethidium bromide fluorescence, analytical ultracentrifugation, and electron microscopy were used. From analytical ultracentrifugation we learned that under physiological salt conditions the siRNA-H1 binding was not cooperative, even though the gel analysis showed disproportionation which would be an indication for a cooperative binding mode. H1 formed very small and stable complexes with siRNA at a molar ratio of 1:1 and 1:2. In order to find out if the observed structural appearance of the H1/siRNA complexes is due to unspecific charge effects only or to special features of H1, polylysine was included in the study. Low molecular weight polylysine (K₁₆) showed also non-cooperative binding with siRNA.     

Structure and histone binding properties of the Vps75-Rtt109 chaperone-lysine acetyltransferase complex

The histone chaperone Vps75 presents the remarkable property of stimulating the Rtt109-dependent acetylation of several histone H3 lysine residues within (H3-H4)(2) tetramers. To investigate this activation mechanism, we determined x-ray structures of full-length Vps75 in complex with full-length Rtt109 in two crystal forms. Both structures show similar asymmetric assemblies of a Vps75 dimer bound to an Rtt109 monomer. In the Vps75-Rtt109 complexes, the catalytic site of Rtt109 is confined to an enclosed space that can accommodate the N-terminal tail of histone H3 in (H3-H4)(2). Investigation of Vps75-Rtt109-(H3-H4)(2) and Vps75-(H3-H4)(2) complexes by NMR spectroscopy-probed hydrogen/deuterium exchange suggests that Vps75 guides histone H3 in the catalytic enclosure. These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109.     

Effect of polyamines and cations on the in vitro methylation of histones

Na⁺ (0.05–0.15 M) increases both the rate and extent of methylation of chromosomal bound histone H4, while spermidine markedly inhibits this reaction. The effects of spermidine could be mimicked by increasing the concentration of Mg²⁺ or Ca²⁺ to 5–10 mM. At the concentrations listed above, these cations have no significant effect on the methylation of free or chromosomal bound histone H3, nor do they affect the rate or extent of methylation of soluble histone H4. Apparently, the accessibility of histone H4 to the methyltransferase is influenced by chromatin structure. Increasing concentrations of Na⁺ alter the conformation of chromatin (DNA) in such a way as to expose lysine residues in the N-terminal region of histone H4 to the methyltransferase, whereas Mg²⁺ or spermidine acts in an opposite manner.     

Characterization of the PR domain of RIZ1 histone methyltransferase

RIZ1 (PRDM2) and PRDI-BF1 (PRDM1) are involved in B cell differentiation and the development of B cell lymphomas. These proteins are expressed in two forms that differ by the presence or absence of a PR domain. The protein product that retains the PR domain is anti-tumorigenic while the product that lacks the PR domain is oncogenic and over-expressed in tumor cells. The conserved PR domain is homologous to the SET domain from a family of histone methyltransferases. RIZ1 is also a histone methyltransferase and methylates lysine 9 in histone H3. This activity has been mapped to the PR domain. In the present study, deuterium exchange mass spectrometry was used to define the structural boundaries of the RIZ1 PR domain and to map sites of missense mutations that occur in human cancers and reduce methyltransferase activity. Flexible segments were selectively deleted to produce protein products that crystallize for structural studies. Segments at the carboxyl terminus of the PR domain that are involved in methylation of H3 were shown to be flexible, similar to SET domains, suggesting that the PR and SET methyltransferases may belong to an emerging class of proteins that contain mobile functional regions.     

Protection of discrete DNA fragments by the complex H1-octamerhistones or H5-octamerhistones after micrococcal nuclease digestion.

Several authors, including ourselves, have reported the existence of chromatosomes with DNA size larger than 166 bp in bird erythrocyte chromatin. It was tempting to correlate this increased DNA size with the presence of histone H5. In order to substantiate this hypothesis, we performed a micrococcal nuclease digestion kinetic on: chicken erythrocyte chromatin, either native, selectively depleted from H1, or from H1 and H5; and rat liver chromatin, either native or partially H1 depleted. The comparative analysis of the lengths of DNA in the chromatosome size region led to the following conclusions: - denaturing gels clearly reveal a first discrete pause at 178 nucleotides in H1 depleted chicken erythrocyte chromatin as well as in partially H1-depleted rat liver chromatin, before the material accumulates at the next intermediate 166 nucleotide chromatosome pause. - the generation of all discrete chromatosome bands is critically dependent on low ionic strength conditions and low Ca++ concentrations during the digestion, suggesting it may result from the protection of DNA cleavage sites by histone H5 or H1, C or N terminal domains.     

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly(ADP-ribosyl)ation on core nucleosome structure

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin was analyzed by gel electrophoresis, electron microscopy, and velocity sedimentation. In parallel, the interaction of automodified poly(ADP-ribose) polymerase with native and H1-depleted chromatin was analyzed. In H1-depleted chromatin histone H2B becomes the major poly(ADP-ribose) histone acceptor protein, whereas in native chromatin histone H1 was the major histone acceptor. Poly(ADP-ribosyl)ation of H1-depleted chromatin prevented the recondensation of polynucleosomes reconstituted with exogenous histone H1. This is probably due to the presence of modified poly(ADP-ribose) polymerase and hyper(ADP-ribosyl)ated histone H2B. Indeed, about 40% of the modified enzyme remained associated with H1-depleted chromatin, while less than 1% of the modified enzyme was bound to native chromatin. The influence of poly(ADP-ribosyl)ation on the chromatin conformation was also studied at the level of nucleosome in using monoclonal and polyclonal antibodies specific for individual histones and synthetic peptides of histones. In native chromatin incubated in the presence of Mg2+ there was a drop in the accessibility of histone epitopes to monoclonal and polyclonal antibodies whereas upon poly(ADP-ribosyl)ation their accessibility was found to remain even in the presence of Mg2+. In poly(ADP-ribosyl)ated H1-depleted chromatin an increased accessibility of some histone tails to antibodies was observed.     

Modulation of chromatin function through linker histone H1 variants

In this review, the structural aspects of linker H1 histones are presented as a background for characterization of the factors influencing their function in animal and human chromatin. The action of H1 histone variants is largely determined by dynamic alterations of their intrinsically disordered tail domains, posttranslational modifications and allelic diversification. The interdependent effects of these factors can establish dynamic histone H1 states that may affect the organization and function of chromatin regions.     

Distinct dynamics and distribution of histone methyl-lysine derivatives in mouse development

Histone methylation acts as an epigenetic regulator of chromatin activity through the modification of arginine and lysine residues on histones H3 and H4. In the case of lysine, this includes the formation of mono-, di-, or trimethyl groups, each of which is presumed to represent a distinct functional state at the cellular level. To examine the potential developmental roles of these modifications, we determined the global patterns of lysine methylation involving K9 on histone H3 and K20 on histone H4 in midgestation mouse embryos. For each lysine target site, we observed distinct subnuclear distributions of the mono- and trimethyl versions in 10T1/2 cells that were conserved within primary cultures and within the 3D-tissue architecture of the embryo. Interestingly, three of these modifications, histone H3 trimethyl K9, histone H4 monomethyl K20, and histone H4 trimethyl K20 exhibited marked differences in their distribution within the neuroepithelium. Specifically, both histone H3 trimethyl K9 and H4 monomethyl K20 were elevated in proliferating cells of the neural tube, which in the case of the K9 modification was limited to mitotic cells on the luminal surface. In contrast, histone H4 trimethyl K20 was progressively lost from these medial regions and became enriched in differentiating neurons in the ventrolateral neural tube. The inverse relationship of histone H4 K20 methyl derivatives is even more striking during skeletal and cardiac myogenesis where the accumulation of the trimethyl modification in pericentromeric heterochromatin suggests a role in gene silencing in postmitotic muscle cells. Importantly, our results establish that histone lysine methylation occurs in a highly dynamic manner that is consistent with their function in an epigenetic program for cell division and differentiation.     

Molecular basis for chromatin assembly and modification by multiprotein complexes

Abstract: Epigenetic regulation of the chromatin landscape is often orchestrated through modulation of nucleosomes. Nucleosomes are composed of two copies each of the four core histones, H2A, H2B, H3, and H4, wrapped in ~150 bp of DNA. We focus this review on recent structural studies that further elucidate the mechanisms used by macromolecular complexes to mediate histone modification and nucleosome assembly. Nucleosome assembly, spacing, and variant histone incorporation are coordinated by chromatin remodeler and histone chaperone complexes. Several recent structural studies highlight how disparate families of histone chaperones and chromatin remodelers share similar features that underlie how they interact with their respective histone or nucleosome substrates. Post‐translational modification of histone residues is mediated by enzymatic subunits within large complexes. Until recently, relatively little was known about how association with auxiliary subunits serves to modulate the activity and specificity of the enzymatic subunit. Analysis of several recent structures highlights the different modes that auxiliary subunits use to influence enzymatic activity or direct specificity toward individual histone residues.     

Recognition of unmodified histone H3 by the first PHD finger of bromodomain-PHD finger protein 2 provides insights into the regulation of histone acetyltransferases monocytic leukemic zinc-finger protein (MOZ) and MOZ-related factor (MORF)

MOZ (monocytic leukemic zinc-finger protein) and MORF (MOZ-related factor) are histone acetyltransferases important for HOX gene expression as well as embryo and postnatal development. They form complexes with other regulatory subunits through the scaffold proteins BRPF1/2/3 (bromodomain-PHD (plant homeodomain) finger proteins 1, 2, or 3). BRPF proteins have multiple domains, including two PHD fingers, for potential interactions with histones. Here we show that the first PHD finger of BRPF2 specifically recognizes the N-terminal tail of unmodified histone H3 (unH3) and report the solution structures of this PHD finger both free and in complex with the unH3 peptide. Structural analysis revealed that the unH3 peptide forms a third antiparallel β-strand that pairs with the PHD1 two-stranded antiparallel β-sheet. The binding specificity was determined primarily through the recognition of arginine 2 and lysine 4 of the unH3 by conserved aspartic acids of PHD1 and of threonine 6 of the unH3 by a conserved asparagine. Isothermal titration calorimetry and NMR assays showed that post-translational modifications such as H3R2me2as, H3T3ph, H3K4me, H3K4ac, and H3T6ph antagonized the interaction between histone H3 and PHD1. Furthermore, histone binding by PHD1 was important for BRPF2 to localize to the HOXA9 locus in vivo. PHD1 is highly conserved in yeast NuA3 and other histone acetyltransferase complexes, so the results reported here also shed light on the function and regulation of these complexes.     

Histone monoubiquitylation position determines specificity and direction of enzymatic cross-talk with histone methyltransferases Dot1L and PRC2

It is well established that chromatin is a destination for signal transduction, affecting many DNA-templated processes. Histone proteins in particular are extensively post-translationally modified. We are interested in how the complex repertoire of histone modifications is coordinately regulated to generate meaningful combinations of "marks" at physiologically relevant genomic locations. One important mechanism is "cross-talk" between pre-existing histone post-translational modifications and enzymes that subsequently add or remove modifications on chromatin. Here, we use chemically defined "designer" nucleosomes to investigate novel enzymatic cross-talk relationships between the most abundant histone ubiquitylation sites, H2AK119ub and H2BK120ub, and two important histone methyltransferases, Dot1L and PRC2. Although the presence of H2Bub in nucleosomes greatly stimulated Dot1L methylation of H3K79, we found that H2Aub did not influence Dot1L activity. In contrast, we show that H2Aub inhibited PRC2 methylation of H3K27, but H2Bub did not influence PRC2 activity. Taken together, these results highlight how the position of nucleosome monoubiquitylation affects the specificity and direction of cross-talk with enzymatic activities on chromatin.     

Linker histone protection of chromatosomes reconstituted on 5S rDNA from Xenopus borealis:a reinvestigation.

The location of the linker histone (LH) on the nucleosome has been the subject of recent controversy. Although previous evidence had supported a location over the dyad axis, some recent experiments suggest an asymmetric, off-axis position. In this paper we show that the DNA sequence used to reconstitute chromatosomes in these experiments is prone to artifacts in nuclease digestion: results interpreted as 'protection' by LHs can be obtained with either naked DNA or with reconstituted core nucleosomes, in the absence of LHs. Consequently, we feel that general interpretation or extrapolation of such results must be regarded with the utmost caution. In addition, we show that the protection data on an alternative, previously unreported major core position on this same DNA sequence support a model of asymmetric, off-axis position of the LH, with linker DNA protection on only one side of the core particle.