Measurement of the intracellular active metabolites of thiopurine drugs to evaluate the enzymatic activity of nudix hydrolase 15 in human blood samples.

Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 µM, 1 µM, and 2 µM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 µM) as a substrate (18.1, 14.9, and 6.43 µM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.

The stability of NPM1 oligomers regulated by acidic disordered regions controls the quality of liquid droplets.

The nucleolus is a membrane-less nuclear body that typically forms through the process of liquid-liquid phase separation (LLPS) involving its components. NPM1 drives LLPS within the nucleolus and its oligomer formation and inter-oligomer interactions play a cooperative role in inducing LLPS. However, the molecular mechanism underlaying the regulation of liquid droplet quality formed by NPM1 remains poorly understood. In this study, we demonstrate that the N-terminal and central acidic residues within the intrinsically disordered regions (IDR) of NPM1 contribute to attenuating oligomer stability, although differences in the oligomer stability were observed only under stringent conditions. Furthermore, the impact of the IDRs is augmented by an increase in net negative charges resulting from phosphorylation within the IDRs. Significantly, we observed an increase in fluidity of liquid droplets formed by NPM1 with decreased oligomer stability. These results indicate that the difference in oligomer stability only observed biochemically under stringent conditions has a significant impact on liquid droplet quality formed by NPM1. Our findings provide new mechanistic insights into the regulation of nucleolar dynamics during the cell cycle.

SET-NUP214 and MLL cooperatively regulate the promoter activity of the HoxA10 gene.

SET-Nup214 is a recurrent fusion gene that is mainly observed in T-cell acute lymphoblastic leukemia (T-ALL). Dysregulation of homeobox (Hox) genes is frequently observed in patients with leukemia. Consistent with this, HoxA genes are upregulated in the SET-Nup214 + T-ALL cell line and patients. Although SET-Nup214 has been reported to be recruited to the promoter regions of HoxA genes, the detailed mechanisms of how SET-Nup214 specifically binds to HoxA gene promoters and regulates HoxA gene expression are not known. In this study, we demonstrated that SET-Nup214 interacts with MLL via the SET acidic region of SET-Nup214. SET-Nup214 and MLL cooperatively enhance the promoter activity of the HoxA10 gene. Neither the SET region alone nor the Nup214 region alone sufficiently enhanced the HoxA10 gene promoter. Our results indicated that the SET portion of the SET-Nup214-fusion protein is important for interactions with MLL and transcription enhancement of the HoxA10 gene. Thus, our study will contribute to the understanding of how SET-Nup214 and MLL disturb the expression of HoxA10 gene in leukemia.

G-patch domain-containing protein 4 localizes to both the nucleoli and Cajal bodies and regulates cell growth and nucleolar structure.

Ribosome formation occurs in the nucleolus through interaction with various trans-acting factors. Therefore, hundreds of nucleolar proteins have a function in ribosome formation, although the precise function of each nucleolar protein in ribosome formation is largely unclear. We have previously identified an uncharacterized protein, G-patch domain-containing protein 4 (GPATCH4 or G4), as a component of the pre-ribosomes purified with either nucleolin (NCL) or NPM1. In this present study, we sought to clarify the localization and function of G4. We identified that G4 localizes to both the nucleolus and the Cajal body. Although knockdown of G4 did not have a significant effect on pre-ribosomal RNA processing, cell growth did decrease. Interestingly, G4 knockdown also decreased the number of fibrillar center and dense fibrillar component regions inside the nucleolus. This data has identified G4 as a novel nucleolar protein involved in the regulation of cell growth and nucleolar structure.

The interaction between nucleophosmin/NPM1 and the large ribosomal subunit precursors contribute to maintaining the nucleolar structure.

Nucleoli are sites where both the large and small ribosomal subunits mature. Biochemical assays have suggested that a multivalent nucleolar protein, NPM1/nucleophosmin contributes to the formation of the outer layer of the nucleolus. Prior works show that NPM1 depletion disorganizes the nucleolar structure. However, the mechanism of how NPM1 regulates the nucleolar structure has been unknown. We demonstrated that NPM1 directly interacts with the large ribosomal subunits and maintains them in the nucleolus. Ectopically localized NPM1 efficiently recruits only the large ribosomal subunit precursors, while ectopically localized large ribosomal subunit by the ribosomal protein RPL4 efficiently recruits NPM1. These results suggest that the nucleolar localization of NPM1 and the large ribosomal subunit precursors are mutually dependent. Furthermore, proteomic and localization analyses suggest that NPM1 plays a crucial role in the accumulation of the late processing machinery of the large ribosomal subunits in the nucleolus. Our results suggest that NPM1 maintains the pre-ribosomes and assembly machinery in the nucleolus, which in turn determines the nucleolar volume.

RNA-recognition motifs and glycine and arginine-rich region cooperatively regulate the nucleolar localization of nucleolin.

Nucleolin (NCL) is a nucleolar protein i.e. involved in the regulation of the nucleolar structure and functions, and consists of three distinct regions: the N-terminal region; the middle region, which contains four RNA-recognition motifs (RRMs); and the C-terminal glycine- and arginine-rich (GAR) region. The primary function of the RRMs and GAR is thought to be specific RNA binding. However, it is not well understood how these RNA-binding regions of NCL separately or cooperatively regulate its nucleolar localization and functions. To address this issue, we constructed mutant proteins carrying point mutations at the four RRMs individually or deletion of the C-terminal GAR region. We found that the GAR deletion and the mutations in the fourth RRM (RRM4) decreased the nucleolar localization of NCL. Biochemical analyses showed that NCL interacted directly with ribosomal RNAs (rRNAs) and G-rich oligonucleotides, and that this interaction was decreased by mutations at RRM1 and RRM4 and GAR deletion. Although GAR deletion decreased the rRNA-binding activity of NCL, the mutant was efficiently coprecipitated with rRNAs and nucleolar proteins from cell extracts. These contradictory results suggest that NCL stably localizes to the nucleoli via the interactions with rRNAs and nucleolar proteins via GAR, RRM1 and RRM4.

Formation of adenovirus DNA replication compartments and viral DNA accumulation sites by host chromatin regulatory proteins including NPM1.

The adenovirus (Ad) genome is believed to be packaged into the virion by forming a chromatin-like structure. The replicated viral genome is likely to be condensed through binding with viral core proteins before encapsidation. Replicated viral genomes accumulate in the central region of the nucleus, which we termed virus-induced postreplication (ViPR) body. However, the molecular mechanism by which the nuclear structure is reorganized and its functional significance in virus production are currently not understood. In this study, we found that viral packaging protein IVa2, but not capsid proteins, accumulated in the ViPR body. In addition, nucleolar chromatin regulatory proteins, nucleophosmin 1 (NPM1), upstream binding factor, and nucleolin accumulated in the ViPR body in late-stage Ad infection. NPM1 depletion increased the nuclease-resistant viral genome and delayed the ViPR body formation. These results suggested that structural changes in the infected cell nucleus depend on the formation of viral chromatin by host chromatin regulatory proteins. Because NPM1 depletion decreases production of the infectious virion, we propose that host factor-mediated viral chromatin remodeling and concomitant ViPR body formation are prerequisites for efficient encapsidation of Ad chromatin.

Selective regulation of type II interferon-inducible genes by NPM1/nucleophosmin.

Nucleophosmin (NPM1) is a multifunctional nucleolar protein. Here, we analyze the role of NPM1 in gene expression using our previous microarray data and find a relationship between NPM1 and interferon (IFN)-γ-inducible genes. We show that NPM1 selectively regulates the expression of a subset of IFN-γ-inducible genes and directly binds to two important transcription factors in the type II IFN pathway: signal transducer and activator of transcription 1 and interferon regulatory factor 1 (IRF1). Furthermore, NPM1 is found to regulate the IFN-γ-inducible promoter activity of major histocompatibility complex class II transactivator (CIITA), and mutation of the IRF1-binding site on the CIITA promoter abolishes the effect of NPM1. Our results suggest a novel mechanism for IFN-γ-mediated gene expression by NPM1.

Assembly and remodeling of viral DNA and RNA replicons regulated by cellular molecular chaperones.

A variety of cellular reactions mediated by interactions among proteins and nucleic acids requires a series of proteins called molecular chaperones. The viral genome encodes relatively few kinds of viral proteins and, therefore, host-derived cellular factors are required for virus proliferation. Here we discuss those cellular proteins known as molecular chaperones, which are essential for the assembly of functional viral DNA/RNA replicons. The function of these molecular chaperones in the cellular context is also discussed.

Internal Associations of the Acidic Region of Upstream Binding Factor Control Its Nucleolar Localization.

Upstream binding factor (UBF) is a member of the high-mobility group (HMG) box protein family, characterized by multiple HMG boxes and a C-terminal acidic region (AR). UBF is an essential transcription factor for rRNA genes and mediates the formation of transcriptionally active chromatin in the nucleolus. However, it remains unknown how UBF is specifically localized to the nucleolus. Here, we examined the molecular mechanisms that localize UBF to the nucleolus. We found that the first HMG box (HMG box 1), the linker region (LR), and the AR cooperatively regulate the nucleolar localization of UBF1. We demonstrated that the AR intramolecularly associates with and attenuates the DNA binding activity of HMG boxes and confers the structured DNA preference to HMG box 1. In contrast, the LR was found to serve as a nuclear localization signal and compete with HMG boxes to bind the AR, permitting nucleolar localization of UBF1. The LR sequence binds DNA and assists the stable chromatin binding of UBF. We also showed that the phosphorylation status of the AR does not clearly affect the localization of UBF1. Our results strongly suggest that associations of the AR with HMG boxes and the LR regulate UBF nucleolar localization.

Function of Nup98 subtypes and their fusion proteins, Nup98-TopIIß and Nup98-SETBP1 in nuclear-cytoplasmic transport.

Nup98 is a component of the nuclear pore complex. The nup98-fusion genes derived by chromosome translocations are involved in hematopoietic malignancies. Here, we investigated the functions of Nup98 isoforms and two unexamined Nup98-fusion proteins, Nup98-TopIIß and Nup98-SETBP1. We first demonstrated that two Nup98 isoforms are expressed in various mouse tissues and similarly localized in the nucleus and the nuclear envelope. We also showed that Nup98-TopIIß and Nup98-SETBP1 are localized in the nucleus and partially co-localized with full-length Nup98 and a nuclear export receptor XPO1. We demonstrated that Nup98-TopIIß and Nup98-SETBP1 negatively regulate the XPO1-mediated protein export. Our results will contribute to the understanding of the molecular mechanism by which the Nup98-fusion proteins induce tumorigenesis.

Efficient DNA binding of NF-κB requires the chaperone-like function of NPM1.

NPM1/nucleophosmin is frequently overexpressed in various tumors, although the oncogenic role of NPM1 remains unclear. Here we revealed the link between NPM1 and nuclear factor-κB (NF-κB), a master regulator of inflammation. We found that NPM1 knockdown decreased NF-κB-mediated transcription of selected target genes by decreasing the recruitment of NF-κB p65 to the gene promoters. NPM1 is directly associated with the DNA binding domain of p65 to enhance its DNA binding activity without being a part of the DNA-NF-κB complex. This result suggests that NF-κB requires the chaperone-like function of NPM1 for DNA binding. Furthermore, we demonstrated that NPM1 was required for efficient inflammatory gene expression induced by tumor necrosis factor alpha (TNF-α) and lipopolysaccharide in fibroblasts and macrophages. The NF-κB-mediated invasion of breast cancer cells was significantly decreased by NPM1 knockdown. Our study suggests a novel mechanistic insight into the NF-κB-mediated transcription and an oncogenic role of NPM1 in both tumor cells and the tumor micro-environment through the regulation of NF-κB.

Functional characterization and efficient detection of Nucleophosmin/NPM1 oligomers.

NPM1/nucleophosmin is a multifunctional and oligomeric phosphoprotein. A number of observations have suggested that changes in the oligomer formation of NPM1 could influence its biological functions, especially its oncogenic functions. To understand the functional meaning of oligomerization of NPM1/nucleophosmin, we have established a novel method to monitor protein oligomerization in cells. We utilized the split synthetic Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) bioluminescence activity and observed the change of NPM1 oligomer levels under various cell culture conditions. Our study provides a method for systematic characterization of NPM1 oligomer formation changes and for screening inhibitors of NPM1 oligomerization.

Tracking adenovirus genomes identifies morphologically distinct late DNA replication compartments.

In adenoviral virions, the genome is organized into a chromatin-like structure by viral basic core proteins. Consequently viral DNAs must be replicated, chromatinized and packed into progeny virions in infected cells. Although viral DNA replication centers can be visualized by virtue of viral and cellular factors, the spatiotemporal regulation of viral genomes during subsequent steps remains to be elucidated. In this study, we used imaging analyses to examine the fate of adenoviral genomes and to track newly replicated viral DNA as well as replication-related factors. We show de novo formation of a subnuclear domain, which we termed Virus-induced Post-Replication (ViPR) body, that emerges concomitantly with or immediately after disintegration of initial replication centers. Using a nucleoside analogue, we show that viral genomes continue being synthesized in morphologically distinct replication compartments at the periphery of ViPR bodies and are then transported inward. In addition, we identified a nucleolar protein Mybbp1a as a molecular marker for ViPR bodies, which specifically associated with viral core protein VII. In conclusion, our work demonstrates the formation of previously uncharacterized viral DNA replication compartments specific for late phases of infection that produce progeny viral genomes accumulating in ViPR bodies.

Regulation of Cellular Dynamics and Chromosomal Binding Site Preference of Linker Histones H1.0 and H1.X.

Linker histones play important roles in the genomic organization of mammalian cells. Of the linker histone variants, H1.X shows the most dynamic behavior in the nucleus. Recent research has suggested that the linker histone variants H1.X and H1.0 have different chromosomal binding site preferences. However, it remains unclear how the dynamics and binding site preferences of linker histones are determined. Here, we biochemically demonstrated that the DNA/nucleosome and histone chaperone binding activities of H1.X are significantly lower than those of other linker histones. This explains why H1.X moves more rapidly than other linker histones in vivo Domain swapping between H1.0 and H1.X suggests that the globular domain (GD) and C-terminal domain (CTD) of H1.X independently contribute to the dynamic behavior of H1.X. Our results also suggest that the N-terminal domain (NTD), GD, and CTD cooperatively determine the preferential binding sites, and the contribution of each domain for this determination is different depending on the target genes. We also found that linker histones accumulate in the nucleoli when the nucleosome binding activities of the GDs are weak. Our results contribute to understanding the molecular mechanisms of dynamic behaviors, binding site selection, and localization of linker histones.

Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system.

C-terminal acidic domain of histone chaperone human NAP1 is an efficient binding assistant for histone H2A-H2B, but not H3-H4.

Nucleosome assembly protein 1 (NAP1) binds both the (H3-H4)2 tetramer and two H2A-H2B dimers, mediating their sequential deposition on DNA. NAP1 contains a C-terminal acidic domain (CTAD) and a core domain that promotes dimer formation. Here, we have investigated the roles of the core domain and CTAD of human NAP1 in binding to H2A-H2B and H3-H4 by isothermal calorimetry and native mass spectrometry and compared them with the roles of yeast NAP1. We show that the hNAP1 and yNAP1 dimers bind H2A-H2B by two different modes: a strong endothermic interaction and a weak exothermic interaction. A mutant hNAP1, but not yNAP1, dimer lacking CTAD loses the exothermic interaction and shows greatly reduced H2A-H2B binding activity. The isolated CTAD of hNAP1 binds H2A-H2B only exothermically with relatively stronger binding as compared with the exothermic interaction observed for the full-length hNAP1 dimer. Thus, the two CTADs in the hNAP1 dimer seem to provide binding assistance for the strong endothermic interaction of the core domain with H2A-H2B. By contrast, in the relatively weaker binding of hNAP1 to H3-H4 as compared with yNAP1, CTAD of hNAP1 has no significant role. To our knowledge, this is the first distinct role identified for the hNAP1 CTAD.

pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA.

Replication of influenza viral genomic RNA (vRNA) is catalyzed by viral RNA-dependent RNA polymerase (vRdRP). Complementary RNA (cRNA) is first copied from vRNA, and progeny vRNAs are then amplified from the cRNA. Although vRdRP and viral RNA are minimal requirements, efficient cell-free replication could not be reproduced using only these viral factors. Using a biochemical complementation assay system, we found a novel activity in the nuclear extracts of uninfected cells, designated IREF-2, that allows robust unprimed vRNA synthesis from a cRNA template. IREF-2 was shown to consist of host-derived proteins, pp32 and APRIL. IREF-2 interacts with a free form of vRdRP and preferentially upregulates vRNA synthesis rather than cRNA synthesis. Knockdown experiments indicated that IREF-2 is involved in in vivo viral replication. On the basis of these results and those of previous studies, a plausible role(s) for IREF-2 during the initiation processes of vRNA replication is discussed.

Reconstitution of human rRNA gene transcription in mouse cells by a complete SL1 complex.

An important characteristic of the transcription of a ribosomal RNA gene (rDNA) mediated by DNA-dependent RNA polymerase (Pol) I is its stringent species specificity. SL1/TIF-IB is a key complex for species specificity, but its functional complex has not been reconstituted. Here, we established a novel and highly sensitive monitoring system for Pol I transcription to reconstitute the SL1 activity in which a transcript harboring a reporter gene synthesized by Pol I is amplified and converted into translatable mRNA by the influenza virus RNA-dependent RNA polymerase. Using this monitoring system, we reconstituted Pol I transcription from the human rDNA promoter in mouse cells by expressing four human TATA-binding protein (TBP)-associated factors (TAFIs) in the SL1 complex. The reconstituted SL1 also re-activated human rDNA transcription in mouse A9 cells carrying an inactive human chromosome 21 that contains the rDNA cluster. Chimeric SL1 complexes containing human and mouse TAFIs could be formed, but these complexes were inactive for human rDNA transcription. We conclude that four human TAFIs are necessary and sufficient to overcome the barrier of species specificity for human rDNA transcription in mouse cells.

Upstream binding factor-dependent and pre-rRNA transcription-independent association of pre-rRNA processing factors with rRNA gene.

The nucleolus is the ribosome biogenesis center. The nucleolar structure is disrupted upon entry into mitosis and is formed in early G1 phase. To understand the molecular mechanisms of nucleolar assembly and disassembly, we have studied the mechanism of association between factors involved in pre-ribosome RNA (rRNA) processing and rRNA gene chromatin (r-chromatin). We found that the pre-rRNA transcription-processing linking factor Nopp140 and pre-rRNA processing factors such as DKC1 and fibrillarin (FBL) associate with r-chromatin during interphase, while Nopp140, DKC1, and FBL were released from r-chromatin in mitosis. The association of these factors with r-chromatin was found to be restored independent of pre-rRNA transcription in early G1 phase, but a mature nucleolar structure was not formed, suggesting that nucleolar assembly can be divided into at least two steps with respect to pre-rRNA transcription. Moreover, we found that the r-chromatin association of Nopp140, DKC1, and FBL was dependent on the transcription factor upstream binding factor (UBF). However, we demonstrated that UBF alone was not sufficient to recruit these pre-rRNA processing factors to r-chromatin. Thus, UBF is necessary but not sufficient for the associations between pre-rRNA processing factors and r-chromatin.