Chromatin decondensation and nuclear reprogramming by nucleoplasmin

Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1beta and TIF1beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.     

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.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

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 structure and function of Xenopus NO38-core, a histone chaperone in the nucleolus

Xenopus NO38 is an abundant nucleolar chaperone and a member of the nucleoplasmin (Np) family. Here, we report high-resolution crystal structures of the N-terminal domain of NO38, as a pentamer and a decamer. As expected, NO38 shares the Np family fold. In addition, NO38- and Np-core pentamers each use highly conserved residues and numerous waters to form their respective decamers. Further studies show that NO38 and Np each bind equal amounts of the four core histones. However, NO38 prefers the (H3-H4)(2) tetramer, while Np probably prefers H2A-H2B dimers. We also show that NO38 and Np will each bind noncognate histones when the preferred partner is absent. We suggest that these chaperones must form decamers in order to bind histones and differentiate between histone tetramers and dimers. When taken together, these data imply that NO38 may function as a histone chaperone in the nucleolus.     

Expression and purification of the full murine NPM2 and study of its interaction with protamines and histones

Mouse nucleoplasmin M.NPM2 was recombinantly expressed and the protein consisting of the complete sequence was purified and characterized. Similar to its Xenopus laevis X.NPM2 counterpart, the protein forms stable pentameric complexes and exhibits an almost undistinguishable hydrodynamic ionic strength-dependent unfolding behavior. The interaction of N.PM2 with histones and mouse P1/P2 protamines revealed that these chromosomal proteins bind preferentially to the distal part of the nucleoplasmin pentamer. Moreover, the present work highlights the critical role played by histones H2B and H4 in the association of the histone H2A-H2B dimers and histone octamer with nucleoplasmin.     

DNA replication in quiescent cell nuclei: regulation by the nuclear envelope and chromatin structure

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.     

Sequential establishment of marks on soluble histones H3 and H4

Much progress has been made concerning histone function in the nucleus; however, following their synthesis, how their marking and subcellular trafficking are regulated remains to be explored. To gain an insight into these issues, we focused on soluble histones and analyzed endogenous and tagged H3 histones in parallel. We distinguished six complexes that we could place to account for maturation events occurring on histones H3 and H4 from their synthesis onward. In each complex, a different set of chaperones is involved, and we found specific post-translational modifications. Interestingly, we revealed that histones H3 and H4 are transiently poly(ADP-ribosylated). The impact of these marks in histone metabolism proved to be important as we found that acetylation of lysines 5 and 12 on histone H4 stimulated its nuclear translocation. Furthermore, we showed that, depending on particular histone H3 modifications, the balance in the presence of the different translocation complexes changes. Therefore, our results enabled us to propose a regulatory means of these marks for controlling cytoplasmic/nuclear shuttling and the establishment of early modification patterns.     

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.     

Regulation of histone H2A and H2B deubiquitination and Xenopus development by USP12 and USP46

Post-translational histone modifications play important roles in regulating gene expression programs, which in turn determine cell fate and lineage commitment during development. One such modification is histone ubiquitination, which primarily targets histone H2A and H2B. Although ubiquitination of H2A and H2B has been generally linked to gene silencing and gene activation, respectively, the functions of histone ubiquitination during eukaryote development are not well understood. Here, we identified USP12 and USP46 as histone H2A and H2B deubiquitinases that regulate Xenopus development. USP12 and USP46 prefer nucleosomal substrates and deubiquitinate both histone H2A and H2B in vitro and in vivo. WDR48, a WD40 repeat-containing protein, interacts with USP12 and USP46 and is required for the histone deubiquitination activity. Overexpression of either gene leads to gastrulation defects without affecting mesodermal cell fate, whereas knockdown of USP12 in Xenopus embryos results in reduction of a subset of mesodermal genes at gastrula stages. Immunohistochemical staining and chromatin immunoprecipitation assays revealed that USP12 regulates histone deubiquitination in the mesoderm and at specific gene promoters during Xenopus development. Taken together, this study identifies USP12 and USP46 as histone deubiquitinases for H2A and H2B and reveals that USP12 regulates Xenopus development during gastrula stages.     

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.     

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.     

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.     

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.     

Xenopus nucleoplasmin: egg vs. oocyte

Nucleoplasmin has been purified from either oocytes or unfertilized eggs of the frog, Xenopus laevis. We find that the pentameric form of egg nucleoplasmin exhibits an apparent molecular mass approximately 15 000 daltons larger than its oocyte counterpart upon sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis. Egg nucleoplasmin monomers are more heterogeneous, substantially more acidic, and overall larger in apparent molecular weight than oocyte nucleoplasmin monomers when analyzed by isoelectric focusing or SDS gel electrophoresis. Protease digestions indicate that the structural differences between egg and oocyte nucleoplasmin are primarily confined to the N-terminal halves of the proteins. The structural diversity observed is accompanied by a difference in the ability of nucleoplasmin from the two sources to act as a nucleosome assembly agent in vitro. Egg nucleoplasmin efficiently promotes the formation of nucleosomes onto circular pBR322 DNA in vitro at physiological ionic strength and at physiological histone:DNA ratios, while oocyte nucleoplasmin is markedly deficient in serving as an in vitro chromatin assembly agent under all conditions which we have tested. Treatment of egg nucleoplasmin in vitro with alkaline phosphatase demonstrates that the structural diversity between egg and oocyte nucleoplasmin results primarily from extensive additional phosphorylation of the egg protein. The relevance of nucleoplasmin phosphorylation in leading to differences in the chromatin assembly activity of this protein both in vitro and in vivo is considered.     

Phosphorylation of Histone H2A.X by DNA-dependent Protein Kinase Is Not Affected by Core Histone Acetylation,but It Alters Nucleosome Stability and Histone H1 Binding

Phosphorylation of the C-terminal end of histone H2A.X is the most characterized histone post-translational modification in DNA double-stranded breaks (DSB). DNA-dependent protein kinase (DNA-PK) is one of the three phosphatidylinositol 3 kinase-like family of kinase members that is known to phosphorylate histone H2A.X during DNA DSB repair. There is a growing body of evidence supporting a role for histone acetylation in DNA DSB repair, but the mechanism or the causative relation remains largely unknown. Using bacterially expressed recombinant mutants and stably and transiently transfected cell lines, we find that DNA-PK can phosphorylate Thr-136 in addition to Ser-139 both in vitro and in vivo. Furthermore, the phosphorylation reaction is not inhibited by the presence of H1, which in itself is a substrate of the reaction. We also show that, in contrast to previous reports, the ability of the enzyme to phosphorylate these residues is not affected by the extent of acetylation of the core histones. In vitro assembled nucleosomes and HeLa S3 native oligonucleosomes consisting of non-acetylated and acetylated histones are equally phosphorylated by DNA-PK. We demonstrate that the apparent differences in the extent of phosphorylation previously observed can be accounted for by the differential chromatin solubility under the MgCl₂ concentrations required for the phosphorylation reaction in vitro. Finally, we show that although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding.