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PubMed2002

Herpes simplex virus 1 ICP0 co-localizes with a SUMO-specific protease.

Early during infection, the herpes simplex regulatory protein ICP0 promotes the proteasome-dependent degradation of a number of cellular proteins and the loss of a number of SUMO-1-modified protein isoforms, including PML. Recently, ICP0 has been shown to induce the accumulation of conjugated ubiquitin and function as a ubiquitin E3 ligase. However, certain aspects of the biochemistry, cell biology and the links between SUMO-1 conjugation/deconjugation and protein degradation remain unclear. For example, it is not currently known whether SUMO-1 deconjugation is a prerequisite for ubiquitination or degradation and, if so, by what mechanism this may occur. To help address these questions, a SUMO-specific protease (SENP1) was cloned and its expression and localization in relation to ICP0 examined. A cell line was established which constitutively expresses SUMO-1 to facilitate studies of localization and biochemistry. SENP1 localized to the nucleus mainly in discrete subdomains, a subset of which co-localized with the PML bodies. Both ICP0 and SENP1 protease promoted the loss of SUMO-1 from the nucleus, observed both for the endogenous species and the cell line expressing the epitope-tagged SUMO-1. The tagged SUMO-1 was recruited into high molecular mass conjugates in the cell line, and expression of SENP1 promoted loss of these species, including the modified species of PML. Finally, in co-transfection experiments ICP0 promoted the recruitment of SENP1 to nuclear domains, a result which was also observed early during infection. The significance of these findings is discussed in relation to the function of ICP0.

Amino Acid Sequence↗
PubMed2006

Actin-based modeling of a transcriptionally competent nuclear substructure induced by transcription inhibition.

During transcription inactivation, the nuclear bodies in the mammalian cells often undergo reorganization. In particular, the interchromatin granule clusters, or IGCs, become colocalized with RNA polymerase II (RNAP II) upon treatment with transcription inhibitors. This colocalization has also been observed in untreated but transcriptionally inactive cells. We report here that the reorganized IGC domains are unique substructure consisting of outer shells made of SC35, ERK2, SF2/ASF, and actin. The apparently hollow holes of these domains contain clusters of RNAP II, mostly phosphorylated, and the splicing regulator SMN. This class of complexes are also the sites where prominent transcription activities are detected once the inhibitors are removed. Furthermore, actin polymerization is required for reorganization of the IGCs. In connection with this, immunoprecipitation and immunostaining experiments showed that nuclear actin is associated with IGCs and the reorganized IGC domains. The study thus provides further evidence for the existence of an actin-based nuclear skeleton structure in association with the dynamic reorganization processes in the nucleus. Overall, our data suggest that mammalian cells have adapted to utilize the reorganized, uniquely shaped IGC domains as the temporary storage sites of RNAP II transcription machineries in response to certain transient states of transcription inactivation.

Actins↗
PubMed2004

Nuclear structure-associated TIF2 recruits glucocorticoid receptor and its target DNA.

Assembly of multi-protein complexes on promoter and enhancer elements is a prerequisite for onset of gene transcription. At the beginning of this process, transcription factors are thought to act as nucleating centers for complex formation through the binding of their target DNA sequences, and thereafter recruit coactivators. Here, we investigated this process of assembly by determining the distribution of the glucocorticoid receptor (GR) and its coactivator, TIF2. Both endogenously and ectopically expressed TIF2 were shown to form foci in the nucleus, and GR could be recruited to the TIF2 foci upon GR agonist but not antagonist treatment. Moreover, we show that the coactivators, p300 and PCAF, are also recruited to the TIF2 foci. The TIF2 foci could recruit GR carrying a microinjected GR responsive element. We propose that TIF2 provides a nuclear compartment that allows the assembly of multi-protein complexes required for GR-mediated gene activation.

Animals↗
PubMed2004

Nuclear stress granules: the awakening of a sleeping beauty?

Nuclear stress granules are subnuclear compartments that form in response to heat shock and other stress stimuli. Although many components of nuclear stress granules have been identified, including HSF1 and pre-mRNA processing factors, their function remains a mystery. A paper in this issue describes the stress-induced transcriptional activation of one of the nuclear stress granule target sites, a heterochromatic region that has been considered silent (Jolly et al., 2004). These intriguing findings will certainly give the research of these structures a new twist.

Animals↗
PubMed2003

P300 transcriptional repression is mediated by SUMO modification.

p300 and CREB binding protein can both activate and repress transcription. Here, we locate the CRD1 transcriptional repression domain between residues 1017 and 1029 of p300. This region contains two copies of the sequence psiKxE that are modified by the ubiquitin-like protein SUMO-1. Mutations that reduce SUMO modification increase p300-mediated transcriptional activity and expression of a SUMO-specific protease or catalytically inactive Ubc9 relieved repression, demonstrating that p300 repression was mediated by SUMO conjugation. SUMO-modified CRD1 domain bound HDAC6 in vitro, and p300 repression was relieved by histone deacetylase inhibition and siRNA-mediated ablation of HDAC6 expression. These results reveal a mechanism controlling p300 function and suggest that SUMO-dependent repression is mediated by recruitment of HDAC6.

Amino Acid Sequence↗
PubMed2003

Stress-induced transcription of satellite III repeats.

Exposure of mammalian cells to stress induces the activation of heat shock transcription factor 1 (HSF1) and the subsequent transcription of heat shock genes. Activation of the heat shock response also correlates with a rapid relocalization of HSF1 within a few nuclear structures termed nuclear stress granules. These stress-induced structures, which form primarily on the 9q12 region in humans through direct binding of HSF1 to satellite III repeats, do not colocalize with transcription sites of known hsp genes. In this paper, we show that nuclear stress granules correspond to RNA polymerase II transcription factories where satellite III repeats are transcribed into large and stable RNAs that remain associated with the 9q12 region, even throughout mitosis. This work not only reveals the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure, but also provides evidence for a transcriptional activity within a locus considered so far as heterochromatic and silent.

Acetylation↗
PubMed2006

Transcriptional regulation is affected by subnuclear targeting of reporter plasmids to PML nuclear bodies.

Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.

Cell Nucleus Structures↗
PubMed2001

The glucocorticoid receptor interacting protein 1 (GRIP1) localizes in discrete nuclear foci that associate with ND10 bodies and are enriched in components of the 26S proteasome.

The glucocorticoid receptor interacting protein-1 (GRIP1) is a member of the steroid receptor coactivator (SRC) family of transcriptional regulators. Green fluorescent protein (GFP) fusions were made to full-length GRIP1, and a series of GRIP1 mutants lacking the defined regulatory regions and the intracellular distribution of these proteins was studied in HeLa cells. The distribution of GRIP1 was complex, ranging from diffuse nucleoplasmic to discrete intranuclear foci. Formation of these foci was dependent on the C-terminal region of GRIP1, which contains the two characterized transcriptional activation domains, AD1 and AD2. A subpopulation of GRIP1 foci associate with ND10s, small nuclear bodies that contain several proteins including PML, SP100, DAXX, and CREB-binding protein (CBP). Association with the ND10s is dependent on the AD1 of GRIP1, a region of the protein previously described as a CBP-interacting domain. The GRIP1 foci are enriched in components of the 26S proteasome, including the core 20S proteasome, PA28alpha, and ubiquitin. In addition, the irreversible proteasome inhibitor lactacystin induced an increase in the total fluorescence intensity of the GFP-GRIP1 expressing cells, demonstrating that GRIP1 is degraded by the proteasome. These findings suggest the intriguing possibility that degradation of GRIP1 by the 26S proteasome may be a key component of its regulation.

Acetylcysteine↗
PubMedDate not supplied

[Splicing. 2. Splicing of mRNA in the cell nucleus].

The mosaic structure of genes is shown for three out of four known types of RNAs, namely transfer, ribosomal and messenger. At least three different splicing mechanisms are involved in maturation of transcripts of these genes, two of them had been described in the previous review. Well-developed effective in vitro splicing systems permit to provide a scheme for the nuclear pre-mRNA maturation processes which take place presumably in specific ribonucleoprotein complex particles. Alternative splicing character of these pre-mRNAs, yields in increased variety of the encoded proteins.

Animals↗
PubMed2001

Cellular proteins localized at and interacting within ND10/PML nuclear bodies/PODs suggest functions of a nuclear depot.

ND10, PML bodies or PODs have become the defining nuclear structure for a highly complex protein complement involved in cell activities such as aging, apoptosis, the cell cycle, stress response, hormone signaling, transcriptional regulation and development. ND10 are present in many but not all cell types and are not essential for cell survival. Here, we review the cellular proteins found in ND10, their few known interactions and their contribution to the ND10 structure per se and to functions elsewhere in the nucleus. The discrepancy between the functions of the ND10 proteins and the nonessential nature of the structure in which they are aggregated at their highest concentrations leads to the conclusion that the proteins function elsewhere. The regulated recruitment of specific proteins into ND10 as well as their controlled release upon external induced stress points to a regulated nuclear depot function for ND10. These nuclear depot functions seem important as nuclear defense against viral attack and other external insults.

Animals↗
PubMed2000

Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes.

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.

Animals↗
PubMed1979

The use of DNA-cellulose for analyzing histone-DNA interactions. Discovery of nucleosome-like histone binding to single-stranded DNA.

In this report, we introduce the use of DNA-cellulose chromatography for evaluating the strength of binding of histones to DNA under a variety of conditions. We have found that histones added directly to DNA-cellulose at physiological salt concentrations bind relatively weakly, with all histones eluting together at about 0.5 M NaCl when a salt gradient is applied. However, much tighter binding of the four nucleosomal histones to DNA-cellulose is obtained if gradual histone-DNA reconstitution conditions are used. In this case, the binding of histones H2A, H2B, H3, and H4 to DNA-cellulose closely resembles their binding to native chromatin. The nativeness of the binding is indicated both by the distinctive sodium chloride elution profile of these histones from DNA-cellulose and by their relative resistance to trypsin digestion when DNA-bound. The binding to DNA-cellulose of histones H2A, H2B, H3, and H4, which have had the first 20 to 30 amino acid residues removed from their NH2 termini, is indistinguishable from the binding to DNA-cellulose of the same intact histones, as judged by their salt elution profile. Thus, even though the NH2 termini contain 40 to 50% of the positively charged amino acid residues (thought to interact with the DNA backbone), a major contribution to the DNA binding comes from the remainder of the histone molecule. Finally, we have discovered that histones can form a "nucleosome-like" complex on single-stranded DNA. The same complex does not appear to form on RNA. Histones H3 and H4 play a predominant role in organizing this histone complex on single-stranded DNA, as they do on double-stranded DNA in normal nucleosomes. We suggest that, in the cell nucleus, nucleosomal structures may form transiently on single strands of DNA, as DNA and RNA polymerases traverse DNA packaged by histones.

Animals↗
PubMed2001

Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina.

The number and complexity of genes encoding nuclear lamina proteins has increased during metazoan evolution. Emerging evidence reveals that transcriptional repressors such as the retinoblastoma protein, and apoptotic regulators such as CED-4, have functional and dynamic interactions with the lamina. The discovery that mutations in nuclear lamina proteins cause heritable tissue-specific diseases, including Emery-Dreifuss muscular dystrophy, is prompting a fresh look at the nuclear lamina to devise models that can account for its diverse functions and dynamics, and to understand its enigmatic structure.

Animals↗