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.     

Co-existence of two different types of soluble histone complexes in nuclei of Xenopus laevis oocytes

The nuclear pool of soluble histones in Xenopus laevis oocytes is organized into two major types of acidic histone complexes separable by sucrose density gradient centrifugation. One type of complex sediments at 5 S (Mr approximately 120,000), is isoelectric at pH 4.6, and contains histones H3 and/or H4 tightly bound to one polypeptide of a pair of very acidic polypeptides, designated N1 and N2 (Kleinschmidt, J. A., and Franke, W. W. (1982) Cell 29, 799-809). This complex can be selectively immunoprecipitated by guinea pig antibodies against purified protein N1/N2. In contrast, a larger complex of 7 S contains four histones and nucleoplasmin (the purified protein exists as a pentamer of a polypeptide of Mr approximately 30,000), is isoelectric over the pH range of 5-7, and can be immunoprecipitated by nucleoplasmin antibodies. Its relative molecular weight of 130,000-170,000, as determined by gel filtration, sucrose density gradient centrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked complexes, excludes the association of a histone octamer with nucleoplasmin. In addition to histones H2A and H2B, two histones (designated H3 and H4) which are similar in their electrophoretic mobilities to histones H3 and H4 but have lower isoelectric pH values are enriched in immuno-precipitates obtained with nucleoplasmin antibodies. Cross-linking of complexes present in intact nuclei, using 1% formaldehyde at near-physiological ionic strength and pH, indicates the coexistence of these two soluble histone complexes in the living cell. In chromatin assembly experiments using SV 40 DNA, both histone fractions are able to transfer histones to DNA, resulting in an increase of DNA superhelicity and the formation of beaded nucleoprotein complexes of nucleosome-like morphology. The common principle governing both types of complexes, i.e. the association of one or two histone molecules with a karyophilic large acidic histone-binding protein is emphasized. We discuss the possible role of these complexes in storing histones utilized in chromatin assembly during early amphibian embryogenesis as well as the possible existence of similar complexes, albeit at lower concentrations, in somatic cells.     

Identification and characterization of nucleoplasmin 3 as a histone-binding protein in embryonic stem cells

Embryonic stem (ES) cells are thought to have unique chromatin structures responsible for their capacity for self-renewal and pluripotency. To examine this possibility, we sought nuclear proteins in mouse ES cells that specifically bind to histones using a pull-down assay with synthetic peptides of histone H3 and H4 tail domain as baits. Nuclear proteins preferentially bound to the latter. We identified 45 proteins associated with the histone H4 tail and grouped them into four categories: 10 chromatin remodeling proteins, five histone chaperones, two histone modification-related proteins, and 28 other proteins. mRNA expression levels of 20 proteins selected from these 45 proteins were compared between undifferentiated and retinoic acid (RA)-induced differentiated ES cells. All of the genes were similarly expressed in both states of ES cells, except nucleoplasmin 3 (NPM3) that was expressed at a higher level in the undifferentiated cells. NPM3 proteins were localized in the nucleoli and nuclei of the cells and expression was decreased during RA-induced differentiation. When transfected with NPM3 gene, ES cells significantly increased their proliferation compared with control cells. The present study strongly suggests that NPM3 is a chromatin remodeling protein responsible for the unique chromatin structure and replicative capacity of ES cells.     

Interaction of nucleoplasmin with core histones

Nucleoplasmin is one of the most abundant proteins in Xenopus laevis oocytes, and it has been involved in the chromatin remodeling that takes place immediately after fertilization. This molecule has been shown to be responsible for the removal of the sperm-specific proteins and deposition of somatic histones onto the male pronuclear chromatin. To better understand the latter process, we have used sedimentation velocity, sedimentation equilibrium, and sucrose gradient fractionation analysis to show that the pentameric form of nucleoplasmin binds to a histone octamer equivalent consisting of equal amounts of the four core histones, H2A, H2B, H3, and H4, without any noticeable preference for any of these proteins. Removal of the histone N-terminal "tail" domains or the major C-terminal polyglutamic tracts of nucleoplasmin did not alter these binding properties. These results indicate that interactions other than those electrostatic in nature (likely hydrophobic) also play a critical role in the formation of the complex between the negatively charged nucleoplasmin and positively charged histones. Although the association of histones with nucleoplasmin may involve some ionic interactions, the interaction process is not electrostatically driven.     

The plant nucleoplasmin AtFKBP43 needs its extended arms for histone interaction

The nucleoplasmin family of histone chaperones is a key player in governing the dynamic architecture of chromatin, thereby regulating various DNA-templated processes. The crystal structure of the N-terminal domain of Arabidopsis thaliana FKBP43 (AtFKBP43), an FK506-binding immunophilin protein, revealed a characteristic nucleoplasmin fold, thus confirming it to be a member of the FKBP nucleoplasmin class. Small-Angle X-ray Scattering (SAXS) analyses confirmed its pentameric nature in solution, and additional studies confirmed the nucleoplasmin fold to be highly stable. Unlike its homolog AtFKBP53, the AtFKBP43 nucleoplasmin core domain could not interact with histones and required the acidic arms, C-terminal to the core, for histone association. However, SAXS generated low-resolution envelope structure, ITC, and AUC results revealed that an AtFKBP43 pentamer with C-terminal extensions interacts with H2A/H2B dimer and H3/H4 tetramer in an equimolar ratio, like AtFKBP53. Put together, AtFKBP43 belongs to a hitherto unreported subclass of FKBP nucleoplasmins that requires the C-terminal acidic stretches emanating from the core domain for histone interaction.     

Purification and initial characterization of a protein which facilitates assembly of nucleosome-like structure from mammalian cells

A protein, which facilitates assembly of a nucleosome-like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem. (Tokyo) 94, 735-744]. The protein has been purified to approximately 80% from FM3A cells by using histone-Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53-kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53-kDa peptide bound to core histones and formed a 12-S complex. This complex contained stoichiometrical amounts of the 53-kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B, H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12-S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53-kDa peptide, formation of a 10.5-S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53-kDa peptide was detected in the complex. From above results it is suggested that the 53-kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53-kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53-kDa peptide and nucleoplasmin is discussed.     

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF-I, and NAP-I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.     

Functional studies of MP62 during male chromatin decondensation in sea urchins

In amphibians, sperm histone transition post‐fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin‐like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin‐like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post‐fertilization and this phosphorylation is dependent on CDK‐cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin‐like activity in sea urchins participating during male pronucleus formation post‐fecundation. J. Cell. Biochem. 114: 1779–1788, 2013. © 2013 Wiley Periodicals, Inc.     

Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts

The composition and function of histone storage complexes of Xenopus eggs have been investigated using monoclonal antibodies. We show that core histones are contained in two distinct complexes: H2A and H2B are associated with nucleoplasmin, and H3 and H4 are associated with nuclear protein N1. Immunodepletion analyses demonstrate that both complexes are required for nucleosome core assembly by extracts in vitro, the product being a simple sum of the histones from each complex. In addition, the majority of the stored H2A is shown to be an unusual form that migrates close to the position of H3 by SDS-polyacrylamide gel electrophoresis and resembles a variant synthesized in a cell-cycle-independent manner in mammalian cells.     

The nuclear transport machinery recognizes nucleoplasmin-histone complexes

The nuclear transport of the chromatin remodeling protein nucleoplasmin and chromatin building histones is mediated by importins. Nucleoplasmin (NP) contains a classical bipartite nuclear localization signal (NLS) that is recognized by the importin α/β heterodimer, while histones present multiple NLS-like motifs that are recognized by importin β family members for nuclear targeting. To explore the possibility of a cotransport of histones and their chaperone NP to the nucleus, we have analyzed the assembly of complexes of NP/histones with importins by means of fluorescence anisotropy, centrifugation in sucrose gradients, and isothermal titration calorimetry. Data show that importin α ΔIBB (a truncated form of importin α lacking the autoinhibitory N-terminal domain) and histones (linker, H5, and nucleosomal core, H2AH2B) can simultaneously bind to NP. Analysis of the binding energetics reveals an enthalpy-driven formation of high affinity ternary, NP/Δα/H5 and NP/Δα/H2AH2B, complexes. We find that different amount of importin α molecules can be loaded on NP/histone complexes dependent on the histone type, linker or core, and the amount of bound histones. We further demonstrate that NP/H5 complexes can also incorporate importin α/β, thus forming quaternary NP/histones/α/β complexes that might represent a putative coimport pathway for nuclear import of histones and their chaperone protein NP, enhancing the histone import efficiency.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones

Background

Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3.

Results

Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus.

Conclusions

Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus.     

Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus

We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.     

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [³H]acetate. The acetylation was enhanced by 10 mm sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem.257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.     

Histone acetylation and heterochromatin content of cultured Peromyscus cells

In order to explore the relationship between unacetylated arginine-rich histones and condensed chromatin structure, the extent of histone acetylation was examined in cultured cell lines derived from three species of deer mice. These species differ considerably in their genomic content of heterochromatin but contain essentially the same euchromatin content. Cells of Peromyscus eremicus, containing 34–36% more constitutive heterochromatin than Peromyscus boylii or Peromyscus crinitus cells were found to contain 28–35% more unacetylated histone H4, 22–29% more unacetylated histone H3, and 18–22% more unacetylated histone H2B. This relationship between unacetylated histones and heterochromatin content was further explored by inducing hyperacetylation of P. eremicus and P. boylii histones through treatment of cells with 15 mM sodium butyrate for 24 h. It was found that the percentages of unacetylated histones H3 and H4 remaining after butyrate treatment were proportional to the amount of constitutive heterochromatin in the genome. These data support the concept that a small core of histones in constitutive heterochromatin is inaccessible to acetylation. It was also found that the acetylated state of isolated histones was sensitive to the method of histone extraction. Thus concern must be given to preparative procedures when studying histone acetylation in order to minimize these acetate losses.     

Crystal structure and function of human nucleoplasmin (npm2): a histone chaperone in oocytes and embryos

Human Npm2 is an ortholog of Xenopus nucleoplasmin (Np), a chaperone that binds histones. We have determined the crystal structure of a truncated Npm2-core at 1.9 ? resolution and show that the N-terminal domains of Npm2 and Np form similar pentamers. This allowed us to model an Npm2 decamer which may be formed by hydrogen bonds between quasi-conserved residues in the interface between two pentamers. Interestingly, the Npm2 pentamer lacks a prototypical A1-acidic tract in each of its subunits. This feature may be responsible for the inability of Npm2-core to bind histones. However, Npm2 with a large acidic tract in its C-terminal tail (Npm2-A2) is able to bind histones and form large complexes. Fluorescence resonance energy transfer experiments and biochemical analysis of loop mutations support the premise that nucleoplasmins form decamers when they bind H2A-H2B dimers and H3-H4 tetramers simultaneously. In the absence of histone tetramers, these chaperones bind H2A-H2B dimers with a single pentamer forming the central hub. When taken together, our data provide insights into the mechanism of histone binding by nucleoplasmins.