Promyelocytic leukemia (PML) nuclear bodies are protein structures that do not accumulate RNA.

The promyelocytic leukemia (PML) nuclear body (also referred to as ND10, POD, and Kr body) is involved in oncogenesis and viral infection. This subnuclear domain has been reported to be rich in RNA and a site of nascent RNA synthesis, implicating its direct involvement in the regulation of gene expression. We used an analytical transmission electron microscopic method to determine the structure and composition of PML nuclear bodies and the surrounding nucleoplasm. Electron spectroscopic imaging (ESI) demonstrates that the core of the PML nuclear body is a dense, protein-based structure, 250 nm in diameter, which does not contain detectable nucleic acid. Although PML nuclear bodies contain neither chromatin nor nascent RNA, newly synthesized RNA is associated with the periphery of the PML nuclear body, and is found within the chromatin-depleted region of the nucleoplasm immediately surrounding the core of the PML nuclear body. We further show that the RNA does not accumulate in the protein core of the structure. Our results dismiss the hypothesis that the PML nuclear body is a site of transcription, but support the model in which the PML nuclear body may contribute to the formation of a favorable nuclear environment for the expression of specific genes.

Topoisomerase II alpha is associated with the mammalian centromere in a cell cycle- and species-specific manner and is required for proper centromere/kinetochore structure.

A study of the distribution of Topoisomerase II alpha (Topo II) in cells of six tissue culture cell lines, human (HeLa), mouse (L929), rat, Indian muntjac, rat kangaroo (PTK-2), and wallaby revealed the following features: (1) There is a cell cycle association of a specific population of Topo II with the centromere. (2) The centromere is distinguished from the remainder of the chromosome by the intensity of its Topo II reactivity. (3) The first appearance of a detectable population of Topo II at the centromere varies between species but is correlated with the onset of centromeric heterochromatin condensation. (4) Detectable centromeric Topo II declines at the completion of cell division. (5) The distribution pattern of Topo II within the centromere is species- and stage-specific and is conserved only within the kinetochore domain. In addition, we report that the Topo II inhibitor ICRF-193 can prevent the normal accumulation of Topo II at the centromere. This results in the disruption of chromatin condensation sub-adjacent to the kinetochore as well as the perturbation of kinetochore structure. Taken together, our studies indicate that the distribution of Topo II at the centromere is unlike that reported for the remainder of the chromosome and is essential for proper formation of centromere/kinetochore structure.

Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments.

Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF-GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF-GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.

The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.

The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

Direct visualization of a protein nuclear architecture.

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.

Organization of highly acetylated chromatin around sites of heterogeneous nuclear RNA accumulation.

Histones found within transcriptionally competent and active regions of the genome are highly acetylated. Moreover, these highly acetylated histones have very short half-lives. Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids. Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities. We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs). The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into "chromonema" fibers. Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm. Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles. The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules. Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb. We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin. An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs.

Association of human DEAD box protein DDX1 with a cleavage stimulation factor involved in 3'-end processing of pre-MRNA.

DEAD box proteins are putative RNA helicases that function in all aspects of RNA metabolism, including translation, ribosome biogenesis, and pre-mRNA splicing. Because many processes involving RNA metabolism are spatially organized within the cell, we examined the subcellular distribution of a human DEAD box protein, DDX1, to identify possible biological functions. Immunofluorescence labeling of DDX1 demonstrated that in addition to widespread punctate nucleoplasmic labeling, DDX1 is found in discrete nuclear foci approximately 0.5 microm in diameter. Costaining with anti-Sm and anti-promyelocytic leukemia (PML) antibodies indicates that DDX1 foci are frequently located next to Cajal (coiled) bodies and less frequently, to PML bodies. Most importantly, costaining with anti-CstF-64 antibody indicates that DDX1 foci colocalize with cleavage bodies. By microscopic fluorescence resonance energy transfer, we show that labeled DDX1 resides within a Förster distance of 10 nm of labeled CstF-64 protein in both the nucleoplasm and within cleavage bodies. Coimmunoprecipitation analysis indicates that a proportion of CstF-64 protein resides in the same complex as DDX1. These studies are the first to identify a DEAD box protein associating with factors involved in 3'-end cleavage and polyadenylation of pre-mRNAs.

Nuclear distribution of histone deacetylase: a marker enzyme for the internal nuclear matrix.

Nuclear matrins are proteins that localize to the internal nuclear matrix. In a previous study, we reported that histone deacetylase is a component of the internal matrix, suggesting that histone deacetylase is a nuclear matrin. Here, we demonstrate that the majority of the histone deacetylase activity is associated with the internal nuclear matrices of chicken and trout liver. Thus, the association of the histone deacetylase with the internal nuclear matrix is neither tissue- nor species-specific. Using histone deacetylase as a marker enzyme for the partitioning of the internal nuclear matrix during nuclear fractionations, we show that in contrast to the internal nuclear matrices of trout liver, trout hepatocellular carcinoma and chicken liver, the stability of the chicken erythrocyte internal nuclear matrix is temperature-dependent. Our results support a model that has the histone deacetylase mediating transient interactions between the internal nuclear matrix and chromatin regions undergoing dynamic acetylation, for example transcriptionally active chromatin regions.

A history of total mercury in edible muscle of fish from lakes in northern Canada.

Subsistence fishing has been an important source of food for Native People in northern Canada since prehistoric time. Measurements of the levels of mercury in edible muscle of northern fish have been undertaken for over three decades in efforts to evaluate the risks of consuming northern fish. This report summarizes the data obtained from 7974 fish of 25 species from sites distributed from the Yukon to Labrador. The most abundant species were lake trout, lake whitefish, arctic char, walleye, northern pike and burbot. The question being asked was essentially "Are the fish safe to eat?" The results were used to support decisions on fishing and consumption of fish. They were sorted in several ways, into concentration ranges corresponding to human consumption guidelines, into political jurisdictions and into types of bedrock geology. Overall walleye, northern pike and lake trout, usually exceeded the subsistence consumption guideline of 0.2 microg g-1 total mercury and often exceeded the higher guideline of 0.5 microg g-1 total mercury for commercial sales of fish. Mercury in burbot, another facultative predator, was often lower but several still exceeding a guideline. Arctic char collections were mostly from anadromous populations and these had very low levels of mercury, presumably reflecting marine food sources. Lake whitefish were among the cleanest fish examined with 69 of 81 collections falling in the lowest range. Most collections were from sites in sedimentary rock. However a few sites were in metamorphic, intrusive or volcanic rocks and these, taken together, tended to have a higher proportion of sites in the higher ranges of mercury. These results indicate a widespread problem with mercury in subsistence fisheries for predator species of fish with the problem being most problematic for Nunavut.

Association of ATM activation and DNA repair with induced radioresistance after low-dose irradiation.

Mammalian cells often exhibit a hyper-radiosensitivity (HRS) to radiation doses <20 cGy, followed by increased radioresistance (IRR) at slightly higher doses (∼20-30 cGy). Here, the influence of DNA double-strand break repair (DSBR) on IRR was examined. The failure of Ataxia telangiectasia (AT) cells to undergo IRR reported by others was confirmed. Flow cytometric analysis indicated that normal cells fail to show a measurable increase in serine 1981 phosphorylated AT-mutated (ATM) protein after 10 cGy up to 4 h post irradiation, but a two- to fourfold increase after 25 cGy. Similarly, more proficient reduction of phosphorylated histone H2AX was observed 24 h after 25 cGy than after 10 cGy, suggesting that DSBR is more efficient during IRR than HRS. A direct examination of the consequences of inefficient DNA repair per se (as opposed to ATM-mediated signal transduction/cell cycle responses), by determining the clonogenic survival of cells lacking the DNA repair enzyme polynucleotide kinase/phosphatase, indicated that these cells have a response similar to AT cells, i.e. HRS but no IRR, strongly linking IRR to DSBR.

Compartmentalization of regulatory proteins in the cell nucleus.

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is considered and their implications for regulation through compartmentalization of proteins and genomic DNA are discussed.

Stoichiometric analysis of protein- and nucleic acid-based structures in the cell nucleus.

We describe a method to image selectively the protein-based architecture in the eukaryotic cell nucleus using nitrogen and phosphorus mapping. In addition, we describe a method to determine total mass as well as stoichiometric relationships between protein and RNA. This method is illustrated using particulate structures in the nucleus called interchromatin granules. In so doing, we demonstrate that these granules contain heterogeneous nuclear RNA, and have an average protein and RNA content of 3.094 and 1.672 MDa, respectively. We also tested the sensitivity of phosphorus detection by exogenously applying purified duplex DNA to the surfaces of thin sections, and have shown that structures as small as single molecules of duplex DNA can be detected in situ using these electron spectroscopic imaging techniques.

PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

Modelling the compartmentalization of splicing factors.

Splicing factor (SF) compartments, also known as speckles, are heterogeneously distributed compartments within the nucleus of eukaryotic cells that are enriched in pre-mRNA SFs. We derive a fourth-order aggregation-diffusion model that describes a possible mechanism underlying the organization of SFs into speckles. The model incorporates two hypotheses, namely (1) that self-organization of dephosphorylated SFs, modulated by a phosphorylation-dephosphorylation cycle, is responsible for the formation and disappearance of speckles, and (2) that an underlying nuclear structure plays a major role in the organization of SFs. A linear stability analysis about homogeneous steady-state solutions of the model reveals how the self-interaction among dephosphorylated SFs can result in the onset of spatial patterns. A detailed bifurcation analysis of the model describes how phosphorylation and dephosphorylation modulate the onset of the compartmentalization of SFs.

Multiple functions of dynamic histone acetylation.

Besides its role in organizing nuclear DNA, the nuclear matrix is involved in specific nuclear functions, including replication, transcription, and RNA splicing. It is becoming increasingly evident that nuclear processes are localized to distinct regions in the nucleus. For example, transcriptionally active genes and RNA transcripts are found in discrete transcription foci. Current evidence suggests that nuclear matrix-bound transcriptionally active DNA sequences are in nucleosomes with dynamically acetylated histones. Histone acetylation, which precedes transcription, alters nucleosome and chromatin structure, decondensing the chromatin fibre and making the nucleosomal DNA accessible to transcription factors. Histone acetyltransferase and histone deacetylase, which catalyze this rapid acetylation and deacetylation, are associated with the internal nuclear matrix. We hypothesize that these enzymes play a role in maintaining the association of the active chromatin domains with the internal nuclear matrix at sites of ongoing transcription.

Dynamically acetylated histones of chicken erythrocytes are selectively methylated.

The relationship between histone acetylation and methylation in chicken immature erythrocytes was investigated. Previous studies have shown that transcriptionally active/competent gene-enriched chromatin fragments are enriched in newly methylated histones H3 and H4. Moreover, newly methylated histone H4 is hyperacetylated. Here, we show that dynamically acetylated histone H4 is selectively engaged in ongoing methylation. While sodium butyrate (an inhibitor of histone deacetylase) does not inhibit ongoing histone methylation, it does affect the acetylation state of newly methylated histone H4 when chicken immature erythrocytes are incubated in its presence or absence. Only one rate of acetylation of labelled newly methylated unacetylated histone H4 with a t1/2 of 8 min is observed. Previous studies have shown that the solubility of transcriptionally active/competent gene chromatin fragments in 0.15 M-NaCl is dependent upon the level of acetylated histone species, with induction of hyperacetylation increasing the solubility of this gene chromatin. Here, we show that the low salt solubility of chromatin fragments associated with newly methylated histones H3 and H4 is also dependent upon the level of acetylated histones. These results provide further support for the hypothesis that histones participating in ongoing methylation are associated with transcriptionally active/competent chromatin and suggest that the processes of histone H4 methylation and dynamic acetylation are partially coupled in terminally differentiated erythrocytes.

Acetylation and methylation of histones H3 and H4 in chicken immature erythrocytes are not directly coupled.

The relationship between histone methylation and dynamic histone acetylation was investigated. Previously, we demonstrated in chicken erythrocytes that dynamically acetylated histones H3 and H4 of transcriptionally active gene chromatin were selectively methylated. However, methylation of these histones was not dependent upon their acetylated states. Here, we tested the hypothesis that methylation tags these histones for participation in dynamic acetylation. Using an inhibitor of protein methylation, adenosine dialdehyde, we show that the processes of histone methylation and dynamic acetylation are not directly coupled. Our results suggest that the selective methylation of dynamically acetylated chromatin reflects features of the organization of transcriptionally active gene chromatin.

CBP, a transcriptional coactivator and acetyltransferase.

The CREB binding protein (CBP) was first identified as a protein that specifically binds to the active phosphorylated form of the cyclic-AMP response element binding protein (CREB). CBP was initially defined as a transcriptional coactivator that, as a result of its large size and multiple protein binding domain modules, may function as a molecular scaffold. More recently, an acetyltransferase activity, both of histones and nonhistones, has been found to be essential for transactivation. In this review, we will discuss the current understanding of the acetyltransferase specificity and activity of the CBP protein and how it may function to coactivate transcription. We will also examine the regulation of the CBP histone acetyltransferase activity in the cell cycle, by signal-transduction pathways and throughout development.

The integration of tissue structure and nuclear function.

Living cells can filter the same set of biochemical signals to produce different functional outcomes depending on the deformation of the cell. It has been suggested that the cell may be "hard-wired" such that external forces can mediate internal nuclear changes through the modification of established, balanced, internal cytoskeletal tensions. This review will discuss the potential of subnuclear structures and nuclear chromatin to participate in or respond to transduction of mechanical signals originating outside the nucleus. The mechanical interactions of intranuclear structure with the nuclear lamina will be examined. The nuclear lamina, in turn, provides a structural link between the nucleus and the cytoplasmic and cortical cytoskeleton. These mechanical couplings may provide a basis for regulating gene expression through changes in cell shape.

Nucleosomal histones of transcriptionally active/competent chromatin preferentially exchange with newly synthesized histones in quiescent chicken erythrocytes.

The incorporation of newly synthesized histones among various chromatin fraction isolated from non-replicating cell-cycle-phase-Go chicken immature erythrocytes was investigated. We find that newly synthesized erythroid-specific histone Hl variant H5, is incorporated randomly into chromatin. In contrast, newly synthesized nucleosomal histones H2A, H2A.Z, H2B, H3.3, and H4 are preferentially found in a fraction that is highly enriched in active/competent gene chromatin fragments and depleted in repressed gene chromatin. Moreover, ubiquitinated species of histones H2A and H2B and hyperacetylated species of H4 and H2B, which are complexed to active DNA, are labelled. These observations provide evidence that newly synthesized histones preferentially exchange with the nucleosomal histones of transcriptionally active/component chromatin domains. The results of this study suggest that nucleosomes of active chromatin may be inherently less stable than bulk nucleosomes in vivo and have implications for chromatin remodelling.