The effect of P38 MAP kinase inhibition in a mouse model of influenza.

PURPOSE: Influenza viruses are a common cause of human respiratory infections, resulting in epidemics of high morbidity and mortality. We investigated the effect of a novel mitogen-activated protein kinase (MAPK) inhibitor in vitro and in a murine influenza model to further explore whether p38 MAPK inhibition could reduce viral replication. METHODS: In vitro, the antiviral effect of p38 MAPK inhibitor BCT194 was evaluated in differentiated human bronchial epithelial cells (HBECs); in vivo, female BALB/c mice were infected intranasally with 150 pfu of influenza H1N1 A/Puerto Rico/8/34 and treated with BCT197 (a closely related p38 MAPK inhibitor with an IC50 value of<1 µM, currently in clinical testing), dexamethasone or oseltamivir (Tamiflu) starting 24 h post infection. Body weight, bronchoalveolar lavage cells, cytokines, total protein and lactate dehydrogenase as well as serum cytokines were measured; a subset of animals was evaluated histopathologically.Results/Key findings. p38MAP kinase inhibition with BCT194 had no impact on influenza replication in HBECs. When examining BCT197 in vivo, and comparing to vehicle-treated animals, reduced weight loss, improvement in survival and lack of impaired viral control was observed at BCT197 concentrations relevant to those being used in clinical trials of acute exacerbations of chronic obstructive pulmonary disease; at higher concentrations of BCT197 these effects were reduced. CONCLUSIONS: Compared to vehicle treatment, BCT197 (administered at a clinically relevant concentration) improved outcomes in a mouse model of influenza. This is encouraging given that the use of innate inflammatory pathway inhibitors may raise concerns of negative effects on infection regulation.

Sequential assembly of the nucleotide excision repair factors in vivo.

Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.

Nuclear domains enriched in RNA 3'-processing factors associate with coiled bodies and histone genes in a cell cycle-dependent manner.

Nuclear domains, called cleavage bodies, are enriched in the RNA 3'-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle-dependent expression of histone genes.

Coiled bodies are predisposed to a spatial association with genes that contain snoRNA sequences in their introns.

Small nucleolar RNAs (snoRNAs) constitute a group of more than 100 different stable RNA molecules that are found concentrated in the nucleolus where they are involved in the maturation of ribosomal RNA. Most snoRNAs are not produced from their own genes but are encoded in the introns of other genes, referred to as snoRNA host genes. Little is known about the mechanisms by which the snoRNAs are produced from introns and how the snoRNAs mature to functional snoRNP complexes in the nucleolus. One class of intron-encoded snoRNAs binds with high specificity to the protein fibrillarin which is found concentrated in the nucleolus, but also in small nuclear domains known as coiled bodies. It has become clear that genes that code for small stable RNAs, e.g., U1, U2 snRNA, and the U3 snoRNA, are often found adjacent to coiled bodies. High concentrations of transcription factors and RNA processing factors in and around coiled bodies indicate that they may be involved in the expression of the adjacent genes. In order to investigate whether coiled bodies could play a role in the synthesis of intron-encoded snoRNAs the distribution of coiled bodies was studied relative to three different snoRNA host genes, i.e., hsc70, RPS3, UHG. All three were found adjacent to coiled bodies at significantly high frequencies (11-19%), compared to control sequences (0-2%), to conclude a preferential association between the snoRNA host genes and coiled bodies. This association could point to a possible role for coiled bodies in the synthesis and/or maturation of snoRNAs. An involvement in snoRNA production could explain the presence of transcription factors, splicing factors, and fibrillarin in coiled bodies.

Spatial organization of four hnRNP proteins in relation to sites of transcription, to nuclear speckles, and to each other in interphase nuclei and nuclear matrices of HeLa cells.

RNA polymerase II transcripts are complexed with heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. These proteins are involved in several aspects of the maturation and transport of hnRNA. We performed a detailed study of the spatial distribution of four hnRNP proteins (hnRNP C, I, L, and U) in HeLa nuclei, using immunofluorescent labeling and confocal microscopy. Despite the fact that hnRNP proteins have been shown to coimmunoprecipitate, a hallmark of hnRNP proteins, we find that hnRNP C, I, and L have a spatial nuclear distribution that is not related to that of hnRNP U. We also examined the distribution of hnRNP proteins in relation to that of nascent transcripts. The four hnRNP proteins that we examined are not enriched at sites of RNA synthesis. Using antibodies against the nuclear poly(A)-binding protein (PAB II) we investigated the relationship between the distribution of hnRNP proteins and that of nuclear domains (nuclear speckles) that are enriched in splicing factors, poly(A)+RNA, and PAB II. We found that the four hnRNP proteins are not enriched in these domains. This indicates that the poly(A)+RNA, present in high concentration in speckles, is not complexed with these hnRNP proteins. This is in agreement with the notion that poly(A)+RNA in speckles is different from ordinary hnRNA. Previously, we have shown that hnRNP proteins are the major protein components of the fibrogranular internal nuclear matrix (K. A. Mattern et al. (1996) J. Cell. Biochem. 62, 275-289; K. A. Mattern et al. (1997) J. Cell. Biochem. 65, 42-52). We observed that in nuclear matrices the spatial distributions of the four hnRNP proteins, like that of nascent RNA and PAB II, are essentially the same as observed in intact nuclei. Moreover, also in nuclear matrix preparations, like in intact nuclei, nascent RNA and PAB II have spatial distributions that differ from those of hnRNP proteins. Our results are compatible with the notion that hnRNP proteins are able to form complexes of many different, probably overlapping, compositions.

Nuclear neighbours: the spatial and functional organization of genes and nuclear domains.

It is becoming clear that the cell nucleus is not only organized in domains but that these domains are also organized relative to each other and to the genome. Specific nuclear domains, enriched in different proteins and RNAs, are often found next to each other and next to specific gene loci. Several lines of investigation suggest that nuclear domains are involved in facilitating or regulating gene expression. The emerging view is that the spatial relationship between different domains and genes on different chromosomes, as found in the nucleolus, is a common organizational principle in the nucleus, to allow an efficient and controlled synthesis and processing of a range of gene transcripts.

Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle.

PTF (PSE-binding transcription factor) activates transcription of snRNA and related genes. We investigated its distribution in HeLa nuclei by immunofluorescence, and found it spread throughout the nucleoplasm in small foci. In some cells, PTF is also concentrated in one, or very few, discrete regions (diameter approximately 1.3 micron) that appear during G1 phase and disappear in S phase. Oct1, a transcription factor that interacts with PTF, is also enriched in these domains; RNA polymerase II, TBP and Sp1 are also present. Each domain typically contains 2 or 3 transcription 'factories' where Br-UTP is incorporated into nascent transcripts. Accordingly, we have christened this region the Oct1/PTF/transcription (OPT) domain. It colocalizes with some, but not all, PIKA domains. It is distinct from other nuclear domains, including coiled bodies, gemini bodies, PML bodies and the perinucleolar compartment. A small region on chromosome 6 (band 6p21) containing only approximately 30 Mbp DNA, and chromosomes 6 and 7, associate with the domain significantly more than other chromosomes. The domains may act like nucleoli to bring particular genes on specific chromosomes together to a region where the appropriate transcription and processing factors are concentrated, thereby facilitating the expression of those genes.

Coiled bodies and U2 snRNA genes adjacent to coiled bodies are enriched in factors required for snRNA transcription.

A significant percentage of the gene clusters that contain the human genes for U1 small nuclear RNA (snRNA) or for U2 snRNA have been found associated with small nuclear domains, known as coiled bodies. We show here, by immunofluorescent labeling of human cells, that coiled bodies are enriched in factors required for the transcription of these snRNA genes. The 45-kDa gamma-subunit of the transcription factor, proximal element sequence-binding transcription factor (PTF), which is specific for the snRNA genes, was found in high concentrations in coiled bodies, along with the general transcription factor TATA-box binding protein and a subset of RNA polymerase II. We show that the transcription factors and RNA polymerase II are concentrated in irregularly shaped domains that not only overlap with coiled bodies but also extend to their immediate surroundings. Fluorescent in situ hybridization showed that these domains can overlap with U2 snRNA genes adjacent to coiled bodies. In addition, we found the domains to contain newly synthesized RNA, visualized by 5-bromo-uridine triphosphate labeling. Our data suggest that coiled bodies are involved in the expression of snRNA genes, which leads us to propose the model that coiled bodies are associated with snRNA genes to facilitate and regulate their transcription. These findings point to a general principle of higher order organization of gene expression in the nucleus.

A subset of poly(A) polymerase is concentrated at sites of RNA synthesis and is associated with domains enriched in splicing factors and poly(A) RNA.

We have performed a detailed study of the spatial distribution of a set of mRNA 3' processing factors in human T24 cells. A key enzyme in RNA 3' processing, poly(A) polymerase (PAP), was found in the cytoplasm and throughout the nucleus in a punctated pattern. A subset of the various isoforms of PAP was specifically concentrated at sites of RNA synthesis in the nucleoplasm. Additionally, the other factors necessary for RNA 3' processing, such as CstF, CPSF, and PABII, were also found at these transcription sites. Our data show that the set of 3' processing factors that are presumed to be necessary for most RNA 3' cleavage and polyadenylation is indeed found at sites of RNA synthesis in the nucleoplasm. Furthermore, sites of RNA synthesis that are particularly enriched in both PAP and PABII are found at the periphery of irregularly shaped domains, called speckles, which are known to contain high concentrations of splicing factors and poly(A) RNA. Disruption of RNA 3' processing by the drug 9-beta-D-arabinofuranosyladenine caused the speckles to break up into smaller structures. These findings indicate that there is a spatial and structural relationship between 3' processing and the nuclear speckles. Our studies reveal a complex and distinct organization of the RNA 3' processing machinery in the mammalian cell nucleus.

RING1 is associated with the polycomb group protein complex and acts as a transcriptional repressor.

The Polycomb (Pc) protein is a component of a multimeric, chromatin-associated Polycomb group (PcG) protein complex, which is involved in stable repression of gene activity. The identities of components of the PcG protein complex are largely unknown. In a two-hybrid screen with a vertebrate Pc homolog as a target, we identify the human RING1 protein as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. Here, we show that RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). Also, RING1 colocalizes with these vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. Finally, we show that RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. Our findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor.

PML-containing nuclear bodies: their spatial distribution in relation to other nuclear components.

The PML protein is a human growth suppressor concentrated in 10 to 20 nuclear bodies per nucleus (PML bodies). Disruption of the PML gene has been shown to be related to acute promyelocytic leukaemia (APL). To obtain information about the function of PML bodies we have investigated the 3D-distribution of PML bodies in the nucleus of T24 cells and compared it with the spatial distribution of a variety of other nuclear components, using fluorescence dual-labeling immunocytochemistry and confocal microscopy. Results show that PML bodies are not enriched in nascent RNA, the splicing component U2-snRNP, or transcription factors (glucocorticoid receptor, TFIIH, and E2F). These results show that PML bodies are not prominent sites of RNA synthesis or RNA splicing. We found that a large fraction of PML bodies (50 to 80%) is closely associated with DNA replication domains during exclusively middle-late S-phase. Furthermore, in most cells that we analysed we found at least one PML body was tightly associated with a coiled body. In the APL cell line NB4, the PML gene is fused with the RAR alpha gene due to a chromosomal rearrangement. PML bodies have disappeared and the PML antigen, i.e., PML and the PML-RAR fusion protein, is dispersed in a punctated pattern throughout the nucleoplasm. We showed that in NB4 cells the sites that are rich in PML antigen significantly colocalize with sites at which nascent RNA accumulates. This suggests that, in contrast to non-APL cells, in NB4 cells the PML antigen is associated with sites of transcription. The implications of these findings for the function of PML bodies are consistent with the idea that PML bodies are associated with specific genomic loci.

The RNA 3' cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA.

The cleavage stimulation factor (CstF), and the cleavage and polyadenylation specificity factor (CPSF) are necessary for 3'-terminal processing of polyadenylated mRNAs. To study the distribution of 3' cleavage factors in the nuclei of human T24 cells, monoclonal antibodies against the CstF 64 kDa subunit and against the CPSF 100 kDa subunit were used for immunofluorescent labelling. CstF 64 kDa and CPSF 100 kDa were distributed in a fibrogranular pattern in the nucleoplasm and, in addition, were concentrated in 1-4 bright foci. Double immunofluorescence labelling experiments revealed that the foci either overlapped with, or resided next to, a coiled body. Inhibition of transcription with alpha-amanitin or 5,6-dichloro-beta-D-ribofuranosyl-benzimidazole (DRB) resulted in the complete co-localization of coiled bodies and foci containing 3' cleavage factors. Electron microscopy on immunogold double-labelled cells revealed that the foci represent compact spherical fibrous structures, we named 'cleavage bodies', intimately associated with coiled bodies. We found that approximately 20% of the cleavage bodies contained a high concentration of newly synthesized RNA, whereas coiled bodies were devoid of nascent RNA. Our results suggest that the cleavage bodies that contain RNA are those that are adjacent to a coiled body. These findings reveal a dynamic and transcription-dependent interaction between different subnuclear domains, and suggest a relationship between coiled bodies and specific transcripts.

Nuclear domains involved in RNA synthesis, RNA processing, and replication.

Two main principles of nuclear organization have been outlined on the basis of contributions by many research groups in recent years. The first principle is that interphase chromosomes occupy discrete territories in the nucleus, with no intermingling of the DNA from different chromosomes. Within a chromosome territory the DNA is organized in chromatin fibers at several levels of folding, that meander through the territory. Transcription and replication take place at the surface of these higher order chromatin fibers, probably on locally unfolded DNA templates. The second principle is that different types of nuclear domains are associated with several specific gene loci. This holds for clusters of interchromatin granules, coiled bodies, RNA 3'-cleavage factor-containing nuclear bodies (cleavage bodies) and probably PML-containing nuclear bodies. These domains may play an important role in the spatial arrangement of genes in the interphase nucleus. Despite these new insights, our knowledge of the function of many nuclear compartments and the molecular interactions responsible for the dynamic organization of a compartmentalized nucleus is still in its infancy.

Nuclear domains and the nuclear matrix.

This overview describes the spatial distribution of several enzymatic machineries and functions in the interphase nucleus. Three general observations can be made. First, many components of the different nuclear machineries are distributed in the nucleus in a characteristic way for each component. They are often found concentrated in specific domains. Second, nuclear machineries for the synthesis and processing of RNA and DNA are associated with an insoluble nuclear structure, called nuclear matrix. Evidently, handling of DNA and RNA is done by immobilized enzyme systems. Finally, the nucleus seems to be divided in two major compartments. One is occupied by compact chromosomes, the other compartment is the space between the chromosomes. In the latter, transcription takes place at the surface of chromosomal domains and it houses the splicing machinery. The relevance of nuclear organization for efficient gene expression is discussed.

Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus.

Several nuclear activities and components are concentrated in discrete nuclear compartments. To understand the functional significance of nuclear compartmentalization, knowledge on the spatial distribution of transcriptionally active chromatin is essential. We have examined the distribution of sites of transcription by RNA polymerase II (RPII) by labeling nascent RNA with 5-bromouridine 5'-triphosphate, in vitro and in vivo. Nascent RPII transcripts were found in over 100 defined areas, scattered throughout the nucleoplasm. No preferential localization was observed in either the nuclear interior or the periphery. Each transcription site may represent the activity of a single gene or, considering the number of active pre-mRNA genes in a cell, of a cluster of active genes. The relation between the distribution of nascent RPII transcripts and that of the essential splicing factor SC-35 was investigated in double labeling experiments. Antibodies against SC-35 recognize a number of well-defined, intensely labeled nuclear domains, in addition to labeling of more diffuse areas between these domains (Spector, D. L., X. -D. Fu, and T. Maniatis. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:3467-3481). We observe no correlation between intensely labeled SC-35 domains and sites of pre-mRNA synthesis. However, many sites of RPII synthesis colocalize with weakly stained areas. This implies that contranscriptional splicing takes place in these weakly stained areas. These areas may also be sites where splicing is completed posttranscriptionally. Intensely labeled SC-35 domains may function as sites for assembly, storage, or regeneration of splicing components, or as compartments for degradation of introns.