Low resolution models of self-assembled histone fibers from X-ray diffraction studies.

X-ray diffraction data from self-assembled histone fibers are presented for three systems: H4, H3-H4, and the four core histones H2A, H2B, H3 and H4. These data have been obtained under conditions of high ionic strength and high protein concentration which are thought to promote histone conformation similar to that found in intact chromatin. The low angle equatorial scattering (R less than .05 A-1) is analysed, and, with additional constraints imposed by electron microscopy data, four low resolution fibrillar models are derived. Two features common to all the possible models are a maximum outer diameter of approximately 60 A and a subfibril diameter of approximately 25 A. It is the interference of the protein subfibrils across a central region of low electron density - a 10 A "hole" - which gives rise to the characteristic diffraction peak at 36 A. Possible relationships of the models of the histone fibers to the structure of the histone component of chromatin are suggested.     

Statistical Models for Detecting Differential Chromatin Interactions Mediated by a Protein

Chromatin interactions mediated by a protein of interest are of great scientific interest. Recent studies show that protein-mediated chromatin interactions can have different intensities in different types of cells or in different developmental stages of a cell. Such differences can be associated with a disease or with the development of a cell. Thus, it is of great importance to detect protein-mediated chromatin interactions with different intensities in different cells. A recent molecular technique, Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET), which uses formaldehyde cross-linking and paired-end sequencing, is able to detect genome-wide chromatin interactions mediated by a protein of interest. Here we proposed two models (One-Step Model and Two-Step Model) for two sample ChIA-PET count data (one biological replicate in each sample) to identify differential chromatin interactions mediated by a protein of interest. Both models incorporate the data dependency and the extent to which a fragment pair is related to a pair of DNA loci of interest to make accurate identifications. The One-Step Model makes use of the data more efficiently but is more computationally intensive. An extensive simulation study showed that the models can detect those differentially interacted chromatins and there is a good agreement between each classification result and the truth. Application of the method to a two-sample ChIA-PET data set illustrates its utility. The two models are implemented as an R package MDM (available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM).     

The anchorosome, a special chromatin granule for the anchorage of the interphase chromosome to the nuclear envelope

Peripheral chromatin granules bound to the nuclear envelope of rat liver nuclei have been further investigated. Judging by the results of Staphylococcal nuclease digestion of nuclei and electron microscopical observations, the peripheral granules have nucleosomal organization. As shown by ultraviolet radiation DNA-protein cross-linkage, the histone-like proteins present in the peripheral chromatin instead of histone H1 (Fais et al., 1982) are in close contact with DNA. The peripheral chromatin contains a DNA firmly bound to the lamina. This DNA, resistant to extraction in high salt, heparin and SDS, is protected against a DNase attack since, as shown by DNA electrophoresis data, high molecular weight molecules (up to 20 kbas) are still present in the lamina residue. However, the high molecular weight DNA disappeared if the nuclear envelope fraction was again DNase-digested after high salt treatment. Altogether, the data of the previous (Fais et al., 1982; Prusov et al., 1980: Prusov et al., 1982) and the present investigations demonstrate that the peripheral chromatin granules are endowed with properties which distinguish them from the bulk chromatin and account for the chromosome bond to the nuclear envelope during interphase. This is why we suggest the term "anchorosome" for the peripheral protein granule attached to the nuclear envelope.     

DNA-protein binding in interphase chromosomes

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased β/α-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (µ-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.     

银鲫(Carassius auratus gibelio)成熟精子染色质的结构和碱性蛋白的研究

银鲫属行雌核发育的遗传系统,其精子入卵后不能形成雄性原核。如同其它种类的精子一样,在精子发生过程中其染色质形成高度浓缩的具有一定结构的DNA和碱性蛋白的复合物。为了研究分析银鲫成熟精子的碱性蛋白、染色质的结构及其与体细胞染色质的异同,我们用聚丙烯酰胺凝胶电泳分析了银鲫精子碱性蛋白的组分,并结合核酸酶解的结果,以电镜铺片和超薄切片法探讨了银鲫染色质的结构。聚丙烯酰胺凝胶电泳分析表明,银鲫精子的碱??性蛋白为含H_1、H_2a、H_2b、H_3及H_4五个主要组分的组蛋白,且与体细胞核组蛋白无明显差异。电镜铺片法观察精子染色质,可见染色质丝的近100A粗细的串珠状结构。小球菌核酸酶水解精子染色质,DNA的琼脂糖电泳呈“阶梯型”电泳条带,证实了银鲫成熟精子保存了核小体基本单元,在低渗处理后的精子超薄切片中,可以见到直径为300A左右的染色质纤维,这个结果提示了精子染色质与体细胞染色体有类似的二级结构。根据以上所得到的结果推测,行雌核发育的银鲫,其精子在入卵后不能原核化,与精子染色质的基本结构及碱性蛋白组分无明显直接关系。     

Analysis of the heterogeneity of chromatin proteins fractionated on hydroxyapatite

The protein composition of the liver chromatin has been studied by two techniques for fractionation of histones. The "lability" fraction of histones H2A-H2B is revealed. In these fractions histones H2B have many modified forms and they are not included into octamer (H3, H4, H2A, H2B)2. Young animals rather than old ones have much quantitative subfractions of histone H2B. The "lability" fraction of histones H2A-H2B is stated to be very significant in the activated and repressed chromatin structure.     

Introduction of superhelical turns into DNA by adenoviral core proteins and chromatin assembly factors.

The interaction in vitro between adenoviral histone-like proteins and DNA in the presence of chromatin assembly factors was investigated. Viral core protein VII or its precursor pVII was incubated with DNA in the presence of an extract of HeLa cell chromatin, which mediates nucleosome assembly from histones and DNA. We have demonstrated that either protein can introduce superhelical turns into relaxed closed-circular DNA and that the presence of chromatin extract is necessary for the supertwisting effect. A greater density of superhelical turns was produced by pVII than by VII, but neither protein-DNA interaction resulted in the "physiological" amount of supertwisting produced by histones. The inhibition of histone-induced supercoiling by both proteins and the protection of turns in supertwisted starting material are also described. The nucleosome assembly factor, nucleoplasmin, fails to mediate the introduction of superhelical turns by VII or pVII.     

Quantitative analysis of HP1alpha binding to nucleosomal arrays

Elucidating how the metazoan genome is organised into distinct functional domains is fundamental to understanding all aspects of normal cellular growth and development. The "histone code" hypothesis predicts that post-translational modifications of specific histone residues regulate genomic function by selectively recruiting nuclear factors that modify chromatin structure. A paradigm supporting this hypothesis is the preferential binding of the silencing protein heterochromatin protein 1 (HP1) to histone H3 trimethylated at K9. However, a caveat to several in vitro studies is that they employed histone N-terminal tail peptides to determine dissociation constants, thus ignoring any potential role of DNA and/or the underlying chromatin structure in the recruitment of HP1. Using a well-defined in vitro chromatin assembly system (employing a 12-208 DNA template), we describe here, the use of a fluorescence spectroscopic method that enabled us to measure and quantify the relative binding affinities of HP1alpha to unmodified and variant nucleosomal arrays. Using this approach, we previously demonstrated that mouse HP1alpha (i) binds with high affinity to naked DNA, (ii) has an intrinsic affinity for highly folded chromatin, (iii) has a 2-fold higher affinity for nucleosomal arrays when H2A is replaced with H2A.Z, and (iv) binds to DNA or chromatin in a non-cooperative manner.     

Purification of a nuclear protein (Receptor Binding Factor-1) associated with the chromatin acceptor sites for the avian oviduct progesterone receptor

The specific high affinity binding of the avian oviduct progesterone receptor (PR) to target cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0–6.0 [Goldberger and Spelsberg (1988),Biochem. 27, 2103–2109]. Using western immunoblots with anti-RBF-1 polyclonal antibodies to monitor the purification, a 10 kD candidate acceptor protein, termed the Receptor Binding Factor-1 (RBF-1), has been purified to apparent homogeneity. RBF-1 has an amino acid composition consistent with a hydrophobic protein having an acidic pI and a unique N-terminal sequence. Two-dimensional polyacrylamide gel electrophoresis and high-performance capillary electrophoresis support the purity of a protein 10 kD in size, having an acidic pI, but with evidence of several differently charged isoforms. Phosphatase treatment provides evidence that charge heterogeneity may result from variable phosphorylation states. A role of this factor as a candidate acceptor protein in the chromatin acceptor sites for the avian oviduct PR is proposed.     

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isamyl alcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein constituent strongly linked to DNA. The specificity in association of the peptide(s) to DNA has also been considered.     

Neutron scatter and diffraction techniques applied to nucleosome and chromatin structure

Neutron scatter and diffraction techniques have made substantial contributions to our understanding of the structure of the nucleosome, the structure of the 10-nm filament, the "10-nm----30-nm" filament transition, and the structure of the "34-nm" supercoil or solenoid of nucleosomes. Neutron techniques are unique in their properties, which allows for the separation of the spatial arrangements of histones and DNA in nucleosomes and chromatin. They have equally powerful applications in structural studies of any complex two-component biological system. A major success for the application of neutron techniques was the first clear proof that DNA was located on the outside of the histone octamer in the core particle. A full analysis of the neutron-scatter data gave the parameters of Table 3 and the low-resolution structure of the core particle in solution shown in Fig. 6. Initial low-resolution X-ray diffraction studies of core particle crystals gave a model with a lower DNA pitch of 2.7 nm. Higher-resolution X-ray diffraction studies now give a structure with a DNA pitch of 3.0 nm and a hole of 0.8 nm along the axis of the DNA supercoil. The neutron-scatter solution structure and the X-ray crystal structure of the core particle are thus in full agreement within the resolution of the neutron-scatter techniques. The model for the chromatosome is largely based on the structural parameters of the DNA supercoil in the core particle, nuclease digestion results showing protection of a 168-bp DNA length by histone H1 and H1 peptide, and the conformational properties of H1. The path of the DNA outside the chromatosome is not known, and this information is crucial for our understanding of higher chromatin structure. The interactions of the flexible basic and N- and C-terminal regions of H1 within chromatin and how these interactions are modulated by H1 phosphorylation are not known. The N- and C-terminal regions of H1 represent a new type of protein behavior, i.e., extensive protein domains that are designed not to fold up into secondary and tertiary protein structures. This behavior is increasingly observed in DNA and chromatin binding proteins, and in the case of the high-mobility group proteins HMG 14 and 17, the entire polypeptide chain is a flexible random coil over a wide range of solution, ionic, and pH conditions. It follows that the native conformations are probably imposed on these flexible domains and molecules by their binding sites in chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

A model for chromatin structure.

A model for chromatin structure is presented. (a) Each of four histone species, H2A (IIbl or f2a2), H2B (IIb2 or f2b), H3 (III or f3) and H4 (IV or f2al) can form a parallel dimer. (b) These dimers can form two tetramers, (H2A)2(H2b)2 and (H3)2(H4)2. (C) These two tetramers bind a segment of DNA and condense it into a "C" segments. (d) The adjacent segments, termed extended or "E" segments, are bound by histone H1 (I or fl) for the major fraction of chromatin; the other "E" regions can be either bound by non-histone proteins or free of protein binding. (e) The binding of histones causes a structural distortion of the DNA which, depending upon the external conditions, may generate the formation of either an open structure with a heterogeneous and non-uniform supercoil or a compact structure with a string of beads. The model is supported by experimental data on histone-histone interaction, histone-DNA interaction and histone subunit-DNA interaction.     

Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria

Changes in DNA supercoiling in response to environmental signals such as osmolarity, temperature, or anaerobicity appear to play an underlying role in the regulation of gene expression in bacteria. Extensive genetic analyses have implicated the osmZ gene in this regulatory process: osmZ mutations are highly pleiotropic and alter the topology of cellular DNA. We have shown that the product of the osmZ gene is the "histone-like" protein H1 (H-NS). Protein H1 is one of the most abundant components of bacterial chromatin and binds to DNA in a relatively nonspecific fashion. These data imply a regulatory role for one of the major components of bacterial chromatin and provide support for the notion that changes in DNA topology and/or chromatin structure play a role in regulating gene expression.     

The HMG-I(Y) A.T-hook peptide motif confers DNA-binding specificity to a structured chimeric protein.

Chromosomal translocations involving genes coding for members of the HMG-I(Y) family of "high mobility group" non-histone chromatin proteins (HMG-I, HMG-Y, and HMG-IC) have been observed in numerous types of human tumors. Many of these gene rearrangements result in the creation of chimeric proteins in which the DNA-binding domains of the HMG-I(Y) proteins, the so-called A.T-hook motifs, have been fused to heterologous peptide sequences. Although little is known about either the structure or biophysical properties of these naturally occurring fusion proteins, the suggestion has been made that such chimeras have probably assumed an altered in vivo DNA-binding specificity due to the presence of the A.T-hook motifs. To investigate this possibility, we performed in vitro "domain-swap" experiments using a model protein fusion system in which a single A. T-hook peptide was exchanged for a corresponding length peptide in the well characterized "B-box" DNA-binding domain of the HMG-1 non-histone chromatin protein. Here we report that chimeric A. T-hook/B-box hybrids exhibit in vitro DNA-binding characteristics resembling those of wild type HMG-I(Y) protein, rather than the HMG-1 protein. These results strongly suggest that the chimeric fusion proteins produced in human tumors as a result of HMG-I(Y) gene chromosomal translocations also retain A.T-hook-imparted DNA-binding properties in vivo.