The nature of forces stabilizing nucleosome structure. Dissociation of histone octamers from DNA

We have used the measurements of the histone fluorescence parameters to study the influence of the ionic strength on histone-DNA and histone-histone interactions in reconstructed nucleosomes. The ionic strength increase lead to the two-stage nucleosome dissociation. The dimer H2A-H2B dissociates at the first stage and the tetramer (H3-H4)2 at the second one. The dimer H2A-H2B dissociation from nucleosome is a two-stage process also. The ionic bonds between (H2A-H2B) histone dimer and DNA break at first and then the dissociation of dimer from histone tetramer (H3-H4)2 occurs. According to the proposed model the dissociation accompanying a nucleosome "swelling" and an increase of DNA curvature radius. It was shown that the energy of electrostatic interactions between histone dimer and DNA is sufficiently less than the energy of dimer-tetramer interaction. We propose that the nucleosome DNA ends interact with the dimer and tetramer simultaneously. The calculated number (approximately 30 divided by 40) of ionic bonds between DNA and histone octamer globular part practically coincides with the number of exposed cationic groups on the surface of octamer globular head. On this basis we have assumed that the spatial distribution of these groups is precisely determined, which explains the high evolutionary conservatism of the histone primary structure.     

Intermolecular histone H4 interactions in core nucleosomes

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.     

盐离子作用下组蛋白变体H2A.Z增强核小体结构的稳定性

真核生物染色质的基本结构组成单元是核小体,基因组DNA被压缩在染色质中,核小体的存在通常会抑制转录、复制、修复和重组等发生在DNA模板上的生物学过程。组蛋白变体H2A.Z可以调控染色质结构进而影响基因的转录过程,但其详细的调控机制仍未研究清楚。为了比较含有组蛋白变体H2A.Z的核小体和常规核小体在盐离子作用下的稳定性差异,本文采用Förster共振能量转移的方法检测氯化钠、氯化钾、氯化锰、氯化钙、氯化镁等离子对核小体的解聚影响。实验对Widom 601 DNA序列进行双荧光Cy3和Cy5标记,通过荧光信号值的变化来反映核小体的解聚变化。Förster共振能量转移检测结果显示:在氯化钠、氯化钾、氯化锰、氯化钙和氯化镁作用下,含有组蛋白变体H2A.Z的核小体解聚速度相比于常规核小体要慢,且氯化钙、氯化锰和氯化镁的影响更明显。电泳分析结果表明,在75℃条件下含有组蛋白变体H2A.Z的核小体的解聚速率明显低于常规核小体。采用荧光热漂移检测(fluorescence thermal shift analysis , FTS)进一步分析含有组蛋白变体H2A.Z核小体的稳定性,发现两类核小体的荧光信号均呈现2个明显的增长期,含有组蛋白变体H2A.Z核小体的第1个荧光信号增速期所对应的温度明显高于常规核小体,表明核小体中H2A.Z/H2B二聚体的解聚变性温度要高于常规的H2A/H2B二聚体,含有组蛋白变体H2A.Z核小体的热稳定性高。研究结果均表明,含有组蛋白变体H2A.Z的核小体的结构比常规核小体的结构稳定。     

Nucleosome assembly in vitro: separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laevis oocytes.

High speed supernatants of Xenopus laevis oocyte nuclei efficiently assemble DNA into nucleosomes in vitro under physiological salt conditions. The assembly activity cofractionates with two histone complexes composed of the acidic protein N1/N2 in complex with histones H3 and H4, and nucleoplasmin in complex with histones H2B and H2A. Both histone complexes have been purified and their nucleosome assembly activities have been analysed separately and in combination. While the histones from the N1/N2 complexes are efficiently transferred to DNA and induce supercoils into relaxed circular plasmid DNA, the nucleoplasmin complexes show no supercoil induction, but can also transfer their histones to DNA. In combination, the complexes act synergistically in supercoil induction thereby increasing the velocity and the number of supercoils induced. Electron microscopic analysis of the reaction products shows fully packaged nucleoprotein structures with the typical nucleosomal appearance resulting in a compaction ratio of 2.8 under low ionic strength conditions. The high mobility group protein HMG-1, which is also present in the soluble nuclear homogenate from X. laevis oocytes, is not required for nucleosome core assembly. Fractionation experiments show that the synergistic effect in the supercoiling reaction can be exerted by histones H3 and H4 bound to DNA and the nucleoplasmin complexes alone. This indicates that it is not the synchronous action of both complexes which is required for nucleosome assembly, but that their cooperative action can be resolved into two steps: deposition of H3 and H4 from the N1/N2 complexes onto the DNA and completion of nucleosome core formation by addition of H2B and H2A from the nucleoplasmin complexes.     

Reconciling the positive and negative roles of histone H2A.Z in gene transcription

The incorporation of variant histone H2A.Z within chromatin is important for proper gene expression and genome stability. H2A.Z is inserted at discrete loci by the Swr1 or Swr1-like remodeling complexes, although very little is known about the nature of the targeting mechanism involved. Replacement of canonical histone H2A for H2A.Z has been shown to modify nucleosome dynamics, although discrepancies still exist in the literature regarding the mechanisms. Recent experiments have shown that H2A.Z can allow nucleosomes to adopt stable translational positions as compared to H2A, which could influence the accessibility to DNA regulatory proteins. This review provides a brief overview of H2A.Z biology and presents hypotheses that could reconcile contradictory reports that are found in the literature regarding the influence of H2A.Z on nucleosome stability.     

Intra- and inter-nucleosomal protein-DNA interactions of the core histone tail domains in a model system

The core histone tail domains are key regulators of eukaryotic chromatin structure and function and alterations in the tail-directed folding of chromatin fibers and higher order structures are the probable outcome of much of the post-translational modifications occurring in these domains. The functions of the tail domains are likely to involve complex intra- and inter-nucleosomal histone-DNA interactions, yet little is known about either the structures or interactions of these domains. Here we introduce a method for examining inter-nucleosome interactions of the tail domains in a model dinucleosome and determine the propensity of each of the four N-terminal tail domains to mediate such interactions in this system. Using a strong nucleosome "positioning" sequence, we reconstituted a nucleosome containing a single histone site specifically modified with a photoinducible cross-linker within the histone tail domain, and a second nucleosome containing a radiolabeled DNA template. These two nucleosomes were then ligated together and cross-linking induced by brief UV irradiation under various solution conditions. After cross-linking, the two templates were again separated so that cross-linking representing inter-nucleosomal histone-DNA interactions could be unambiguously distinguished from intra-nucleosomal cross-links. Our results show that the N-terminal tails of H2A and H2B, but not of H3 and H4, make internucleosomal histone-DNA interactions within the dinucleosome. The relative extent of intra- to inter-nucleosome interactions was not strongly dependent on ionic strength. Additionally, we find that binding of a linker histone to the dinucleosome increased the association of the H3 and H4 tails with the linker DNA region.     

Conformations of the core nucleosome: effects of ionic strength and high mobility group protein 14 and 17 binding on the fluorescence emission and polarization of dansylated methionine-84 of histone H4

Chicken histone H4 labeled at Met-84 with the fluor N-[(acetylamino)ethyl]-8-naphthyl-amine-1-sulfonic acid has been incorporated into a nucleosome which has physical characteristics virtually identical with those of native core nucleosomes. The fluorescence emission and polarization properties of the labeled nucleosome were measured as a function of ionic strength and the binding of high mobility group (HMG) proteins 14 and 17. Also, the accessibility of the fluor to the quenching agent acrylamide was determined. It was found that the fluorescence emission changes in the range 0.1-1000 mM NaCl are rather small and indicate that no major unfolding of the octamer structure occurs around Met-84 on H4 at least. Five or perhaps six discrete states were found in that ionic strength range. Each has a different accessibility to the quenching agent. The range of accessibilities varied from 9 X 10(-7) to 32 X 10(-7) mol-1 s-1 for 0.1-1000 mM NaCl, respectively. Polarization measurements showed that there was little change in the rotational relaxation lifetime of the fluor at ionic strengths less than 50 mM NaCl. Above this value, the rotational relaxation lifetimes decreased from 107 to 25 ns at 600 mM NaCl, indicating a moderately increased rotational freedom for the fluor. It is suggested that the histone octamer changes its degree of compaction in the range 0.1-600 mM NaCl but that no major protein unfolding occurs.(ABSTRACT TRUNCATED AT 250 WORDS)     

The histone variant macro-H2A preferentially forms "hybrid nucleosomes"

The histone domain of macro-H2A, which constitutes the N-terminal one third of this histone variant, is only 64% identical to major H2A. We have shown previously that the main structural differences in a nucleosome in which both H2A moieties have been replaced by macro-H2A reside in the only point of contact between the two histone dimers, the L1-L1 interface of macro-H2A. Here we show that the L1 loop of macro-H2A is responsible for the increased salt-dependent stability of the histone octamer, with implications for the nucleosome assembly pathway. It is unknown whether only one or both of the H2A-H2B dimers within a nucleosome are replaced with H2A variant containing nucleosomes in vivo. We demonstrate that macro-H2A preferentially forms hybrid nucleosomes containing one chain each of major H2A and macro-HA in vitro. The 2.9-A crystal structure of such a hybrid nucleosome shows significant structural differences in the L1-L1 interface when comparing with homotypic major H2A- and macro-H2A-containing nucleosomes. Both homotypic and hybrid macro-nucleosome core particles (NCPs) are resistant to chaperone-assisted H2A-H2B dimer exchange. Together, our findings suggest that the histone domain of macro-H2A modifies the dynamic properties of the nucleosome. We propose that the possibility of forming hybrid macro-NCP adds yet another level of complexity to variant nucleosome structure and function.     

H2A.Z stabilizes chromatin in a way that is dependent on core histone acetylation

The functional and structural chromatin roles of H2A.Z are still controversial. This work represents a further attempt to resolve the current functional and structural dichotomy by characterizing chromatin structures containing native H2A.Z. We have analyzed the role of this variant in mediating the stability of the histone octamer in solution using gel-filtration chromatography at different pH. It was found that decreasing the pH from neutral to acidic conditions destabilized the histone complex. Furthermore, it was shown that the H2A.Z-H2B dimer had a reduced stability. Sedimentation velocity analysis of nucleosome core particles (NCPs) reconstituted from native H2A.Z-containing octamers indicated that these particles exhibit a very similar behavior to that of native NCPs consisting of canonical H2A. Sucrose gradient fractionation of native NCPs under different ionic strengths indicated that H2A.Z had a subtle tendency to fractionate with more stabilized populations. An extensive analysis of the salt-dependent dissociation of histones from hydroxyapatite-adsorbed chromatin revealed that, whereas H2A.Z co-elutes with H3-H4, hyperacetylation of histones (by treatment of chicken MSB cells with sodium butyrate) resulted in a significant fraction of this variant eluting with the canonical H2A. These studies also showed that the late elution of this variant (correlated to enhanced binding stability) was independent of the chromatin size and of the presence or absence of linker histones.     

Diffusion-enhanced energy transfer investigation of histone H5 in chromatin with a fluorescently-labelled antibody fragment Fab'.

The location of chicken erythrocyte H5 histone relative to the axis the 30 nm chromatin fibre axis has been investigated by diffusion-enhanced energy transfer. In this investigation, a neutral lanthanide chelate as donor and a fluorescent probe specific to H5 as acceptor have been used. The acceptor probe consists of H5 antibody Fab' fragment, which has been labeled with 5-iodoacetamidofluorescein (5-IAF). Using H5 fragments we have shown by ELISA that the antibodies recognized the N- and C-terminal ends of this histone. A neutral chelate of terbium (TbHED3A) was chosen as a suitable donor for energy transfer with IAF-labelled Fab' (Fab'-IAF) bound to H5 in various chromatin structures. The ionic strength dependence of the energy transfer from TbHED3A to chromatin-bound Fab'-IAF was used to estimate the accessibility and the location of the Fab' in chromatin. The rate constants for energy transfer, obtained from the lifetimes of the TbHED3A excited state in presence and absence of acceptor, indicated a decrease in transfer efficiency upon increase of salt concentration from 5 to 80 mM NaCl. This can be correlated with the chromatin folding occurring in this ionic strength range and is consistent with the location of at least some of the N and C-termini of H5 within the condensed chromatin structure.     

Clipping of Flexible Tails of Histones H3 and H4 Affects the Structure and?Dynamics of the Nucleosome

Förster resonance energy transfer was used to monitor the dynamic conformations of mononucleosomes under different chromatin folding conditions to elucidate the role of the flexible N-terminal regions of H3 and H4 histones. The H3 tail was shown to partake in intranucleosomal interactions by restricting the DNA breathing motion and compacting the nucleosome. The H3 tail effects were mostly independent of the ionic strength and valency of the ions. The H4 tail was shown to not greatly affect the nucleosome conformation, but did slightly influence the relative population of the preferred conformation. The role of the H4 tail varied depending on the valency and ionic strength, suggesting that electrostatic forces play a primary role in H4 tail interactions. Interestingly, despite the H4 tail’s lack of influence, when H3 and H4 tails were simultaneously clipped, a more dramatic effect was seen than when only H3 or H4 tails were clipped. The combinatorial effect of H3 and H4 tail truncation suggests a potential mechanism by which various combinations of histone tail modifications can be used to control accessibility of DNA-binding proteins to nucleosomal DNA.     

Structure of histone octamers in reconstituted polynucleosomes

The salt-dependent structural changes of the histone octamer in complex with high-molecular-weight DNA have been studied by fluorescent spectroscopy. Changes in both the spectra maximum position and anisotropy of the histone tyrosine fluorescence reveal structural transitions in nucleosome within the ranges of 0.5-3 mM and 20-30 mM NaCl. Comparison of the octamer fluorescent parameters in complex with DNA as well as in a free state permits to interpret the revealed structural transitions as a change in degree of contacts stability between (H2A-H2B) dimer and (H3-H4)2 tetramer. More pronounced conformational changes in histone octamer are observed under the conditions of polynucleosome fibers interaction within the range of physiological ionic strength (100-600 mM NaCl). As far as fluorescent parameters are concerned, the aforementioned changes are connected with entire destruction of (H2A-H2B) dimer specific contacts with (H3-H4)2 tetramer. The obtained results suggest the possibility of existence of different structural states of histone octamer in the chromatin composition including those which are quite dissimilar from the octamer structure in the 2M NaCl solution.     

Labelling of histone H5 and its interaction with DNA. 2. Cooperative binding of histone H5 to DNA as probed by steady-state fluorescence and diffusion-enhanced energy transfer

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

基于组蛋白甲基化信息的H2A.Z和H2A核小体识别

组蛋白H2A的变体H2A.Z在基因的表达过程中发挥着重要的作用。根据H2A.Z和H2A核小体中组蛋白甲基化修饰方式的不同,作者应用多样性增量二次判别方法(increment of diversity with quadratic discriminant,IDQD)成功地对H2A.Z和H2A核小体进行了识别,说明了以组蛋白甲基化信息作为特征参数的IDQD模型对H2A.Z和H2A核小体识别的有效性。通过计算DNA序列的柔性,发现H2A.Z核小体对应的DNA序列的平均柔性比常规H2A核小体对应的DNA序列的平均柔性弱。