Nucleoplasmin Research Articles

Characterization of bovine long non-coding RNAs, OOSNCR1, OOSNCR2 and OOSNCR3, and their roles in oocyte maturation and early embryonic development

In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.

Loss of NPM2 expression is a potential immunohistochemical marker for malignant peritoneal mesothelioma: a single-center study of 92 cases

BackgroundMalignant peritoneal mesothelioma (MPM) is a rare malignant tumor with a high mortality rate and extremely poor prognosis. In-depth pathological analysis is essential to assess tumor biological behaviors and explore potential therapeutic targets of MPM. Nucleoplasmin 2 (NPM2) is a molecular chaperone that binds histones and may play a key role in the development and progression of tumors. This study aimed to analyze the correlation between the expression level of NPM2 and the main clinicopathological characteristics and prognosis of MPM.MethodsNinety-two postoperative specimens from MPM patients following cytoreductive surgery were collected. Postoperative specimens were stained with immunohistochemistry. The expression level of NPM2 was quantitatively analyzed by QuPath-0.3.2 software. Univariate and multivariate analyses were conducted to investigate the correlation between NPM2 expression and other conventional clinicopathological characteristics.ResultsAmong the 92 MPM patients, there were 47 males (48.9%) and 45 females (51.1%), with a median age of 56 (range: 24–73). There were 70 (76.0%) cases with loss of NPM2 protein expression, 11 (12.0%) cases with low expression, and 11 (12.0%) cases with high expression. Univariate analysis showed that NPM2 protein expression level (negative vs. low expression vs. high expression) was negatively correlated with the following three clinicopathological factors: completeness of cytoreduction (CC) score, vascular tumor emboli, and serious adverse events (SAEs) (all P < 0.05). Multivariate analysis showed that NPM2 protein expression level (negative vs. low expression vs. high expression) was independently negatively correlated with the following two clinicopathological factors: CC score [odds ratio (OR) = 0.317, 95% CI: 0.317–0.959, P = 0.042] and vascular tumor emboli (OR = 0.092, 95% CI = 0.011–0.770, P = 0.028). Survival analysis showed that loss of NPM2 protein expression (negative vs. positive) was associated with poor prognosis of MPM.ConclusionsLoss of NPM2 expression is a potential immunohistochemical marker for MPM.

T-NHL subtypes and clinical perspective

Peripheral T-cell lymphomas (PTCL) are a distinct pathological entity with clinical advancements lagging behind its B-cell lymphoma counterpart. A significant proportion (15–20%) of PTCL cases are misdiagnosed as reactive or a different lymphoma subtype. An accurate diagnosis routinely requires analysis of the immunophenotype in conjunction with cellular morphology, analysis of lymph node architecture and molecular genetic analysis. Recent major advances in genomic studies examining gene expression profiling (GEP) and the genetic landscape of T-cell and NK-cell neoplasms have resulted in diagnostic, prognostic and therapeutic implications and has consequently led to revisions in the classification of PTCLs and introduction of new provisional entities. Incorporating these molecular and genetic markers, the latest 2016 WHO classification, an update of the current fourth edition, has identified 25 definitive and 6 provisional entities and further groups these lymphomas according to their usual presentation into disseminated disease (leukaemias), predominantly nodal, predominantly extra-nodal or cutaneous lymphomas. Nodal PTCLs represent the most prevalent PTCL subtype and include peripheral T-cell lymphoma - not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), follicular T-cell lymphoma (FTCL), nodal PTCL with T follicular helper (TFH) phenotype, and systemic anaplastic large cell lymphoma (sALCL) either ALK-positive or ALK-negative. Due to better understanding of the cell of origin in T-cell lymphomas, AITL, FTCL and PTCL with TFH phenotype have been recognised as being derived from TFH cells as these neoplastic cells express at least two or three TFH cell antigens which include CD279/PD1, CD10, BCL6, CXCL13, ICOS, SAP and CCR5. In addition to the common cell of origin, these lymphomas share pathological and clinical features as well as recurrent genetic abnormalities such as mutations in TET2, IDH2, DNMT3A, RHOA and CD28, and gene fusions such as ITK-SYK and CTLA-CD28. Such features suggest that these lymphomas belong to the same spectrum of disease and are therefore now unified under a common heading. PTCL-NOS encompasses those which do not match the classification of other PTCL categories. This subtype mostly has a CD4+/CD8– phenotype and shows extreme cytological and phenotypical heterogeneity but GEP have identified at least three subgroups characterised by overexpression of GATA3, TBX21 and cytotoxic genes associated with varied clinical behaviour. Systemic ALCLs have a primarily aggressive nodal presentation and are further subdivided to ALK-positive or ALK-negative based on whether they carry the nucleoplasmin (NPM) anaplastic lymphoma kinase (ALK) translocation between chromosome 2 and 5, designated as t(2;5). We now know that a subset of ALCL, ALK-negative cases harbour rearrangement at the locus containing DUSP22 and IRF4 in chromosome 6p25, which induces down regulation of DUSP22, a dual specific phosphatase that inhibits TCR signalling. Another subset of ALK-negative ALCL has rearrangements in TP63. Clear distinction of PTCL with high CD30 expression from ALCL, ALK-negative have also been possible through GEP and as such, the former is no longer considered a provisional entity. Breast implant associated ALCL, a unique form of ALCL, ALK-negative arising in association with breast implants, is increasingly being recognised and has been included as a provisional entity. Cutaneous ALCL (cALCL), ALK-negative, the fourth subtype, is an indolent form limited to the skin. Whilst notorious to have an aggressive clinical course and an extremely poor overall prognosis, clinicians are constantly challenged with low rates of complete remissions, early relapses and failures to achieve long-term remissions despite aggressive first-line chemotherapy, in the majority of patients. More recently, improved understanding of disease biology and the molecular profile of specific PTCL subtypes has opened the door to the potential of more targeted therapies with a number of new biological therapies approved by regulatory agencies in the last few years.

Structure-function relationship of H2A-H2B specific plant histone chaperones.

Studies on chromatin structure and function have gained a revived popularity. Histone chaperones are significant players in chromatin organization. They play a significant role in vital nuclear functions like transcription, DNA replication, DNA repair, DNA recombination, and epigenetic regulation, primarily by aiding processes such as histone shuttling and nucleosome assembly/disassembly. Like the other eukaryotes, plants also have a highly orchestrated and dynamic chromatin organization. Plants seem to have more isoforms within the same family of histone chaperones, as compared with other organisms. As some of these are specific to plants, they must have evolved to perform functions unique to plants. However, it appears that only little effort has gone into understanding the structural features of plant histone chaperones and their structure-function relationships. Studies on plant histone chaperones are essential for understanding their role in plant chromatin organization and how plants respond during stress conditions. This review is on the structural and functional aspects of plant histone chaperone families, specifically those which bind to H2A-H2B, viz nucleosome assembly protein (NAP), nucleoplasmin (NPM), and facilitates chromatin transcription (FACT). Here, we also present comparative analyses of these plant histone chaperones with available histone chaperone structures. The review hopes to incite interest among researchers to pursue further research in the area of plant chromatin and the associated histone chaperones.

Structural insights into the ability of nucleoplasmin to assemble and chaperone histone octamers for DNA deposition

Nucleoplasmin (NP) is a pentameric histone chaperone that regulates the condensation state of chromatin in different cellular processes. We focus here on the interaction of NP with the histone octamer, showing that NP could bind sequentially the histone components to assemble an octamer-like particle, and crosslinked octamers with high affinity. The three-dimensional reconstruction of the NP/octamer complex generated by single-particle cryoelectron microscopy, revealed that several intrinsically disordered tail domains of two NP pentamers, facing each other through their distal face, encage the histone octamer in a nucleosome-like conformation and prevent its dissociation. Formation of this complex depended on post-translational modification and exposure of the acidic tract at the tail domain of NP. Finally, NP was capable of transferring the histone octamers to DNA in vitro, assembling nucleosomes. This activity may have biological relevance for processes in which the histone octamer must be rapidly removed from or deposited onto the DNA.

Re-evaluating the Localization of Sperm-Retained Histones Revealed the Modification-Dependent Accumulation in Specific Genome Regions

The question of whether retained histones in the sperm genome localize to gene-coding regions or gene deserts has been debated for years. Previous contradictory observations are likely caused by the non-uniform sensitivity of sperm chromatin to micrococcal nuclease (MNase) digestion. Sperm chromatin has a highly condensed but heterogeneous structure and is composed of 90%∼99% protamines and 1%∼10% histones. In this study, we utilized nucleoplasmin (NPM) to improve the solubility of sperm chromatin by removing protamines invitro. NPM treatment efficiently solubilized histones while maintaining quality and quantity. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses using NPM-treated sperm demonstrated the predominant localization of H4 to distal intergenic regions, whereas modified histones exhibited a modification-dependent preferential enrichment in specific genomic elements, such as H3K4me3 at CpG-rich promoters and H3K9me3 in satellite repeats, respectively, implying the existence of machinery protecting modified histones from eviction.

Reconstitution of the oocyte nucleolus in mice through a single nucleolar protein, NPM2

The mammalian oocyte nucleolus, the most prominent subcellular organelle in the oocyte, is vital in early development, yet its key functions and constituents remain unclear. We show here that the parthenotes/zygotes derived from enucleolated oocytes exhibited abnormal heterochromatin formation around parental pericentromeric DNAs, which led to a significant mitotic delay and frequent chromosome mis-segregation upon the first mitotic division. A proteomic analysis identified nucleoplasmin 2 (NPM2) as a dominant component of the oocyte nucleolus. Consistently, Npm2-deficient oocytes, which lack a normal nucleolar structure, showed chromosome segregation defects similar to those in enucleolated oocytes, suggesting that nucleolar loss, rather than micromanipulation-related damage to the genome, leads to a disorganization of higher-order chromatin structure in pronuclei and frequent chromosome mis-segregation during the first mitosis. Strikingly, expression of NPM2 alone sufficed to reconstitute the nucleolar structure in enucleolated embryos, and rescued their first mitotic division and full-term development. The nucleolus rescue through NPM2 required the pentamer formation and both the N- and C-terminal domains. Our findings demonstrate that the NPM2-based oocyte nucleolus is an essential platform for parental chromatin organization in early embryonic development.

Maternal effect gene expression in porcine metaphase II oocytes and embryos in vitro: effect of epidermal growth factor, interleukin-1β and leukemia inhibitory factor.

Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1β or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1β 10 ng/ml or LIF 50 ng/ml. After maturation for 44-46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1β did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1β decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1β in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.

A Quantitative Characterization of Nucleoplasmin/Histone Complexes Reveals Chaperone Versatility.

Nucleoplasmin (NP) is an abundant histone chaperone in vertebrate oocytes and embryos involved in storing and releasing maternal histones to establish and maintain the zygotic epigenome. NP has been considered a H2A–H2B histone chaperone, and recently it has been shown that it can also interact with H3-H4. However, its interaction with different types of histones has not been quantitatively studied so far. We show here that NP binds H2A–H2B, H3-H4 and linker histones with Kd values in the subnanomolar range, forming different complexes. Post-translational modifications of NP regulate exposure of the polyGlu tract at the disordered distal face of the protein and induce an increase in chaperone affinity for all histones. The relative affinity of NP for H2A–H2B and linker histones and the fact that they interact with the distal face of the chaperone could explain their competition for chaperone binding, a relevant process in NP-mediated sperm chromatin remodelling during fertilization. Our data show that NP binds H3-H4 tetramers in a nucleosomal conformation and dimers, transferring them to DNA to form disomes and tetrasomes. This finding might be relevant to elucidate the role of NP in chromatin disassembly and assembly during replication and transcription.

Zygote arrest 1, nucleoplasmin 2, and developmentally associated protein 3 mRNA profiles throughout porcine embryo development in vitro

Maternal effect genes (MEGs) are expressed in oocytes and embryos and play an important role in activation of the embryonic genome. An abnormality in the expression of these genes may lead to arrest of embryonic cleavage or to altered transcription of factors responsible for further embryonic development. In vitro–produced porcine embryos have a lower developmental potential than embryos produced in vivo. We hypothesized that in vitro embryo culture conditions have an effect on the expression of MEGs at various developmental stages, which may affect their developmental potential. Here, using real-time polymerase chain reaction, we examined mRNA profiles of the MEGs, zygote arrest 1 (ZAR-1), nucleoplasmin 2 (NPM2), and developmentally associated pluripotency protein 3 (DPPA3), in porcine oocytes and embryos produced in vitro and in vivo. Further, we evaluated the effect of the combined addition of EGF, interleukin 1β, and leukemia inhibitory factor to the porcine in vitro embryo production system on mRNA profiles of selected MEGs. Finally, we studied localization of the MEG protein products in in vitro–obtained oocytes and embryos using confocal microscopy. We found that the ZAR-1 mRNA profile differed throughout in vitro and in vivo embryo development. In the embryos produced in vitro, the decrease in ZAR-1 mRNA levels was observed at the 2-cell stage, whereas in in vivo embryos, ZAR-1 mRNA levels declined significantly starting at the 4-cell stage (P < 0.05). In vitro culture conditions affected transiently also DPPA3 mRNA levels at the 4-cell stage (P < 0.05). There was no difference in the NPM2 mRNA profile during in vitro and in vivo embryo development. The ZAR-1 and DPPA3 proteins were localized in the cytoplasm of the oocytes and embryos, whereas the NPM2 protein was found both in the cytoplasm and in the nucleus. All proteins were expressed until blastocyst stage. The addition of EGF and cytokines to the culture medium decreased DPPA3 mRNA levels in 8-cell embryos (P < 0.05). This study indicated that IVC conditions affect ZAR-1 mRNA levels before the 4-cell stage, which may disturb the activation of the embryonic genome in pigs. The expression of the proteins after the 4-cell to 8-cell transition indicates that these factors play a role beyond activation of the embryonic genome. Supplementation of the culture media with EGF and cytokines affects DPPA3 mRNA levels after maternal to embryonic transition.

The intrinsically disordered distal face of nucleoplasmin recognizes distinct oligomerization states of histones

The role of Nucleoplasmin (NP) as a H2A-H2B histone chaperone has been extensively characterized. To understand its putative interaction with other histone ligands, we have characterized its ability to bind H3-H4 and histone octamers. We find that the chaperone forms distinct complexes with histones, which differ in the number of molecules that build the assembly and in their spatial distribution. When complexed with H3-H4 tetramers or histone octamers, two NP pentamers form an ellipsoidal particle with the histones located at the center of the assembly, in stark contrast with the NP/H2A-H2B complex that contains up to five histone dimers bound to one chaperone pentamer. This particular assembly relies on the ability of H3-H4 to form tetramers either in solution or as part of the octamer, and it is not observed when a variant of H3 (H3C110E), unable to form stable tetramers, is used instead of the wild-type protein. Our data also suggest that the distal face of the chaperone is involved in the interaction with distinct types of histones, as supported by electron microscopy analysis of the different NP/histone complexes. The use of the same structural region to accommodate all type of histones could favor histone exchange and nucleosome dynamics.

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.

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.

Molecular cloning and expression of bovine nucleoplasmin 2 (NPM2): a maternal effect gene regulated by miR-181a

BackgroundNucleoplasmin 2 (NPM2) is an oocyte-specific nuclear protein essential for nuclear and nucleolar organization and early embryonic development. The aims of this study were to clone the bovine NPM2 gene, determine its temporal expression during oocyte development and early embryogenesis, and evaluate the potential role of miRNA-181a in regulation of its expression.MethodsA 329 bp cDNA fragment was amplified from bovine fetal ovary using primers designed based on the conserved regions of the human and mouse NPM2 cDNA sequences. RACE experiments were performed to obtain the 5' and 3' ends of the bovine NPM2 cDNA. Real time PCR and Western blot analysis were used to examine the expression of bovine NPM2 in oocytes and early embryos. Co-expression of bovine NPM2 and miRNA-181a in Hela cells was performed to determine if expression of bovine NPM2 is regulated by miRNA-181a.ResultsThe bovine NPM2 cDNA is 851 bp in length encoding a protein of 200 amino acids. The protein contains the conserved bipartite nuclear localization sequence and shows 53% and 62% identity with mouse and human NPM2, respectively. Expression of bovine NPM2 mRNA is restricted to ovaries. NPM2 mRNA is abundant in GV and MII stage oocytes, decreases in early cleavage stage embryos, and barely detectable in morula and blastocyst stage embryos. Similarly, expression of NPM2 protein is high in oocytes and early embryos but extremely low in blastocysts. The abundance of NPM2 mRNA is significantly lower in oocytes isolated from persistent versus growing dominant follicles (P < 0.05). A miR-181a binding site in the 3'UTR of the NPM2 transcript was identified. Transfection experiments showed that bovine NPM2 protein expression is reduced in Hela cells expressing miR-181a compared to control cells without miR-181a, indicating that translation of NPM2 is repressed by miR-181a.ConclusionsOur data suggest that expression of bovine NPM2 is temporally regulated during early embryogenesis and miR-181a may play a role in its regulation.

Involvement of Mouse Nucleoplasmin 2 in the Decondensation of Sperm Chromatin after Fertilization1

The tightly condensed chromatin of spermatozoa is rapidly decondensed after the spermatozoa enter oocytes. Although no factor involved in sperm chromatin decondensation (SCD) has been identified in mammals, it has been suggested that a factor related to SCD activity is present in the germinal vesicle (GV) of oocytes. Here, we found that the nucleolus-like body (NLB), which is a component of the GV, is involved in SCD in murine oocytes. When NLBs were microsurgically removed from GV-stage oocytes, SCD was significantly retarded in the paternal genome after fertilization following meiotic maturation. We found that the retardation of SCD in the NLB-removed oocytes was restored by the microinjection of mRNA encoding nucleoplasmin 2 (NPM2), a component of NLBs. Furthermore, SCD was retarded in the fertilized oocytes from Npm2-knockout females, and recombinant NPM2 alone could induce the SCD in vitro. These data provide evidence that NPM2 is involved in sperm chromatin remodeling in mammals.

Recognition of Nucleoplasmin by Its Nuclear Transport Receptor Importin α/β: Insights into a Complete Import Complex

Nuclear import of the pentameric histone chaperone nucleoplasmin (NP) is mediated by importin α, which recognizes its nuclear localization sequence (NLS), and importin β, which interacts with α and is in charge of the translocation of the NP/α/β complex through the nuclear pore. Herein, we characterize the assembly of a functional transport complex formed by full-length NP with importin α/β. Isothermal titration calorimetry (ITC) was used to analyze the thermodynamics of the interactions of importin α with β, α with NP, and the α/β heterodimer with NP. Our data show that binding of both importin α and α/β to NP is governed by a favorable enthalpic contribution and that NP can accommodate up to five importin molecules per NP pentamer. Phosphomimicking mutations of NP, which render the protein active in histone chaperoning, do not modulate the interaction with importin. Using small-angle X-ray scattering, we model the α/β heterodimer, NP/α, and NP/α/β solution structures, which reveal a glimpse of a complete nuclear import complex with an oligomeric cargo protein. The set of alternative models, equally well fitting the scattering data, yields asymmetric elongated particles that might represent consecutive geometries the complex can adopt when stepping through the nuclear pore.

Investigating the Potential of Genes Preferentially Expressed in Oocyte to Induce Chromatin Remodeling in Somatic Cells

The oocyte capacity to rejuvenate a differentiated nucleus to restart the proper embryonic program has been highly conserved between vertebrate species. In view of the recent progress to induce pluripotency in somatic cells with stemness genes, we investigated the potential of oocyte genes to contribute to chromatin rearrangements in somatic cells. We selected conserved genes that are naturally expressed mainly in oocytes and that were susceptible to play a role in reprogramming during early embryogenesis. We induced their expression by transient transfection in HEK293 cells. We then assessed whether they had a global impact on epigenetic events such as histone core modifications, and also on transcription and expression of pluripotency-associated transcription factors. Nucleoplasmin 2 (NPM2), activation-induced cytidine deaminase (AICDA), and Geminin (GMNN) overexpression induced differences in histone core modifications (methylation and acetylation). AICDA and NPM2 also influenced RNA neosynthesis. NPM2, GMNN, and STELLA induced overexpression of well-known pluripotency transcription factors. Overall, AICDA, GMNN, NPM2, and STELLA influenced at least one of the aspects analyzed. Their potential could be useful in increasing the cell receptivity to pluripotency induction.

Nucleoplasmin Binds Histone H2A-H2B Dimers through Its Distal Face*

Nucleoplasmin (NP) is a pentameric chaperone that regulates the condensation state of chromatin extracting specific basic proteins from sperm chromatin and depositing H2A-H2B histone dimers. It has been proposed that histones could bind to either the lateral or distal face of the pentameric structure. Here, we combine different biochemical and biophysical techniques to show that natural, hyperphosphorylated NP can bind five H2A-H2B dimers and that the amount of bound ligand depends on the overall charge (phosphorylation level) of the chaperone. Three-dimensional reconstruction of NP/H2A-H2B complex carried out by electron microscopy reveals that histones interact with the chaperone distal face. Limited proteolysis and mass spectrometry indicate that the interaction results in protection of the histone fold and most of the H2A and H2B C-terminal tails. This structural information can help to understand the function of NP as a histone chaperone.

Karyopherin α7 (KPNA7), a divergent member of the importin α family of nuclear import receptors

BackgroundClassical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin α and importin β. NLS cargo is recognized by importin α, which is bound by importin β. Importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin β triggers disassembly of the complex. To date, six importin α family members, encoded by separate genes, have been described in humans.ResultsWe sequenced and characterized a seventh member of the importin α family of transport factors, karyopherin α 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin β (IBB) is divergent, and shows stronger binding to importin β than the IBB domains from of other importin α family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic.ConclusionKPNA7 is a novel importin α family member in humans that belongs to the importin α2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin α family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation.

Role of the nucleoplasmin 2 C‐terminal domain in the formation of nucleolus‐like bodies in mouse oocytes

Nucleolus-like bodies (NLBs) are characteristic structures found in the germinal vesicles of mammalian oocytes. Although these structures are essential for embryonic development, their composition, precise function, and mechanism of formation have not been elucidated. Here, we used immunoblotting and EGFP fusion protein fluorescence to demonstrate that murine nucleoplasmin 2 (NPM2) is a component of mouse NLBs and that the targeting of NPM2 to NLBs is regulated by a lysine-rich, 16-aa C-terminal motif (K-rich motif). When the K-rich motif was fused to another nuclear protein, MafG, the resultant fusion protein accumulated in NLBs but not in the nucleoli of somatic cells, suggesting that the K-rich motif functions to target NPM2 specifically to NLBs. To investigate the role of the K-rich motif in NLB formation, we replaced the endogenous NPM2 in growing oocytes with a mutant NPM2 protein lacking the K-rich motif (NPM2(C16del)). Growing oocytes surrounded by granulosa layers were coinjected with NPM2(C16del) mRNA and with small-interfering RNA targeting NPM2 (siNpm2), which was used to degrade the endogenous NPM2 mRNA. After culture in vitro, the NLBs in the resulting full-grown oocytes were significantly smaller than those in control oocytes that had been coinjected with siNpm2 and NPM2 mRNA, indicating that the K-rich motif is necessary for NLB development. Together, these results suggest that NPM2 targeting of NLBs is regulated by the K-rich motif and is essential for the formation of NLBs.