Wilhelm bernhard workshop on the cell nucleus

REGULATION OF PROCESSIVE TRANSCRIPTION

Zhou M.¹, Deng L.¹, Kashanchi F.¹, Shatkin A.² and Kumar A.<sup>1

1</sup>Department of Biochemistry & Molecular Biology, George Wahington University, Washington, D.C. and ²Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, USA.
An understanding of the molecular mechanism of transcription elongation is important not only for basic biology but also for developing specific inhibitors of gene expression. Our studies are based on transactivation of HIV-1 promoter. Tat protein is known to induce transcription elongation of HIV-1 promoter by recruiting positive elongation factor, P-TEFb (composed of cyclin dependent kinase, CDK9 and cyclin T1) to the TAR RNA structure at the 5’end of nascent viral transcripts. We asked, (i) how does Tat modify general transcription factor P-TEFb, to specifically induce processive transcription from HIV-1 promoter, and (ii) would an inhibitor of the Tat/TAR dependent P-TEFb kinase selectively block viral replication? Earlier studies suggested that HIV Tat stimulates transcription elongation by binding cyclin T1 to the TAR RNA domains of nascent viral transcripts. Since the escape from paused transcripts, which appears to be an important check point for RNA Pol II genes, has been linked to P-TEFb mediated phosphorylation of RNAP II CTD repeats, we asked whether the Tat/TAR RNA interaction induced P-TEFb kinase. Main points of the results that will be discussed are as follows: (i) Tat/TAR induced P-TEFb kinase specifically phosphorylates RNAP II CTD serine 5; (ii)Tat/TAR dependent P-CTD serine 5 stimulates co-transcriptional capping of nascent viral transcripts; (iii) phosphorylation of the transcription elongation factors, SPT5 and Tat-SF1, is dependent upon the Tat modified P-TEFb kinase; (iv) the CDK inhibitor, Flavopiridol blocks phosphorylation of SPT5 and Tat-SF1 as well as P-CTD serine 5 at identical kinetics (IC₅₀=1nM); (v) Chromatin immunoprecipitation assays showed that the capped mRNA, along with SPT5, Tat-SF1 and P-CTD serine 5 associate with the promoter proximal region of HIV-1 gene and are highly sensitive to Flavopiridol; and (vi) at the low concentrations of Flavopiridol that attenuates HIV-1 replication, there is negligible change in the expression levels of host cell genes as seen in microarray assays. Overall, the results argue that the Tat modified transcription factors provide the basis for specific induction of viral gene expression. One prediction of the results, that viral replication would be uniquely dependent on the transcription elongation factors, SPT5 and Tat-SF1 was tested by RNAi mediated post-transcriptional gene silencing. Results show that ablation of either SPT5 or Tat-SF1 with synthetic siRNA complementary to SPT5 and Tat-SF1 mRNAs, severely attanuates HIV-1 replication. These studies show that HIV-1 encoded Tat protein modifies transcription factors to stimulate viral gene expression and that the step can be selectively inhibited by a CDK9 inhibitor to block viral replication.. This approach opens the possibility of developing novel inhibitors that are based on host cell enzymes and thus unlikely to engender resistant viral strains.


THE MULTIFUNCTIONAL TUMOUR SUPPRESSOR ZINC FINGER PROTEIN WT1 BINDS TO TRANSCRIPTS IN VIVO

Ladomery M.¹, Hastie N.¹, Woolner S.¹, Slight J.¹ and Sommerville J.²

¹MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, UK; ²School of Biology, Bute Medical Buildings, University of St Andrews, St Andrews, Scotland, UK

The Wilms tumour suppressor gene WT1 encodes a protein involved in urogenital development and disease. The salient feature of WT1 is the presence of four ‘Krüppel’ type C₂-H₂ zinc fingers in the C-terminus. Uniquely to WT1, an evolutionarily conserved alternative splicing event inserts three amino-acids (KTS) between the third and fourth zinc fingers which disrupts DNA binding. The ratio of +/-KTS isoforms is crucial for normal development. Previous work has shown that WT1 (+KTS) interacts with splice factors, and that WT1 zinc fingers (+/-KTS), particularly zinc finger one, bind to RNA in vitro. In this study we investigate the role of zinc finger one and the +KTS splice in vivo by expressing tagged proteins in mammalian cells and Xenopus oocytes. We find that both full length +/-KTS isoforms and deletion constructs that include zinc finger one co-sediment with RNP on density gradients. In Xenopus oocytes both isoforms located to the lateral loops of lampbrush chromosomes. Strikingly, only the +KTS isoform was detected in B-snurposomes, but not if co-expressed with WT1 (-KTS). However co-expression of the C-terminus (amino-acids 233-449, +KTS) resulted in snurposome staining, consistent with an in vivo interaction between isoforms via the N-terminus. Expressed WT1 was also detected in the RNA-rich granular component of nucleoli, and co-immunoprecipitated with oocyte transcripts. Full length WT1 was most stably bound to transcripts, followed by the C-terminus; whereas the least stably bound was CTF1 (C-terminus minus zinc finger one). Expression of the transcription factor EGR1, whose three zinc fingers correspond to WT1 zinc fingers 2-4, caused general chromosomal loop retraction and transcriptional shut-down. However a construct in which WT1 zinc finger one was added to EGR1 now reflected the properties of WT1 (-KTS). We suggest that in evolution, WT1 has acquired the ability to interact with transcripts and splice factors due to the modification of zinc finger one and the +KTS alternative splice.

References:

Ladomery et al., 2003, J Cell Sci. 116, pp. 1539-1549.


THE DEAD BOX RNA HELICASE P68: A TRANSCRIPTIONAL CO-ACTIVATOR AND REPRESSOR

Bates G., Wilson, B., Nicol S. and Fuller-Pace F.

Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee,Scotland, UK.
p68 is a prototypic member of the ‘DEAD box’ family of RNA helicases and shows the characteristic RNA helicase activity associated with DEAD box proteins. It has been reported to be involved in both pre-mRNA and ribosomal RNA processing processes which are consistent with its acting as an RNA helicase. However recent data have indicated that p68 is also a potent transcriptional activator of Estrogen Receptor alpha (ER), a function that is independent of helicase activity. These findings suggest that p68 is a multifunctional protein, which plays a role in several cellular processes. The reports that p68 can co-activate ER function prompted us to examine its ability to co-activate other transcription factors. Accordingly we have shown that p68 can stimulate the activity of several other transcription factors in addition to ERincluding the tumour suppressor p53. In separate experiments we investigated whether p68 has an intrinsic ability to activate or repress transcription. In experiments involving co-transfection of cell lines with GAL4-p68 fusion expression plasmids, together with relevant reporter plasmids containing GAL4 DNA-binding elements, we showed that, in some contexts, p68 can also act as a transcriptional repressor and have mapped domains within p68 which are involved in transcriptional activation/repression. Our data highlight novel functions for the p68 RNA helicase, which are independent of helicase activity.

THE SUPRASPLICEOSOME: A MACRO-MOLECULAR MACHINE FOR MAKING FUNCTIONAL mRNA

Sperling J.,¹ and Sperling R.²

¹The Weizmann Institute of Science, Rehovot, Israel; ²The Hebrew University of Jerusalem, Jerusalem, Israel
Processing of pre-mRNA in the nucleus occurs while the transcript is packaged with spliceosomal U snRNPs and protein splicing factors in a large supraspliceosome complex, which has overall dimensions of 50x50x35 nm and a mass of 21 mDa. By a combination of molecular and electron microscopy (EM) techniques we have now demonstrated that the complex is composed of four similar spliceosomal subunits and an additional substructure, which are associated with a single pre-mRNA molecule that connects the substructures. Structural analysis of the supraspliceosome in its native aqueous state by cryo-EM revealed holes within the spliceosomal subcomplexes, whose role is yet unclear. Cryo-EM also revealed fibrils formed by the pre-mRNA, which interconnect the subunits. The additional fifth substructure is attributed to additional pre-mRNA processing activities, since we have shown that 5’- and 3’-end components, as well as A-to-I editing enzymes, are associated with the supra-spliceosome. The supraspliceosome can thus be viewed as the native pre-mRNA processing machine. The supraspliceosome is a molecular machine required to process the pre-mRNA in an accurate, and regulated manner, yielding functional mRNA. It provides a frame onto which the pre-mRNA is folded, allowing juxtaposing exons about to be spliced, while introns are looped out of each of the respective spliceosomes. It enables “communication” between the spliceosomes and coordinated splicing of a multiintronic pre-mRNA. It can also account for regulated alternative splicing, which is a major source of protein versatility in mammals.


CDC2L5, A NUCLEAR CDC2-RELATED KINASE CONTAINING AN RS DOMAIN IS INVOLVED IN PRE-MRNA SPLICING

Durieux S.#, Even Y., Géraud M.L., Weil D.*, Genevière A.M.

Laboratoire Arago, Université Paris VI-CNRS UMR 7628, Banyuls-sur Mer, France *CNRS UPR1983, Institut André Lwoff, BP8, Villejuif, France
CDC2L5 belongs to a new subgroup of high molecular weight CDC2-like kinases (Marquès et al., 2000). These proteins are characterised by a conserved kinase domain with high similarity with the cyclin-dependent kinases (CDKs). This central domain is surrounded by large sequences displaying conserved motifs among which a sequence rich in serine-arginine residues (RS-domain). RS-domain are frequently found in metazoan proteins involved in pre-mRNA splicing. Northern blotting analysis of CDC2L5 shows that the mRNA is widely yet differentially expressed across a variety of human tissues with the highest expression in liver and placenta. In agreement with the presence of a putative NLS in the N-ter domain, the CDC2L5 protein is expressed in the nucleus of human cultured cells: HeLa, HEK 293 and U2 OS. The CDC2L5 subnuclear distribution is cell cycle dependent. To identify cellular proteins capable of interacting with CDC2L5 a two-hybrid screen was designed in yeast using the full-length CDC2L5 ORF as bait and a human liver cDNA library. Eight clones fulfilling the criteria for interaction of gene products were sequenced and checked against the GenBank-EMBL database. All of them encoded full-length or partial sequences of the same ASF/SF2 splicing factor binding protein. Confirmation of the interaction was provided by coimmunoprecipitation from HeLa cell lysates. A yeast two-hybrid mapping indicates that the N-terminal RS motif containing domain is responsible for this interaction. Moreover overexpression of a CDC2L5 N-terminal domain in transiently transfected HeLa cells severely inhibits the maturation of lymphotoxin transcripts. The above results strongly argue for a role of CDC2L5 in the regulation of premessenger RNA splicing. In that case, CDC2L5 would be the first example of a kinase of the Cdk type involved in the control of gene expression at the mRNA maturation step.

This work was supported by grants from the Association pour la Recherche sur le Cancer (contract n° 5290) and the Ligue contre le Cancer (Pyrénées orientales).

#S. Durieux was recipient of a fellowship from the Fondation Singer-Polignac

MODULATION OF TRANSCRIPTION IN WCH-17 CELLS AFTER HYPOTERMIC STIMULUS

Vecchio L.¹, Baldelli B.², Malatesta M.² and Biggiogera M.<sup>1

1</sup>Dipartimento di Biologia Animale, Lab. Biologia Cellulare, University of Pavia, Pavia; ²Istituto di Istologia e Analisi di Laboratorio, University of Urbino “Carlo Bo”, Italy
DADLE ([D-Ala²-D-Leu⁵]-Enkephalin) is a meta-bolically stable analog of the endogenus δ opioid peptide enkephalin that may initiate its metabolic effects through specific membrane receptor. Since it is able to induce hibernation when injected in active ground squirrels, DADLE is supposed to mimic the action of the putative Hibernation Triggering Factor, i.e. a protein which is thought to drive vertebrates into hibernation. Recent data (Malatesta et al., 1999, 2001; Biggiogera and Pellicciari, 2000) show that, in different vertebrate tissues, RNP-containing structures undergo significant changes during hibernation. Moreover synthetic peptide can dramatically extend the organ survival time in multiorgan block preparation including lung, liver, heart and kidney. Consequently, we have hypothesized that DADLE could modulate both cell proliferation and transcription. The aim of the present investigation was to monitor the modifications in nuclear and nucleolar trascription after treating WCH-17 cells from a hibernating animal either at 4°C (hypotermia) with 10^-3M DADLE or only kept at 4°C, and finally incubated with 10mM BrU. The association of the peptide with hypotermic treatment seems to further affect transcription. When the cells are only kept at 4°C, BrU incorporation is present, although reduced in comparison with controls, while after DADLE treatment an accumulation of RNA as well as RNPs can be visualized in the nuclei.

Supported by a grant from MIUR (Cofin 2002 – grant n. 20022057484)

References:

Biggiogera M. and Pellicciari C.: FASEB J., 14: 828-34, 2000

Malatesta M. et al., Anat Rec., 254: 389-95, 1999

Malatesta M. et al, Chromosoma, 110: 471-7, 2001

GENE EXPRESSION &

NUCLEUS-TO-CYTOPLASM TRANSPORT


TOPOLOGICAL ORGANIZATION OF GENE EXPRESSION IN DINOFLAGELLATES

Alverca E^1,2, Franca S² and Moreno Díaz de la Espina, S¹.

¹Nuclear Matrix Laboratory, CIB CSIC, Madrid, Spain, and ²Microbiology and Ecotoxicology Laboratory, INSA, Lisbon, Portugal
Dinoflagellates are the only eukaryotes lacking histones and nucleosomes. They contain low concentrations of basic proteins (HCc) without homology with histones, have condensed chromosomes in interphase and a unique nuclear organization. RNAPII activity is present in them, but the molecular characterization of the chromatin remodelling and transcription mechanisms is rudimentary. Only three proteins of Dinoflagellate transcription complexes (Dinap1, Dip1 and DapC) and a new class of transcription initiation factors (CcTBP) have been described, although snRNAs are conserved in them. Dinoflagellates are powerful models for the study of eukaryotic transcription without nucleosomes, and for the organization of the RNA nuclear network in their atypical nuclei. Chromatin remodelling, transcription, and RNA processing are spatially organized in dynamic nuclear domains that experience a constant flow of proteins. We investigated the spatial organisation of the transcription-splicing complexes in several Dinoflagellate species by western blot, confocal- and EM, and flow cytometry. Our data demonstrate for the first time the conservation of Cajal Bodies and of the sDMA protein domains, that distribute either in a perichromosomal layer around the condensed chromosomal cores, or in a diffuse pattern in the inter-chromosomal spaces, and in highly reactive CBs, but do not form speckles. Their distribution varies with the species and with the life cycle stages. EM revealed RNP fibrils intermingled with the decondensed DNA loops extruding from the chromosome cores, abundant interchromosomal granules, enriched in highly phosphorylated proteins, PGs and CBs. Cytochemical stainings for DNA, RNPs and immunogoldlabelling revealed that these structures are the counterpart of those detected by confocal microscopy. Our results confirm that in spite of the differences at the molecular level, the topological organization of the transcription/splicing complexes is conserved in Dinoflagellates.

HSP70 AND HSP90 ARE DIFFERENTIALLY EXPRESSED AND RELOCATED IN THE NUCLEUS AFTER INDUCTION OF MICRO-SPORE EMBRYOGENESIS BY STRESS

Seguí J.M., Testillano P.S., and Risueño M.C.

Plant Development and Nuclear Organization. Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
The microspore can switch the gametophytic developmental program towards embryogenesis, it can be induced in vitro by stress (heat shock). This reprogramming of the microspore is accompanied by changes in the structural organization of the nucleus, including alterations in the presence and localization of various molecules related to signalling of stress responses. In Brassica napus, microspore embryogenesis is efficiently induced by a stress treatment of 32ºC for at least 8 hours. After induction, some microspores started symmetric divisions and became haploid embryos after a few days, whereas other microspores which were not sensitive to induction, followed their original gametophytic development. In this work the expression, distribution and ultrastructural localization of two heat shock proteins (Hsp70 and Hsp90) throughout key stages before and after embryogenesis induction were studied. Both Hsp proteins were rapidly induced, localizing in the nucleus and the cytoplasm. Immunogold labeling showed changes in the distribution pattern of these proteins, being these changes assessed by a quantitative analysis. Inside the nucleus, Hsp70 was found in association with RNP structures in the interchromatin region and in the nucleolus, whereas nuclear Hsp90 was mostly found in the interchromatin region. For Hsp70, the accumulation after the inductive treatment was accompanied by a reversible translocation from the cytoplasm to the nucleus, in both induced (embryogenic) and non-induced (gametophytic) microspores. However, the translocation was higher in embryogenic microspores suggesting a possible additional role of Hsp70 in the switching to embryogenesis pathway. In contrast, Hsp90 increase was similar in all microspores, occurring faster than for Hsp70 and suggesting a more specific role for Hsp90 in the stress response. Hsp70 and Hsp90 colocalized in clusters in the cytoplasm and the nucleus, but not in the nucleolus. Results indicated that stress proteins are involved in the process of microspore embryogenesis induction.

Work supported by Spanish MCyT, project BOS2002-03572.

References:

Seguí-Simarro J M, Testillano, P.S., Risueño, M.C.: Hsp70 and Hsp90 Change Their Expression and in situ Localization After Microspore Embryogenesis Induction in Brassica napus cv. Topas. J. Struct. Biol. 2003 ( In press)
Lack of epigenetic reprogramming in early sheep embryos produced by somatic cell nuclear transfer

Beaujean N.¹, Taylor J.², Gardner J.², Wilmut I.², Meehan R.³ and Young L.<sup>4

1</sup>Biologie du Développement et Reproduction, INRA Jouy-en-Josas, France, ²Department of Gene Expression and Development, Roslin Institute, Midlothian, UK, ³Department of Biomedical Sciences, University of Edinburgh, UK ⁴School of Human Development, Queens Medical Centre, University of Nottingham, UK
Low success rates have been universally reported for cloning by nuclear transfer, with most of the resulting animals presenting abnormalities. Cloning by nuclear transfer usually involves taking the nucleus from a somatic cell and transferring this into an egg previously stripped of its genetic content. The simplest explanation for the problems encountered with nuclear transfer is that they arise from incomplete reprogramming of the transferred nucleus. When the somatic nucleus is transferred into the enucleated egg, it needs to switch from its ‘somatic’ status to an ‘embryonic’ status and start the development program of an embryo. Addition of a methyl group to DNA (methylation) or removal (demethylation) are key regulators of gene expression. If somatic methylation marks are not removed and replaced by embryonic marks, this could interfere with embryonic gene expression and successful development. In this work we investigated DNA methylation in early sheep embryos. From examination of normal sheep embryos between zygote (Day 1) and blastocyst (Day 7), we found few changes in DNA methylation patterns. This is in contrast to the mouse where total demethylation is observed. However, in cloned sheep embryos global methylation was even higher than in normal embryos, suggesting that some of the somatic methylation marks had been retained. By the blastocyst stage they also showed very intense methylation in the trophectoderm cells, the first differentiated cells found in embryos which go on to form extra-embryonic tissues. This may be one of the causes of the abnormal placental formation which is frequently observed in cloned animals. These results match recent investigations of mouse and cattle cloned embryos suggesting abnormal reprogramming of DNA methylation is widespread in cloned animals, although the exact nature of the disturbance varies between species.


MAMMALIAN RRN3 BECOMES INACTIVATED IN THE PROCESS OF TRANSCRIPTION

Hirschler-Laszkiewicz, I., Cavanaugh, A., and Rothblum, L.

Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania, USA
The human homologue of yeast Rrn3, a 72 kDa protein, is essential for ribosomal DNA (rDNA) transcription. While the importance of Rrn3 function in rDNA transcription is well established, its mechanism of action has not been determined. It has been suggested that the phosphorylation of either yeast RNA polymerase I or mammalian Rrn3 regulates the formation of RNA polymerase I·Rrn3 complexes that can interact with the committed template. These and other reported differences have important implications with respect to the mechanism by which Rrn3 functions in transcription. For example, in the yeast rDNA transcription system, Rrn3 might function catalytically, but in the mammalian system it might function stoichiometrically. Thus, we examined the question as to whether Rrn3 functions catalytically or stoichiometrically. We report that mammalian Rrn3 becomes the limiting factor as transcription reactions proceed. Moreover, we demonstrate that Rrn3 is inactivated during the transcription reactions. For example, Rrn3 isolated from a reaction that had undergone transcription cannot activate transcription in a subsequent reaction. We also show that this inactivated Rrn3 not only dissociates from RNA polymerase I, but is not capable of forming a stable complex with RNA polymerase I. Using immobilized template assays, we have also demonstrated that Rrn3 is required for recruitment of RNA polymerase I to the committed template. Our results indicate that Rrn3 functions stoichiometrically in rDNA transcription, that its ability to associate with RNA polymerase I is lost upon transcription, and that in the absence of functional Rrn3, RNA polymerase I can not be recruited to the committed template.

ACTIVE MAPKs TRANSLOCATE TO THE NUCLEUS AND ARE TARGETED TO DEFINED DOMAINS DURING PLANT DIFFERENTIA-TION AND PROLIFERATION

Coronado MJ, Seguí JM, González-Melendi P, Ramírez C, Barany I, Testillano PS, and Risueño M.C.

Plant Development and Nuclear Organization. Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Developmental processes involve both proliferation and differentiation events, cellular and nuclear dynamics varying during developmental processes and in response to factors like stress conditions. Mitogen-activated protein kinases (MAPKs) are involved in the signalling of extracellular stimuli in eukaryotes, including plants. Different MAPKs have been shown to be expressed during plant cell proliferation and developmental processes such as pollen development and embryogenesis, but the structural subdomain where these MAPKs are targeted in the nucleus has not yet been characterised. In this work the expression and subcellular localization of ERK1/2 homologues, proteins belonging to the MAPK family, and MAPK-active forms were studied in two plant developmental processes: stress-induced pollen embryogenesis (which involved an initial proliferation), and pollen maturation (which involved differentiation). Immuno-fluorescence and immunogold labelling in various plant species showed that the progression of differentiation and proliferation was accompanied by an increase in the expression of ERKs and MAPK activation together with a translocation to the nucleus. The combination of ultrastructural cytochemistry and immunogold for RNA and phosphorylated proteins indicated that the nuclear sites housing these MAPKs were areas of the interchromatin region enriched in RNA and phosphoproteins, including clusters of interchromatin granules. This could suggest a role of these MAPKs in the early events of activation of the transcription and processing machinery, via phosphorylation, which subsequently would be recruited to the transcription sites. The association of the nuclear localization of MAPKs with the stress-induced progression through the cell cycle and the commitment towards differentiation in the two plant developmental processes can be correlated .

Work supported by Spanish MCyT, project BOS2002-03572.

References:

Coronado MJ, González-Melendi P, Seguí JM, Ramírez C, Barany I, Testillano PS, Risueño MC: J Struct. Biol. 140, 200-213, 2002


Enhancement of nuclear uptake of therapeutic genes and macro-molecules by using NLS and PTD containing viral peptides

Sanders N, Vandenbroucke R, De Smedt S, Vanderleyden E, Lucas B, Remaut K Demeester J

Laboratory of General Biochemistry and Physical Pharmacy, Gent University, Gent, Belgium
Gene therapy is a novel approach to treat genetic based diseases by bringing therapeutic genes or oligonucleotides inside the nucleus or cytoplasm of target cells. Two types of DNA carriers are currently used: viral and non-viral. Because of the importance of safety in clinical applications, much attention has been given to non-viral carriers (e.g. cationic polymers and liposomes). However, these gene carriers are less effective in vivo. Indeed, it is widely recognized that in vivo many biological barriers complicate the delivery of exogenous pDNA to the cell nucleus. At the moment, the nuclear membrane is considered as a major barrier for pDNA. Therefore, we evaluated whether attachment of HIV-1 TAT (37-72) and other NLS containing peptides to fluorescent labeled pDNA and BSA can cause nuclear localization of these macromolecules. After manufacturing Cy5-pDNA and FITC-BSA with and without NLS peptide we microinjected these constructs, into Vero cells and followed, using a confocal scanning laser microscope, the fluorescence enhancement in the nucleus. As an internal control high molecular weight fluorescent, labeled dextrans were co-injected into the cells. When the nuclear membrane is intact, it is known that these dextrans are excluded from the nucleus. Micro-injected pDNA or BSA did not locate in the nucleus after 20 min. NLS conjugated to pDNA by electrostatic interaction did not cause nuclear localization of the pDNA. However, covalent linking of TAT to BSA resulted into a very rapid nuclear localization after micro-injection. These observations indicate that a covalent linking of an NLS petides to its cargo may, probably, be a requisite for an efficient nuclear localization. Moreover, we also observed that FITC-BSA covalently coupled with TAT was able to cross the cell membrane and the nuclear membrane in a few minutes. In further experiments we want to evaluate whether covalently linked NLS peptides to pDNA can cause a nuclear localization of the pDNA.

evidence that The nuclear lamina is an elastic structure which exerts pressure on intranuclear macro-molecules

Hancock R.

Laval Univ. Cancer Research Centre, Québec, Canada
The nuclear lamina is usually thought of as a rigid structure which surrounds the nuclear contents and also projects into the interior forming nuclear channels. Here experiments will be presented which show that the external surface area of the lamina can increase reversibly or decrease. Using nuclei from K562 cells attached to polylysine-coated slides, a reversible increase of the surface area was seen in two conditions. This area increased ≈1.5-fold (≈two-fold increase of nuclear volume) upon transfer from a physiological buffer slightly modified from that of Cook to a buffer containing KCl and Na₂HPO₄ at 1/100 of their concentration, and the nuclei contracted instantaneously to their normal size upon return to physiological buffer. Second, nuclei expanded to about twice their volume when microinjected with physiological buffer (containing fluorescent 77 kDa dextran to verify the absence of leakage) and they contracted instantaneously to their normal size when the injection pressure was released. Inversely, when the concentration of macromolecules in nuclei was reduced by incubation with restriction enzymes and electroelution of the excised chromatin fragments, the nuclei contracted as more chromatin was removed, implying that the nuclear envelope exerted pressure on the remaining nuclear contents. All these responses are due to properties of the nuclear lamina, since they were seen after membranous components of the envelope were extracted with Triton X-100. Immunolabelling studies will be shown to distinguish between two mechanisms for these changes of lamina area: elasticity and stretching of the lamina itself, or externalisation/internalisation of lamina material in nuclear channels. These experiments suggest that the nuclear lamina is a quite flexible elastic structure which normally exerts pressure on the intranuclear macromolecules.

Funded by the Canadian Institutes of Health Research, Grant MOP-14351

NUCLEOLUS


UBF INDUCES CHROMATIN REMODELING

Chen D.¹, Belmont A. S. ², and Huang S. ¹

¹Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois, USA;  ²Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign, USA.
The upstream binding factor (UBF) is involved in RNA polymerase I transcription.  In addition to its binding to the promoter, it has recently been shown to associate with sequences across the rDNA repeats (O'Sullivan et al., 2002).  We were interested in determining whether UBF also plays a role in chromatin remodeling.  We used the A03_1 cell line containing a ~90 Mbp, condensed, late replicating chromosome region labeled lac operator repeats.  This region appears as a condensed spot throughout most of the interphase cell cycle.  A fusion protein of lac repressor and a strong transactivator, VP16, localizes to the lac operator repeats and induces large-scale chromatin decondensation (Tumbar et al., 1999).  We have fused UBF1 to the lac repressor and found that targeting UBF1 to the lac operator repeats also induces chromatin decondensation.  The UBF fusion protein appears to behave similarly as endogenous UBF, including localization to the nucleolus and dimerizing with wild type UBF, but have much stronger affinity to the lac operator repeats.  The decondensed chromatin induced by UBF targeting colocalizes with acetylated core histones and some chromatin remodeling proteins.  These findings demonstrate that association of UBF to condensed repetitive chromatin is sufficient to induce chromatin remodeling, suggesting a novel role of UBF in regulation of rDNA transactivation.

ARTIFICIALLY UNRAVELED NUCLEOLI AS A MODEL TO STUDY THE NUCLEOLUS FUNCTIONAL ORGANIZATION

Dudnik O.A. and Smirnova O.Iu.

A. N. Belozersky Institute of Physical and Chemical Biology, Department of Electron Microscopy, Moscow State University, Moscow, Russia.
It is well known that fibrillar centers (FC) constitute an essential structural component of the active nucleolus in mammalian cells, yet their role in regulation of ribosomal gene transcription still remains an open question. Here, we studied the activity of endogenous RNA polymerase I upon partial and complete unraveling of nucleoli and FCs. The pattern of BrUTP incorporation in nuclei of hypotonically-treated cells was shown to be essentially the same as in the control untreated cells. Moreover, the sites of BrUTP incorporation, which revealed the active PNA polymerase I, were completely coincident with UBF-binding sites. These observations allow to conclude that structural integrity of FCs is not a prerequisite for maintenance of the active RNA polymerase I transcriptional complex. When the action of hypotonic shock was ceased and the cells were transferred to a complete cultural medium, the swollen nucleoli recovered to the control state. Therefore it is possible to conclude that none of the main morphological nucleolar counterparts, such as FCs, dense fibrillar component or the pars granulosa, is responsible for the maintenance of the nucleolar structural and functional integrity. A suggestion is made that this role may be played by the nucleolar matrix associated with the RNA polymerase I transcriptional complex.

This work was supported by INTAS Fellowship Grant for Young Scientists - YSF 2002-0356/F4


EFFECT OF ACTINOMYCIN-D UPON UP-STREAM BINDING FACTOR LOCALIZATION: A (3D + TIME) STUDY

Elias E., Tchélidzé P., Benassarou A.¹, Sauvage C., Bobichon H., Ploton D. and O’Donohue M.F.

Unité MéDIAN, CNRS UMR 6142 and ¹LERI, Reims, France.
Upstream Binding Factor (UBF), is present within the nucleolus as two closely related isoforms, UBF1 and UBF2. The fixation of a dimer of UBF1 remodels the promoter region, which increases RNA polymerase I (RNAP 1) binding affinity. In addition, UBF2 plays an anti-repressor role by binding to enhancer elements. In order to visualize both variants, which are not discriminated by specific anti-UBF antibodies, we previously developed chimeric proteins between UBF1/UBF2 and GFP. In the present work, we addressed the localisation of UBF1 and UBF2 both by electron microscopy and by confocal microscopy within living KB cells (4D study) during the inhibition of RNAP 1 by actinomycin D (AMD; 0.05 µg/ ml). The most sensitive method for ultrastructural immunolocalisation of GFP molecules was performed before embedding by using nanogold labelling, silver enhancement and observation of 100 nm ultrathin sections. In control cells, both proteins are mainly localised within fibrillar centers (FC) either as coils, for UBF1 (as for RNAP1, see T. Cheutin et al. J Cell Sci. 115, 3297, 2002) or with a more diffused pattern, for UBF2. During AMD treatment, both proteins remain within FC and are frequently organised as coils. For study on living cells by confocal microscopy, transfected cells were mounted in a perfusion chamber equipped with a heat controller and 40 optical sections were performed every five minutes for 8 hours. By developing a new software, we were able to study the complex 3D trajectories of the FC, identified by UBF-GFP, during all the steps of nucleolar segregation. This approach allowed us to demonstrate that segregation occurs by successive fusions of FC.

CONNECTION OF NUCLEOLAR TRANS-CRIPTION AND PROCESSING MACHINERY IS ENERGY-DEPENDENT

Louvet E., Junéra H.R., Le Panse S. and Hernandez-Verdun D.

Institut Jacques Monod, UMR 7592 CNRS, University of Paris VI and Paris VII, Paris, France
Nucleolar organization is the consequence of ribosomal gene (rDNA) transcription followed by processing of the ribosomal RNAs (rRNAs). How the coordination between transcription and processing in the nucleolus is maintained, is presently poorly understood. To address this question, we induced reversible disconnection of the nucleolar processing machinery from the rRNA transcription sites. This was achieved by treatment with 5, 6 dichloro-1--D-ribofuranosylbenzimidazole (DRB), a kinase inhibitor. During DRB treatment rDNA transcription is maintained and transcription sites are unraveled in the so-called nucleolar necklace. In the present study, we demonstrate that in addition, DRB generates distinct masses containing the late nucleolar processing machinery. This disconnection between transcription and processing is corroborated by the fact that 28S rRNA production is impaired. Removing DRB reverses this disconnection, and induces recovery of compact nucleoli and rRNA processing. Observations of nucleolar recovery in living cells indicate that recovery occurs in two successive steps, dynamic relocalization of the processing proteins around transcription sites, followed by compaction of the rDNA. We demonstrate that relocalization of the processing machinery on transcription sites is ATP-dependent and that nucleolar compaction is sensitive to temperature suggesting an energy-dependent process.

TOPOGRAPHY OF RIBOSOMAL DNA (rDNA) DETECTABLE BY FISH IN EARLY MOUSE EMBRYOS

¹Korobova F.V., ²Noniashvili E.M., ³Schoefer C., ¹Romanova L.G., ³Wachtler F., ²Dyban A.P., and ¹Zatsepina O.V.

¹Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAN, Moscow, and ²Institute of Experimental Medicine RAMN, St.-Petersburg, Russia; ³Histologisch-Embryologishes Institut, University of Vienna, Vienna, Austria.
It is well known that in preimplantation mouse embryos active ribosomal RNA (rRNA) genes are associated with the surface of so called nucleolar precursor bodies (NPBs), the characteristic structures, which are thought to serve for assembly of the functional nucleolus in early mammalian development. However, the question whether the NPBs are associated with all rDNA repeats and whether the topography of rDNA fluctuates respectively to its transcription and replication status still remains open. We applied fluorescence in situ hybridization (FISH) with mouse rDNA probes labeled with digoxigenin in order to map rDNA repeats in spreads of one- and two-cell mouse embryos. Before use, some one-cell embryos were incubated with okadaic acid, a potent phosphatase inhibitor that induced premature chromosome condensation. G1-, S-, and G2-type prematurely condensed chromosomes were found in pronuclei of embryos of different age, which reflects the embryo progression through the first cell cycle. Our data show that (1) irrespective to the transcription and replication activity of rDNA, the NPBs are not equal in their ability to support recruitment of rDNA repeats; (2) a part of rRNA genes is not associated with NPBs, but is “freely” located within the nucleoplasm; (3) resumption of rDNA transcription in late two-cell embryos is accompanied by significant rDNA unraveling as compared with early (transcriptionally inert) two-cell embryos; (4) in one-cell embryos, i.e. when rRNA genes are not transcribed, rDNA replication apparently occurs during the entire S-period and is asynchronous between individual chromosomal nucleolus organizing regions (NORs); (5) in one-cell mouse embryos, NPBs are involved in general arrangement of chromosomes within the pronuclei and are probably associated with the chromosomal NORs via the centromeric heterochromatin.

The research was supported by the INTAS (grant 96-1638) and Russian Foundation of Basic Researches (grants 02-04-49373 and 01-04-49810).