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PubMed1983

Association of thyroid hormone receptors with chromatin.

A large body of circumstantial evidence indicates that receptors located in nuclei of T3 responsive tissues represent a site of initiation of thyroid hormone action at the cellular level. Partial characterization of T3 receptors indicates that these proteins are monomeric structures in nuclei and are chromatin-associated non-histone proteins. Treatment of rat liver nuclei with either pancreatic DNase I or micrococcal nuclease releases T3 receptors from nuclei in two forms: a predominant (95 400 Mr; 5.5-6.0S) and a minor (265 000-365 000 Mr; 12.5S) nucleoprotein complex. Similar structures are excised from rat kidney, brain, and heart nuclei and from GH1 pituitary cell nuclei by micrococcal nuclease digestion. These endonuclease-excised receptor-containing complexes are significantly larger than the salt-extracted receptor (50 000 Mr; 3.5S). The presence of DNA and other non-receptor proteins in these structures indicates that T3 receptors probably function within multimeric complexes in vivo. Although T3 receptors appear to be associated with DNA between nucleosomes, i.e. linker DNA, it is not entirely clear whether all or only a fraction of T3 receptors interact with nucleosomal components. The 12.5S receptor-containing nucleoprotein complex may represent T3 receptors in association with linker DNA and nucleosomal components. T3 receptors do not appear to be uniformly distributed to all chromatin fractions, but are associated with structures having characteristics of transcriptionally active chromatin. They are found in a region of chromatin which is enriched in RNA polymerase activity, rapidly labeled RNA and non-histone proteins, and depleted of histone Hl. This region is also highly sensitive to both micrococcal nuclease and pancreatic DNase I digestion. The association of receptors with transcriptionally active chromatin, however, must be considered provisional until additional details of the precise receptor-chromatin interaction have been established. The recent demonstration of a 20-fold increase in a specific hepatic mRNA four hours following administration of T3 to hypothyroid rats indicates that thyroid hormone potentially has very rapid effects on hepatic gene expression. However, significant changes in nuclear protein phosphorylation, nuclear protein composition, and chromatin structure have not been detected within this four-hour period. Thus, effects of T3 on hepatic gene expression are brought about by local and presumably subtle changes in nuclear function.

Animals↗

PubMed1996

DNA-binding properties of the yeast transcriptional activator, Gcr1p.

In Saccharomyces cerevisiae the GCRI gene product is required for high-level expression of genes encoding glycolytic enzymes. In this communication, we extend our analysis of the DNA binding properties of Gcr1p. The DNA-binding domain of Gcr1p binds DNA with high affinity. The apparent dissociation constant of the Gcr1p DNA-binding domain for one of its specific binding sites (TTTCAGCTTCCTCTAT) is 2.9 x 10(-10) M. However, competition experiments showed that Gcr1p binds this site in vitro with a low degree of specificity. We measured a 33-fold difference between the ability of specific competitor and DNA of random sequence to inhibit the formation of nucleoprotein complexes between Gcr1p and a radiolabeled DNA probe containing its binding site. DNA band-shift experiments, utilizing probes of constant length in which the positions of Gcr1p-binding sites are varied relative to the ends, indicated that Gcr1p-DNA nucleoprotein complexes contain bent DNA. The implications of these findings in terms of the combinatorial interactions that occur at the upstream activating sequence elements of genes encoding glycolytic enzymes are discussed.

Base Sequence↗

PubMed2006

DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities.

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA-protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12microM porphyrin induced almost complete cell killing after 30min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.

DNA↗

PubMed1999

Connections between virus movement, macromolecular signaling and assimilate allocation.

Studies originating with plant viruses led to the concept that plasmodesmata potentiate the cell-to-cell trafficking of viral and endogenous proteins and nucleoprotein complexes. In this article, we develop the theme that, at the tissue/organ level, cell-to-cell trafficking of information molecules enables non-cell-autonomous control over a range of processes, whereas at the organismal level, the phloem serves as an information superhighway. The capacity to deliver proteins and nucleoprotein complexes, over long distances, allowed for the development of a viral surveillance/resistance mechanism, as well as the integration of processes at the whole-plant level.

Biological Transport↗

PubMed2002

Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins.

Transcription by RNA polymerase can stimulate localized DNA supercoiling in Escherichia coli. In vivo, there is extensive experimental support for a "twin-domain" model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase complex and negative supercoils are formed behind it. Negative supercoils accumulate in the template DNA because the positive supercoils are preferentially removed by cellular topoisomerase action. Yet, in vitro, clear and convincing support for the twin-domain mechanism has been lacking. In this article, we reconcile this inconsistency by showing that, in a defined in vitro system with plasmid DNA templates, a variety of sequence-specific DNA-binding proteins, such as the bacteriophage lambda O replication initiator or the E. coli lactose or galactose repressors, strikingly stimulate transcription-coupled DNA supercoiling. We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase. Our data are most consistent with a model in which specific DNA-binding proteins facilitate a twin-domain mechanism to enhance DNA supercoiling during transcription. More precisely, we suggest that some nucleoprotein complexes, perhaps those that contain sharply bent DNA, can form barriers that impede the diffusion and merger of independent chromosomal supercoil domains. Localization of DNA supercoils by nucleoprotein complexes may serve as a general mechanism for modulating DNA transactions that are sensitive to DNA superhelicity.

Bacteriophage lambda↗

PubMed2004

Formation of nuclear foci of the herpes simplex virus type 1 regulatory protein ICP4 at early times of infection: localization, dynamics, recruitment of ICP27, and evidence for the de novo induction of ND10-like complexes.

Herpes simplex virus type 1 (HSV-1) has an intricate association with cellular nuclear structures known as ND10 or promyelocytic leukemia protein (PML) nuclear bodies. Parental viral genomes initially become juxtaposed to ND10, and then viral replication compartments develop from the ND10-associated genomes. Viral immediate-early (IE) regulatory protein ICP0 colocalizes with ND10 and then induces the degradation of critical ND10 component protein PML and therefore the release and dispersal of other ND10 proteins. The IE transcriptional regulatory protein ICP4 also forms foci at early times of infection, many of which are juxtaposed to ND10 and later develop into replication compartments, indicating that at least some of the initial ICP4 foci contain parental viral genomes. Here we report that the ICP4 foci also contain ICP27 and that their formation occurs extremely rapidly at locations just inside the nuclear envelope. By examining developing plaques or thinly seeded cells infected at high multiplicity, we found evidence to suggest that at least some of the ND10-viral nucleoprotein complex association could be attributed to de novo formation of ND10-like structures in response to incoming viral genomes. The ICP4 complexes associated efficiently with ND10 in cells infected with an ICP0-null mutant virus at high but not at low multiplicity, and the degree of association was reduced by the proteasome inhibitor MG132. Therefore, the interaction between viral nucleoprotein complexes and ND10 is in part due to a dynamic response by the cell. This response is modulated by functional ICP0, and cells that are productively or nonproductively infected in the absence of functional ICP0 can be distinguished by the relative locations of ICP4 foci and ND10 proteins.

Animals↗

PubMed1984

Salt-stable binding of the large T protein to DNA in polyoma virus chromatin.

Nucleoprotein complexes extracted from the nuclei of mouse cells lytically infected with polyoma virus contain an ATPase activity which appears to correspond to that of the viral large T protein, as it exhibits the same characteristic properties; in particular, the activity is extensively inhibited by polyclonal antibodies from animals bearing polyoma tumors (anti-T antigen antibodies) and by monoclonal antibodies against large T. Significant amounts of DNA were immunoprecipitated by adding these antibodies to the nucleoprotein complex, suggesting that the protein is tightly bound to DNA in the viral chromatin. Since one of the monoclonal antibodies quantitatively immunoprecipitated the pulse-labeled replicative intermediates, we conclude that some large T protein remains physically associated with the DNA throughout its replication cycle. After exposure to salt concentrations higher than 1 M KCl, about half of the large T-specific ATPase activity was still observed to co-sediment with 21S form I viral DNA. The observations that the sedimentation coefficient of the salt-stable complexes was shifted to 16S after a limited endonucleolytic digestion, and that both the viral DNA and the ATPase activity were co-precipitated in the presence of polyethylene glycol at high ionic strength, further demonstrated that the protein is engaged in an unusually stable complex with DNA in the viral chromatin.

Adenosine Triphosphatases↗

PubMed1983

Demonstration of stepwise coiling of nucleoprotein in adenovirus core by application of critical point drying.

Mild destruction of a virus particle to observe the organized structure of the nucleoprotein complex in a virion was achieved by application of the critical point drying method. Adenovirus type 12 (ad12) virions have been treated by this method after the particles had been fixed with glutaraldehyde on an electron microscope grid. With 15 min prefixation, the capsids (shells) and the cores were in various stages of unfolding. The core was unfolded in the filamentous structure. The thickness of these filaments was 6.7, 13.3, +23 nm, or more. Some pictures showed that the thicker filaments consisted of super-coiling of two thinner filaments, for example two 6.7-nm filaments coiled up to give the 13.3-nm filaments. This suggests that the nucleoprotein complex of a circular double-stranded DNA and inner proteins of ad12 virus was folded in a stepwise fashion to produce the compacted form of the core.

Adenoviruses, Human↗

PubMed2000

An enhanceosome containing the Jun B/Fra-2 heterodimer and the HMG-I(Y) architectural protein controls HPV 18 transcription.

Recent studies have reported new mechanisms that mediate the transcriptional synergy of strong tissue-specific enhancers, involving the cooperative assembly of higher-order nucleoprotein complexes called enhanceosomes. Here we show that the HPV18 enhancer, which controls the epithelial-specific transcription of the E6 and E7 transforming genes, exhibits characteristic features of these structures. We used deletion experiments to show that a core enhancer element cooperates, in a specific helical phasing, with distant essential factors binding to the ends of the enhancer. This core sequence, binding a Jun B/Fra-2 heterodimer, cooperatively recruits the architectural protein HMG-I(Y) in a nucleoprotein complex, where they interact with each other. Therefore, in HeLa cells, HPV18 transcription seems to depend upon the assembly of an enhanceosome containing multiple cellular factors recruited by a core sequence interacting with AP1 and HMG-I(Y).

Binding Sites↗

PubMed2001

HIV-1 infection requires a functional integrase NLS.

HIV-1 is able to infect nondividing cells productively in part because the postentry viral nucleoprotein complexes are actively imported into the nucleus. In this manuscript, we identify a novel nuclear localization signal (NLS) in the viral integrase (IN) protein that is essential for virus replication in both dividing and nondividing cells. The IN NLS stimulates the efficient nuclear accumulation of viral DNA as well as virion-derived IN protein during the initial stages of infection but is dispensable for catalytic function. Because this NLS is required for infection irrespective of target cell proliferation, we suggest that interactions between uncoated viral nucleoprotein complexes and the host cell nuclear import machinery are critical for HIV-1 infection of all cells.

Active Transport, Cell Nucleus↗

PubMed2000

Novel negative regulatory element in the platelet-derived growth factor B chain promoter that mediates ERK-dependent transcriptional repression.

Platelet-derived growth factor (PDGF), which consists of an A and/or B chain, stimulates migration and proliferation in vascular smooth muscle cells as well as a large number of other cell types. Investigations over recent years have defined roles for several positive regulatory transcription factors in the PDGF-B promoter. However, little is known about the transcriptional mechanisms that negatively regulate this gene. Here, we used transient transfection and 5' deletion analysis to define a specific region in the PDGF-B promoter-mediating repression in vascular smooth muscle cells. Gel retardation assays revealed this region is bound by nuclear protein(s) in a specific manner. Supershift assays excluded the direct association of Sp1, Sp3, and Egr-1. Mutation of the negative regulatory element no longer supported nucleoprotein complex formation and, when introduced into the PDGF-B promoter, rescued the promoter from repression. Promoter activity was also restored by transfection of oligonucleotide decoys bearing the repressor binding site. The MEK1/2 inhibitor, PD98059, and a dominant negative construct generating inactive ERK1 increased reporter expression driven by the PDGF-B promoter. In contrast, the MEK inhibitor had no effect on the activity of the mutant PDGF-B promoter. These effects were cell type-specific, since neither suppression of the PDGF-B promoter nor nucleoprotein complex formation was observed in vascular endothelial cells. These findings define a distinct negative regulatory element in the PDGF-B promoter that interacts with nuclear protein(s) and inhibits PDGF-B promoter-dependent gene expression in an ERK-dependent manner.

Base Sequence↗

PubMed2004

Molecular interplay between RNA polymerase and two transcriptional regulators in promoter switch.

Transcription regulation relies in the molecular interplay between the RNA polymerase (RNAP) and regulatory factors. Phage phi29 promoters A2c, A2b and A3 are coordinately regulated by the transcriptional regulator protein p4 and the histone-like protein p6. This study shows that protein p4 binds simultaneously to four sites: sites 1 and 2 located between promoters A2c and A2b and sites 3 and 4 between promoters A2b and A3, placed in such a way that bound p4 is equidistant from promoters A2c and A2b and one helix turn further upstream from promoter A3. The p4 molecules bound to sites 1 and 3 reorganise the binding of protein p6, giving rise to the nucleoprotein complex responsible for the switch from early to late transcription. We identify the positioning of the alphaCTD-RNAP domain at these promoters, and demonstrate that the domains are crucial for promoter A2b recognition and required for full activity of promoter A2c. Since binding of RNAP overlaps with p4 and p6 binding, repression of the early transcription relies on the synergy of the regulators able to antagonize the stable binding of the RNAP through competition for the same target, while activation of late transcription is carried out through the stabilization of the RNAP by the p4/p6 nucleoprotein complex. The control of promoters A2c and A2b by feed-back regulation is discussed.

Bacteriophages↗

PubMed1982

Transcription and accurate polyadenylation in vitro of RNA from the major late adenovirus 2 transcription unit.

Nucleoprotein complexes with in vitro transcription activity were isolated from HeLa cells late in lytic infection with Adenovirus type 2 (Ad2). Both polymerase II and polymerase III were active in these extracts, and greater than 85% of the labeled RNA was Ad2-specific. Electrophoretic analyses and Southern blot analyses demonstrated that RNA complementary to the entire 30 kb late transcription unit including RNA near the presumed termination site was synthesized. The addition of DRB (5,6,dichloro-1-beta-D-ribofuranosyl-benzimidazole) in vivo prior to the isolation of the complexes resulted in accumulation of polymerase II at the promoter proximal sites, but nascent chains started in vivo in DRB were successfully elongated in vitro. Approximately 10% of the RNA labeled in vitro contained poly(A), and the length of poly(A) was very similar to that of nuclear RNA isolated from Ad2-infected cells. The in vitro sites of poly(A) addition were specific--labeled poly(A)-terminated RNA molecules ended at a point on the genome coincident with previously mapped poly(A) sites of mRNAs produced in vivo. In addition, the polyadenylation enzyme (or enzymes) cosediment with the nucleoprotein complexes during sucrose gradient centrifugation since gradient purified complexes synthesize poly(A) containing RNA in vitro in the absence of any added nuclear extract.

Adenoviruses, Human↗

PubMed1999

Oligomeric structures of the phage phi29 histone-like protein p6.

Protein p6 of Bacillus subtilis phage phi29 has been described as a histone-like protein, playing a role in genome organization and compaction, on the basis of its high intracellular abundance, its pleiotropic effect, and its ability to bind and highly compact the whole phi29 DNA in vitro. Protein p6 forms large multimeric nucleoprotein complexes in which a right-handed superhelical DNA wraps toroidally around the protein core. Analytical ultracentrifugation analysis, at the concentration estimated in vivo (at least 1 mM), showed that protein p6 self-associates into elongated oligomers, suggesting that, in the absence of DNA, the protein could form a scaffold for DNA binding. In this work we have studied the structure of these oligomers by transmission electron microscopy and image processing. The results show that protein p6 aggregates into crooked-shaped oligomers, compatible with a helical structure. The oligomers could interact head-to-tail to form doughnut-shaped structures or they could grow into right-handed double-helical filaments by a nucleation-dependent polymerization process. The dimensions of the crooked-shaped structures are in agreement with that of the DNA in the nucleoprotein complex previously described. We propose that the crooked-shaped structures could act as a scaffold imposing the right-handed path followed by the DNA, and thus it could be considered a non-transient DNA chaperone.

Bacillus Phages↗

PubMed1996

Role of the karyopherin pathway in human immunodeficiency virus type 1 nuclear import.

The interaction of the human immunodeficiency virus type 1 (HIV-1) nucleoprotein complex with the cell nuclear import machinery is necessary for viral replication in macrophages and for the establishment of infection in quiescent T lymphocytes. The karyophilic properties of two viral proteins, matrix (MA) and Vpr, are keys to this process. Here, we show that an early step of HIV-1 nuclear import is the recognition of the MA nuclear localization signal (NLS) by Rch1, a member of the karyopherin-alpha family. Furthermore, we demonstrate that an N-terminally truncated form of Rch1 which binds MA but fails to localize to the nucleus efficiently blocks MA- but not Vpr-mediated HIV-1 nuclear import. Correspondingly, NLS peptide inhibits the nuclear migration of MA but not that of Vpr and prevents the infection of terminally differentiated macrophages by vpr-defective virus but not wild-type virus. These results are consistent with a model in which Rch1 or another member of the karyopherin-alpha family, through the recognition of the MA NLS, participates in docking the HIV-1 nucleoprotein complex at the nuclear pore. In addition, our data suggest that Vpr governs HIV-1 nuclear import through a distinct pathway.

Amino Acid Sequence↗

PubMed1994

Telomeric protein-DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine.

The Oxytricha telomere protein specifically recognizes single-stranded telomeric DNA, forming an extremely salt resistant and kinetically stable nucleoprotein complex. The absence of information on how this heterodimeric protein binds to DNA prompted this photo-cross-linking study. Multiple protein-DNA photo-cross-links are formed upon UV irradiation of Oxytricha telomeres reconstituted with a synthetic oligonucleotide terminating in 5'-T16T15T14T13G12G11G10G9T8T7T6T5G4G3G2G1-3'. Site-specific substitution of certain nucleotides with 5-bromodeoxyuridine (BrdU) greatly increased the photo-cross-linking yield, each substitution favoring a specific protein-DNA cross-link. For example, substitution of BrdU for T7 resulted in 25% cross-linking of the bound DNA, a 10-fold increase over the unsubstituted DNA. Both subunits of the telomere protein cross-link to, and are therefore near, the DNA. Three point contacts within this nucleoprotein complex, involving the alpha subunit, were established using BrdU substitution: Tyr239, Tyr142, and His292 cross-link to G3, T15, and T7, respectively. One photo-cross-link, Tyr239-G3, occurs amid a short acidic stretch of the alpha subunit, counter to expectations for amino acids that approach the polyanionic DNA. The two remaining cross-links are to amino acids in hydrophobic regions of the primary polypeptide sequence, consistent with the hypothesis that hydrophobic interactions account for the salt resistance (> 2 M NaCl) of this protein-DNA complex. These two photo-cross-links suggest that the telomere protein may bind telomeric single-stranded DNA by intercalation of aromatic residues into a nucleotide lattice.

Amino Acid Sequence↗

PubMed2001

The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers.

BACKGROUND: The discovery of histone-like proteins in Archaea urged studies into the possible organization of archaeal genomes in chromatin. Despite recent advances, a variety of structural questions remain unanswered. RESULTS: We have used the atomic force microscope (AFM) with traditional nuclease digestion assays to compare the structure of nucleoprotein complexes reconstituted from tandemly repeated eukaryal nucleosome-positioning sequences and histone octamers, H3/H4 tetramers, and the histone-fold archaeal protein HMf. The data unequivocally show that HMf reconstitutes are indeed organized as chromatin fibers, morphologically indistinguishable from their eukaryal counterparts. The nuclease digestion patterns revealed a clear pattern of protection at regular intervals, again similar to the patterns observed with eukaryal chromatin fibers. In addition, we studied HMf reconstitutes on mononucleosome-sized DNA fragments and observed a great degree of similarity in the internal organization of these particles and those organized by H3/H4 tetramers. A difference in stability was observed at the level of mono-, di-, and triparticles between the HMf particles and canonical octamer-containing nucleosomes. CONCLUSIONS: The in vitro reconstituted HMf-nucleoprotein complexes can be considered as bona fide chromatin structures. The differences in stability at the monoparticle level should be due to structural differences between HMf and core histone H3/H4 tetramers, i.e., to the complete absence in HMf of histone tails beyond the histone fold. We speculate that the existence of core histone tails in eukaryotes may provide a greater stability to nucleosomal particles and also provide the additional ability of chromatin structure to regulate DNA function in eukaryotic cells by posttranslational histone tail modifications.

Animals↗

PubMed1997

Cooperation of binding sites for STAT6 and NF kappa B/rel in the IL-4-induced up-regulation of the human IgE germline promoter.

Ig heavy chain class switching is directed by cytokines inducing transcription from unrearranged CH genes. Subsequently, such primed cells can undergo switch recombination to express the selected new isotype. In the case of IgE class switching, IL-4 activates the IgE germline promoter by inducing the interaction of the transcription factor STAT6 (IL-4STAT) with a responsive DNA element in the proximal region of the promoter. This study describes the characterization of two additional cis-acting elements that interact with members of the NF kappa B/rel transcription factor family in an IL-4-independent fashion. Electrophoretic mobility shift assays show that the nucleoprotein complex formed on the upstream site (NF kappa B1) contains the classical p50/p65 heterodimer. The complex on the proximal site (NF kappa B2) appears to be composed of p50 and relB. IgE germline promoter reporter gene constructs carrying point mutations in the NF kappa B2 site were largely unresponsive to IL-4 stimulation in transient transfection experiments, while plasmids with similar mutations in the NF kappa B1 site responded to cytokine stimulation better than the wild-type promoter. The NF kappa B2 effect was dependent on the presence of the STAT6 binding site, demonstrating that the NF kappa B2 motif is necessary but not sufficient for mediating cytokine up-regulation. In addition, the combination of a NF kappa B/rel binding site and the STAT6 response element conferred IL-4 inducibility to a heterologous minimal promoter, while the individual sites had no effect. The available data suggest that the NF kappa B2 nucleoprotein complex may cooperate with DNA-bound STAT6 to achieve IL-4-dependent activation of the human IgE germline gene.

B-Lymphocytes↗