Histones and DNA Compete for Binding Polyphosphoinositides in Bilayers

Recent discoveries on the presence and location of phosphoinositides in the eukaryotic cell nucleoplasm and nuclear membrane prompted us to study the putative interaction of chromatin components with these lipids in model membranes (liposomes). Turbidimetric studies revealed that a variety of histones and histone combinations (H1, H2AH2B, H3H4, octamers) caused a dose-dependent aggregation of phosphatidylcholine vesicles (large unilamellar vesicle or small unilamellar vesicle) containing negatively charged phospholipids. 5 mol % phosphatidylinositol-4-phosphate (PIP) was enough to cause extensive aggregation under our conditions, whereas with phosphatidylinositol (PI) at least 20 mol % was necessary to obtain a similar effect. Histone binding to giant unilamellar vesicle and vesicle aggregation was visualized by confocal microscopy. Histone did not cause vesicle aggregation in the presence of DNA, and the latter was able to disassemble the histone-vesicle aggregates. At DNA/H1 weight ratios 0.1–0.5 DNA- and PIP-bound H1 appear to coexist. Isothermal calorimetry studies revealed that the PIP-H1 association constant was one order of magnitude higher than that of PI-H1, and the corresponding lipid/histone stoichiometries were ∼0.5 and ∼1, respectively. The results suggest that, in the nucleoplasm, a complex interplay of histones, DNA, and phosphoinositides may be taking place, particularly at the nucleoplasmic reticula that reach deep within the nucleoplasm, or during somatic and nonsomatic nuclear envelope assembly. The data described here provide a minimal model for analyzing and understanding the mechanism of these interactions.     

Interactions of Aflatoxin with Histones and DNA

The interactions of aflatoxin B₁ with certain histone fractions and DNA were investigated by means of viscosity measurements and equilibrium dialysis.     

Polyphosphoinositides in myelin

1. On fractionation of guinea-pig forebrain homogenates by differential and gradient-density centrifugation most of the polyphosphoinositides were recovered in the myelin-rich particles. 2. The phospholipids of pure preparations of myelin contained di- and tri-phosphoinositide in proportions 2-3 times greater than in the whole-brain phospholipids. 3. Di- and tri-phosphoinositide appeared in young rat brain during the period of myelination. 4. After the administration of [(32)P]phosphate to guinea pigs the labelling of the polyphosphoinositides in isolated pure myelin was as great as in the whole brain, whereas little synthesis of the other myelin phospholipids had occurred. 5. When brain subcellular fractions were incubated with [gamma-(32)P]ATP, some triphosphoinositide labelling occurred in the myelin-rich fraction whereas the active labelling of diphosphoinositide was localized mainly in the mitochondrial fraction. 6. The Na(+), K(+) and Mg(2+) plus Ca(2+) concentrations in purified myelin have been determined. The Mg(2+) plus Ca(2+) content present showed close acid-base equivalence to the polyphosphoinositides. 7. It is concluded that di- and tri-phosphoinositide are rapidly-metabolizing components of the myelin sheath or intimately associated structures.     

Binding of Steroid Hormones to Histones of Various Origins in Vitro

Kinetin increased the formation of scopoletin and scopolin in tobacco tissue cultures. The formation of scopolin was increased more in the presence of both kinetin (KIN) and l-phenylalanine (Phe) in the medium than in the case of either KIN or Phe alone. The activity of l-phenylalanine ammonia-lyase (PAL) was increased by the addition of KIN. The increase of PAL activity was inhibited by actinomycin-D and cycloheximide added at the same time as the KIN, but actinomycin-D did not inhibit the increase when added at 8 hr after the addition of KIN at which time cycloheximide inhibited it still. The incorporation of [U-¹⁴C]-Phe into scopoletin and scopolin was stimulated by KIN, but its incorporation into protein and lignin was not affected. These results suggest that KIN increased de novo synthesis of PAL through its mRNA, and the enhanced activity of PAL resulted in the increase of scopoletin and scopolin formation.     

Polyphosphoinositides in normal and neoplastic rodent astrocytes

Polyphosphoinositides were identified in dispersed cell cultures of normal newborn hamster astrocytes and of a chemically transformed adult rat astrocytoma (C6) and are therefore presumed to be constituents of immature astrocytes in brain. Small amounts were also detected in astrocytomas grown as subcutaneous tumors. These lipids were metabolically highly active, accounting for a substantial fraction of ³²P_i incorporated into phospholipids. Astrocytes may thus contain a small pool of polyphosphoinositides metabolically distinct from that in myelin.     

Cooperative Binding of Annexin A2 to Cholesterol- and Phosphatidylinositol-4,5-Bisphosphate-Containing Bilayers

Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca²⁺-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with K_d values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.     

组蛋白与转录因子在hAMFR基因启动子序列上的结合及相互作用

采用从鸡红细胞中分离纯化的组蛋白Ｈ１,核心组蛋白Ｈ２Ａ＋Ｈ２Ｂ和Ｈ３＋Ｈ４,以及从ＨｅＬａ细胞中萃取的含有ＲＮＡ聚合酶Ⅱ和多种Ⅱ类基因转录因子的可溶性ＨｅＬａ细胞核抽提物,通过凝胶迟滞电泳,对组蛋白和ＨｅＬａ细胞核抽提物中的转录因子在人自泌移动因子受体（Ｈｕｍａｎａｕｔｏｃｒｉｎｅｍｏｔｉｌｉｔｙｆａｃｔｏｒ,简称ｈＡＭＦＲ）基因上游启动子序列的相互作用关系进行了初步研究,得到以下结论,组蛋白Ｈ１     

Cooperative Binding of Annexin A2 to Cholesterol- and Phosphatidylinositol-4,5-Bisphosphate-Containing Bilayers

Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca²⁺-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with K_d values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.     

Histones bind more tightly to bromodeoxyuridine-substituted DNA than to normal DNA.

Using a membrane filter assay, we have obtained results from both kinetic and competition experiments indicating that histones bind more strongly to bromodeoxyuridine-substituted DNA than to normal DNA. At 37 degrees C in our standard buffer of 0.2 M ionic strength, the rate of dissociation of histones H1, H2, and h4 from BrdU-substituted DNA is respectively 7, 4, and 2 times slower than it is from normal DNA. Competition experiments show an even greater difference between BrdU-substituted and normal DNA with respect to histone binding. The tighter binding of histones to BrdU-substituted DNA is of interest because of the known effects of BrdU on eukaryotic chromosome condensation and staining, virus induction, and the inhibition of differentiation.     

Histones in perspective

Histones occur in equal amounts to DNA in the cell nucleus and are largely responsible for the compaction of the genome into chromatin via the formation of nucleosomes and higher-order structures. Whereas two of the five histone types exhibit little structural variation, the remaining three occur in many variant tissue- or species-specific forms. Multiple postsynthetic enzymatic modifications accompanying virtually any type of genome activity, together with the programmed appearance of many histone variants during sea urchin embryogenesis (and other differentiation events in a number of organisms) make histones a challenging enigma in eukaryotic molecular biology.     

Histones in crenarchaea

Archaeal histone-encoding genes have been identified in marine Crenarchaea. The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya.     

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.