Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Abstract Full Text (PDF, 684K) PPT slides of all figures Supplememtal Material Index Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Spencer, C. A. Articles by Bazett-Jones, D. P. Search for Related Content PubMed PubMed Citation Articles by Spencer, C. A. Articles by Bazett-Jones, D. P. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Social Bookmarking                            What's this?

© The Rockefeller University Press, 0021-9525/2000//13 $5.00
The Journal of Cell Biology, Volume 150, Number 1, , 2000 13-26

Mitotic Transcription Repression in Vivo in the Absence of Nucleosomal Chromatin Condensation

^a Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2
^b Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta T6G 1Z2.(780) 432-8892(780) 432-8906

charlotte.spencer{at}cancerboard.ab.ca

All nuclear RNA synthesis is repressed during the mitotic phase of the cell cycle. In addition, RNA polymerase II (RNAP II), nascent RNA and many transcription factors disengage from DNA during mitosis. It has been proposed that mitotic transcription repression and disengagement of factors are due to either mitotic chromatin condensation or biochemical modifications to the transcription machinery. In this study, we investigate the requirement for chromatin condensation in establishing mitotic transcription repression and factor loss, by analyzing transcription and RNAP II localization in mitotic cells infected with herpes simplex virus type 1. We find that virus-infected cells enter mitosis and that mitotic viral DNA is maintained in a nucleosome-free and noncondensed state. Our data show that RNAP II transcription is repressed on cellular genes that are condensed into mitotic chromosomes and on viral genes that remain nucleosome free and noncondensed. Although RNAP II may interact indirectly with viral DNA during mitosis, it remains transcriptionally unengaged. This study demonstrates that mitotic repression of transcription and loss of transcription factors from mitotic DNA can occur independently of nucleosomal chromatin condensation.

Key Words: transcription • RNA polymerase II • chromosomes • mitosis • chromatin

© 2000 The Rockefeller University Press

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In higher eukaryotes, mitosis is accompanied by global repression of nuclear RNA synthesis. Studies performed over 30 years ago reveal that incorporation of radioactive precursors into RNA ceases from mid-prophase until late telophase (Taylor 1960; Prescott and Bender 1962; Gottesfeld and Forbes 1997). More recent data from run-on transcription and in situ hybridization assays indicate that transcription on specific RNA polymerase (RNAP) I, II, and III transcribed genes is profoundly repressed during M-phase (Shermoen and O'Farrell 1991; Weisenberger and Scheer 1995; White et al. 1995; Martinez-Balbas et al. 1995; Gebrane-Younes et al. 1997; Parsons and Spencer 1997). The global repression of RNAP II transcription is coupled to the dissociation of transcription activators, RNAP II and nascent RNA molecules from condensing mitotic chromatin (Shermoen and O'Farrell 1991; Martinez-Balbas et al. 1995; Segil et al. 1996; Kim et al. 1997; Parsons and Spencer 1997). RNAP II, TFIID and activators such as HSF1, Sp1 and C/EBP disengage from condensing chromosomes and enter the cytoplasm after breakdown of the nuclear membrane in mid-prophase (Martinez-Balbas et al. 1995; Parsons and Spencer 1997; Segil et al. 1996). At the end of telophase, RNAP II and transcription factors reassociate with decondensing chromatin and transcription resumes.

Several hypotheses have been proposed to explain how transcription might be repressed during mitosis. These fall into two general categories: chromosome condensation hypotheses and transcription factor inactivation hypotheses (Gottesfeld and Forbes 1997). Chromosome condensation hypotheses suggest that compaction of mitotic chromatin may physically block the association of transcription factors with promoters, thereby repressing transcription initiation. Similarly, mitosis-specific modifications to core histones or binding of condensation factors such as condensins and XCAP proteins to mitotic DNA may interfere with transcription initiation or elongation. Although chromosome condensation has been proposed as a potential repression mechanism, in vitro biochemical data support the transcription factor inactivation hypotheses. During mitosis, cells exhibit high levels of protein phosphorylation directly or indirectly triggered by cdc2-cyclin B kinase (Sahasrabuddhe et al. 1984). As a result, RNAP II and many basal and activator transcription factors acquire mitosis-specific phosphorylations (Gottesfeld and Forbes 1997). In vitro transcription assays using purified factors show that cdc2-cyclin B activity inhibits RNAP II transcription. Cdc2-cyclin B–dependent phosphorylation of the cdk7 subunit of TFIIH or the carboxy-terminal domain of the large subunit of RNAP II is sufficient to repress transcription initiation on naked DNA templates in vitro (Leresche et al. 1996; Gebara et al. 1997; Akoulitchev and Reinberg 1998; Long et al. 1998). In addition, mitosis-specific phosphorylation of some RNAP II transcription activators, such as Oct-1 and Sp1, interferes with their DNA binding in vitro (Martinez-Balbas et al. 1995; Segil et al. 1991). In sum, data from in vitro transcription and factor binding assays suggest that hyperphosphorylation of RNAP II transcription factors may be sufficient to prevent transcription initiation on RNAP II promoters during mitosis.

Although mitosis-specific phosphorylation of RNAP II transcription factors may be sufficient to repress transcription initiation in vitro, it is not clear whether the same mechanisms lead to transcription repression and to displacement of RNAP II elongation complexes, transcription factors and nascent RNA from mitotic chromatin in vivo. In this study, we explore the question of how RNAP II transcription is repressed during mitosis in vivo, and what role chromosome condensation plays in triggering either mitotic transcription repression or dissociation of transcription factors from mitotic DNA. To do this, we have developed a system that allows us to separate nucleosomal chromatin condensation from other biochemical effects triggered by cdc2-cyclin B activity as cells enter mitosis. We find that RNAP II transcription is repressed on DNA that is either condensed or noncondensed during mitosis. In addition, loss of RNAP II and basal transcription factors can occur independently of nucleosomal chromatin condensation. We conclude that mitotic repression of RNAP II and displacement of transcription elongation complexes may be triggered by events distinct from mitotic chromosome condensation. Although our data are consistent with the hypothesis that mitosis-specific biochemical modifications to the RNAP II transcription apparatus may be sufficient to bring about mitotic transcription repression, they do not exclude a role for template modification independent from chromosome condensation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells, Viruses, and Infections
HeLa S3 (human epithelioid cervical carcinoma), U2OS (human osteosarcoma), and SK-N-SH (human neuroblastoma) cells were obtained from American Type Culture Collection and grown as monolayers in DME + 10% fetal bovine serum. Herpes simplex virus type 1 (HSV-1) wild-type strain KOS1.1 and the ICP0 mutant strain n212 (kindly provided by Dr. Priscilla Schaffer, University of Pennsylvania School of Medicine) were propagated and titers were obtained as previously described (Rice et al. 1994). Cells were infected at a multiplicity of infection (MOI) of 10 plaque-forming units per cell as described (Rice et al. 1995).

Antibodies and Immunostaining
The following monoclonal antibodies were used for immunostaining: anti-ICP4 (H1101, Goodwin Institute) at a 1:2,000 dilution, anti–phospho-histone H3 (Upstate Biotechnology) at 1:1,000, anti-BrdU (Sigma) at 1:200, anti-BrdU (Boehringer) at 1:50, 8WG16 (Thompson et al. 1989) at 1:200 and ARNA-3 (Research Diagnostics) at 1:20 dilution. Rabbit polyclonal antibodies H4 penta and K8 (Upstate Biotechnology) were used at a 1:500 dilution. Monoclonal antibody anti-ICP4 was conjugated to either Texas red or Oregon green fluorochrome (Molecular Probes) and used at a dilution of 1:200. Secondary antibodies were: goat anti–mouse Alexa 488 and goat anti–rabbit Alexa 488 (Molecular Probes), goat anti–rabbit Cy5, goat anti–mouse Cy3 and goat anti–mouse lissamine rhodamine (Jackson ImmunoResearch Laboratories). For Western blots, the ARNA-3 antibody was used at a dilution of 1:50 and H1101 at a dilution of 1:5,000.

Immunostaining was performed on cycling populations of HeLa S3 cells. Cells were either grown on coverslips or deposited onto coverslips using a cytospin centrifuge. Cells were fixed for 10 min in 1% paraformaldehyde in PBS, then washed in PBS. After fixation, cells were permeabilized by immersing coverslips in 0.5% Triton X-100/PBS for 5 min, then washing in PBS. Coverslips were incubated for 30 min at 37°C in dilutions of primary antibodies in PBS/0.5% BSA. After washing in PBS, cells were incubated for 30 min at 37°C in dilutions of secondary antibodies in PBS/0.5% BSA. To visualize DNA, secondary antibody dilutions contained 5 µg/ml of 4',6-diamidine-2-phenylindole (DAPI). Anti–ICP4-Texas red and anti–ICP4-Oregon green conjugates were used as tertiary stains after secondary antibody staining. After washing in PBS, coverslips were mounted in glycerol and cells were visualized with a Zeiss LSM510 confocal microscope using sequential laser scans for each fluorochrome. Images were converted to TIFF files and figures were assembled and labeled using Adobe Photoshop software. Control stains included: secondary antibody only, secondary plus tertiary antibodies only, single primary antibody and secondary antibodies only. Bleedthrough staining and cross-reactions were undetectable using confocal microscopy and sequential laser scans.

BrdU Labeling and HSV In Situ Hybridization
HeLa S3 cells were synchronized in early S-phase by incubating in 2.5 mM thymidine for 24 h. Thymidine was removed and cells were infected 3 h later with HSV-1, at an MOI of 10.

For BrdU labeling, 5 mM BrdU was added at 4 h postinfection (7 h after thymidine washout). At this time point, cells were predominantly in G2-phase. Cells were harvested at 6 h postinfection. During the 2-h labeling period, 30% of the G2-phase–infected cells progressed into mitosis. Cytospins were prepared and cells were fixed, permeabilized, and stained as described above, using antibodies that recognize BrdU (Boehringer) and ICP4.

For HSV in situ hybridization, the synchronized infected cells were harvested at 6 h postinfection and cytospins were prepared. Cells were fixed with 3% paraformaldehyde for 10 min, washed, and permeabilized with 0.5% Triton X-100/PBS for 10 min. Cells were hybridized to a biotin-labeled HSV DNA probe following instructions provided by ENZO Diagnostics (PathoGene HSV DNA Probe Assay). In situ hybridization was performed at 85°C for 5 min. Hybridized probe was visualized using the Fluorescent Streptavidin In Situ Detection System (ENZO Diagnostics). Cells were then stained with anti–ICP4-Texas red and DAPI.

For both BrdU labeling and in situ hybridization experiments, control cytospins were treated with 1 mg/ml DNase I in PBS for 10 min at 37°C before staining or in situ hybridization.

Whole-Cell Run-on Transcription Assays
U2OS cells were synchronized and harvested for run-on transcription assays as follows. Monolayers at 20% confluency were grown for 24 h in the presence of 2.5 mM thymidine, to block cells in early S-phase. Thymidine was washed out and cells were incubated for 7 h in DME medium. Cells were either mock infected or infected with HSV-1 strain n212 at an MOI of 10, in the presence of 0.5 µg/ml nocodazole. At 7 h postinfection (14 h after thymidine washout), mitotic cells were removed by mitotic shake-off. At this time point 16% of cells in the culture had progressed into M-phase (Table ). The remaining adherent cells (mid- to late-G2) were harvested by trypsinization. Mitotic shake-offs yielded populations that were >95% mitotic as assessed by propidium iodide staining and visual scoring. G2-phase cells were scored at <3% mitotic. Mitotic and G2 populations were stained with anti–ICP4-Oregon green in order to assess the efficiency of infection. Greater than 80% of mitotic and G2 cells showed clearly visible viral replication compartments. After harvesting, cells were resuspended in DME/10% glycerol and frozen at –70°C.

Immunodetection of Nascent RNA and Viral Replication Compartments
In vivo labeling with fluorouridine was performed as follows. SK-N-SH cells were cultured directly on glass coverslips under conditions recommended by American Type Culture Collection. Cells were incubated for 50 min in fresh medium containing wild-type virus, KOS1.1. Infection medium was removed and cells were incubated for an additional 4 h in fresh medium. Cells were pulsed with fluorouridine at a final concentration of 2 mM for 10 min before being fixed with 1% paraformaldehyde in 1x PBS, pH 7.5, at room temperature for 5 min. Cells were washed and permeabilized in PBS containing 0.5% Triton X-100 for 5 min. Cells were immunolabeled first with a monoclonal antibody recognizing the halogenated nucleotide (mouse anti-BrdU; Sigma-Aldrich), then with goat anti–mouse IgG conjugated with Alexa 488 (Molecular Probes), and finally with the anti–ICP4-Texas red conjugate. Cells were incubated a minimum of 1 h at room temperature with each antibody and washed between each incubation step. After rinsing, samples were mounted in 1 mg/ml para-phenylenediamine in PBS/90% glycerol, containing 1 µg/ml DAPI. Cells were visualized using a Leica DMRE epifluorescence microscope and images collected using a digital camera containing a 14-bit cooled CCD detector (Princeton Instruments). Image processing was done using Adobe Photoshop 5.0.

Electron Spectroscopic Imaging and Correlative Fluorescence Microscopy
HeLa S3 cells were synchronized in S-phase by incubating for 24 h in culture medium containing 2.5 mM thymidine. 3 h after thymidine washout, cells were infected with wild-type virus KOS1.1. At 7 h postinfection, mitotic and interphase-infected cells were harvested and deposited onto coverslips using a cytospin centrifuge. Cells were fixed in 1% paraformaldehyde, permeabilized and stained with anti-ICP4 and anti-histone H4 antibodies. Secondary antibodies were goat anti–mouse Cy3 and goat anti–rabbit Cy5. Cells were fixed in 2% glutaraldehyde, dehydrated in ethanol, and embedded in Quetal 651 as described (Hendzel et al. 1999; Boisvert et al. 2000). Sections were cut to 30-nm thickness using an ultramicrotome with a diamond knife (Drukker), and were picked up onto finder grids. Sections were first visualized by fluorescence microscopy in order to identify and image viral replication compartments and host cell chromosomes in individual cells. The same specimen was then visualized by Electron Spectroscopic Imaging (ESI; Hendzel and Bazett-Jones 1996; Hendzel et al. 1998; Bazett-Jones and Hendzel 1999). Electron micrographs were obtained with a Gatan 14-bit slow scan cooled CCD detector on a Zeiss EM902 transmission electron microscope equipped with an imaging spectrometer. Phosphorus-enhanced images were recorded at 155 eV, and mass-sensitive reference images were recorded at 120 eV of energy loss and nitrogen-enhanced maps were recorded at 415 eV and reference image for nitrogen at 385 eV, as described previously (Hendzel and Bazett-Jones 1996; Bazett-Jones and Hendzel 1999; Hendzel et al. 1999; Boisvert et al. 2000). Net phosphorus maps were formed by subtracting the 120 eV image from the 155 eV image and net nitrogen maps by subtracting the 385 eV image from the 415 eV image using Digital Micrograph v. 2.5 software. Resultant images, both IF and EM, were processed and aligned using Adobe Photoshop 5.0.

Quantitation of Phosphorus and Nitrogen Content of HSV Replication Compartments and Host Chromatin
Integrated intensities of defined regions of the nucleus were calculated and the numbers were normalized to background values. The net phosphorus and net nitrogen values were computed as described (Locklear et al. 1990; Hendzel and Bazett-Jones 1996; Bazett-Jones and Hendzel 1999; Bazett-Jones et al. 1999) using Ergo Vista 4.0 (Atlantis) image analysis software. The normalized phosphorus/nitrogen ratio for host chromatin was divided by the phosphorus/nitrogen ratio for HSV replication compartment fibers.

In Vivo DNA-Protein Cross-linking
Mitotic and interphase U2OS cells were harvested as described above for run-on transcription assays. Proteins were cross-linked to DNA as described previously (Parsons and Spencer 1997). In brief, 1 x 10⁷ cells were incubated in 0.1% formaldehyde in DME for 10 min at room temperature. After cross-linking, cells were washed in PBS, resuspended in nuclear lysis buffer and lysed by passing through a 26-gauge needle. Lysates were loaded onto cesium chloride gradients and centrifuged at 77,000 g in a SW 60Ti rotor for 20 h at 20°C. Fractions were collected and those fractions containing DNA cross-linked to protein were pooled, dialyzed, and precipitated. DNA–cross-linked proteins were digested with DNase I, RNase A, and S1 nuclease to remove nucleic acids. The proteins from DNA–cross-linked fractions and proteins from the free protein fractions at the top of the gradients were analyzed by SDS-PAGE and Western blotting. Western blots were probed with ARNA-3, stripped, and reprobed with anti-ICP4. Autoradiographs were scanned and images were assembled and labeled using Adobe Photoshop software, as described above.

Online Supplemental Material
Three-dimensional (3-D) animated confocal images were generated as follows. Cycling HeLa S3 cells were fixed and stained with anti-ICP4 antibody, lissamine rhodamine-conjugated secondary antibody and DAPI to visualize DNA. A stack of 30 images, from top to bottom of each cell, was assembled with an LSM510 confocal microscope using two laser wavelengths. Z-resolution was 0.2 µm. The fluorescence signals from both fluorochromes were superimposed. 3-D images were rendered from z-stack data using 3-D for LSM510 software. Movies were created from 3-D images using Metamorph software. Videos are available at http://www.jcb.org/cgi/content/full/150/1/13/DC1.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Viral DNA Remains in a Nucleosome-free, Noncondensed State during Mitosis
Herpes simplex virus type 1 (HSV-1) is a >150-kD nuclear DNA virus whose genome is transcribed by host RNAP II (Smiley et al. 1991). Within 3–4 h postinfection, RNAP II and host transcription factors are recruited from the host genome onto the viral genome, and become concentrated in subnuclear foci known as viral replication compartments which are sites of viral DNA replication and transcription (de Bruyn Kops and Knipe 1988; Rice et al. 1994; Phelan et al. 1997). BrdU is rapidly incorporated into viral replication compartments and viral DNA remains within replication compartments after its synthesis (de Bruyn Kops and Knipe 1988; Phelan et al. 1997). Formation of replication compartments requires viral DNA synthesis and compartments are absent when viral DNA synthesis is inhibited by DNA polymerase inhibitors or when cells are infected with viruses mutant in genes encoding one of the seven essential virus-encoded DNA replication proteins (Godowski and Knipe 1983; Uprichard and Knipe 1997). Replication compartments disperse after treatment with DNase I (Rixon et al. 1983; Quinlan et al. 1984).

HSV replication compartments are also sites of viral and cellular regulatory protein accumulation. In keeping with their role in viral gene transcription, these structures recruit host RNA polymerase II, TBP and basal transcription factors, as well as the viral transcription activator protein ICP4 (Randall and Dinwoodie 1986; Knipe et al. 1987; Rice et al. 1994; de Bruyn Kops et al. 1998; Spencer, C., unpublished data). ICP4 is a sequence-specific and nonspecific DNA-binding protein, and DNA binding is essential for its gene activation function (Kattar-Cooley and Wilcox 1989; Michael and Roizman 1989; Allen and Everett 1997). Treatment with DNase I or inhibition of viral DNA replication eliminates recruitment of ICP4 into replication compartments. Consistent with the involvement of replication compartments in viral DNA replication, viral replication compartments accumulate virus-encoded replication proteins including DNA polymerase and the DNA-binding protein, ICP8 (Knipe and Spang 1982; Quinlan et al. 1984). ICP8 is required for viral DNA replication and for replication compartment formation and it binds preferentially to single-stranded and double-stranded DNA, holding it in an extended conformation (Leinbach and Casto 1983; Ruyechan 1983; Lee and Knipe 1985; Gourves et al. 2000). ICP8 and the other six essential viral DNA replication proteins colocalize in viral replication compartments at sites of BrdU labeling (Quinlan et al. 1984; de Bruyn Kops and Knipe 1988). DNase I treatment abolishes recruitment of these proteins to replication compartments. Electron microscopic immunocytochemical analysis of viral DNA Miller spreads shows that ICP8 comprises a major component of 17-nm thick viral double-stranded DNA molecules that are nucleosome free (Muller et al. 1980; Puvion-Dutilleul et al. 1985). In addition, nascent viral RNAs have been detected in replication compartments, and more prominently in interchromatin granule clusters and cytoplasm (Besse et al. 1995).

Interestingly, subnuclear structures resembling viral replication compartments form in uninfected cells after transfection with plasmids encoding the seven essential HSV-1 replication proteins, along with a plasmid containing the HSV-1 origin of replication. These replication compartment structures are sites of DNA synthesis and do not form in the presence of HSV DNA polymerase inhibitors or in the absence of one of the viral DNA replication proteins (Lukonis and Weller 1997; Zhong and Hayward 1997). Hence, HSV replication compartments are comprised of viral DNA, nascent RNA, cellular and viral proteins.

As virus infection proceeds, host transcription is repressed and viral gene transcription is activated in a controlled temporal order (Godowski and Knipe 1986; Rice et al. 1994; Spencer et al. 1997). Because viral and host cell genes contain similar RNAP II promoter elements and use the same transcription machinery, this global change in transcription may be due, in part, to nucleotide sequence-independent mechanisms, such as differences between host and viral chromatin structure (Smiley et al. 1991). In support of this idea, data from fractionation and nuclease digestion experiments suggest that parental and newly replicated HSV-1 DNA are free of nucleosomes during lytic infection (Mouttet et al. 1979; Leinbach and Summers 1980; Muller et al. 1980; Pignatti and Cassai 1980; Sinden et al. 1982; Puvion-Dutilleul et al. 1985; Muggeridge and Fraser 1986; Lentine and Bachenheimer 1990). Electron microscopic studies of Miller spreads show a preponderance of DNA in non-nucleosomal form in HSV-infected cells (Muller et al. 1980; Puvion-Dutilleul et al. 1985). This DNA takes three forms: 3–5-nm protein-free strands, strands with sparse 10–20-nm large granules different from nucleosomes and 17-nm uniformly thick heavily stained strands.

We reasoned that if HSV-1 DNA remains nucleosome free and virus infection does not interfere with entry of cells into mitosis, viral DNA may be present in a noncondensed state during mitosis. The presence of both noncondensed and condensed chromatin in vivo would allow us to determine whether chromosome condensation was required for either transcription repression or dissociation of transcription factors during mitosis in intact mitotic cells.

We first confirmed that human cells infected with HSV-1 progress through G2-phase and enter mitosis. HeLa S3 cells were synchronized in early S-phase by growth for 24 h in the presence of thymidine. 3 h after thymidine washout, cells were infected with HSV-1. 6 h after infection, cells were fixed, stained with propidium iodide, and scored for mitotic index. Table shows that HeLa cells entered mitosis when infected with HSV-1, although the rate of mitotic entry was slowed compared with mock infected cells. The rate of mitotic entry was similar in mock infected U2OS cells and in U2OS cells infected with the HSV-1 mutant virus, n212 (Table ).

We next confirmed that infected mitotic cells contained viral replication compartments. Fig. 1 a shows interphase and mitotic HeLa cells, fixed and stained with the DNA stain DAPI and an antibody that recognizes the viral immediate-early protein ICP4. ICP4 binds to viral DNA and is the main transcriptional activator protein encoded by HSV-1 (Kattar-Cooley and Wilcox 1989; Smiley et al. 1991; Allen and Everett 1997). Viral replication compartments were present in both interphase and mitotic infected cells (Fig. 1 a).

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Interphase and mitotic cells contain viral replication compartments. Cycling HeLa cells were infected with HSV-1 strain KOS1.1 for 6 h, fixed, and immunostained with an antibody that recognizes viral immediate-early protein, ICP4, and a secondary antibody conjugated to rhodamine (red). DNA was visualized by DAPI staining (blue). Arrows in a indicate replication compartments within mitotic cells. (b) Frames taken from 3-D animation of an interphase infected cell, showing spatial arrangement of viral replication compartments and host chromatin. (c) Frames taken from 3-D animation of a mitotic infected cell. Optical sections were collected with a confocal microscope and 3-D animations were generated as described in Materials and Methods. Frames are taken from the beginning, middle and end of the animation series. Videos 1 and 2 are available at http://www.jcb.org/cgi/content/full/150/1/13/DC1. Bars, 10 µm.

In all transcription and cross-linking experiments described in this report, >80% of mitotic infected cells contained clearly visible replication compartments. Together, Table and Fig. 1 indicate that human cells infected with HSV-1 enter mitosis, condense host chromatin, and contain discrete extrachromosomal viral replication compartments.

Although it has been established that viral replication compartments in interphase cells contain newly replicated viral DNA (de Bruyn Kops and Knipe 1988; Knipe 1990; Phelan et al. 1997), we wanted to determine whether viral replication compartments in mitotic cells also contain newly replicated viral DNA. To do this, we used two methods: BrdU incorporation and HSV in situ hybridization.

For BrdU labeling experiments, HeLa S3 cells were arrested in early S-phase, released from arrest for 3 h, and infected with HSV-1. The synchronized infected cells were incubated in the presence of BrdU for 2 h, beginning at 4 h postinfection. At 4 h postinfection (7 h after thymidine washout), cells were predominantly in G2-phase; therefore, incorporation of BrdU into nascent DNA would be due to the actions of the HSV-1 DNA polymerase. Viral DNA polymerase is necessary for formation of viral replication compartments (Quinlan et al. 1984; de Bruyn Kops et al. 1998). Mitotic and G2 cells were harvested at 6 h postinfection and cytospins were prepared. Cells were fixed and stained with anti-BrdU and anti-ICP4 antibodies. Both interphase and M-phase cells contained replication compartments that reacted with anti-BrdU and anti-ICP4 antibodies (Fig. 2 a). The BrdU and DAPI signals were undetectable in cells that were treated with DNase I before staining. These data indicate that viral replication compartments in both interphase and mitotic cells contain newly replicated DNA.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 2 HSV-1 replication compartments contain newly replicated viral DNA. HeLa S3 cells were synchronized in S-phase, released for 3 h and infected with wild-type HSV-1. (a) At 4 h postinfection (when the majority of cells were in G2-phase), 5 mM BrdU was added to the culture medium. Cells were harvested at 6 h postinfection, fixed, and stained with anti-BrdU and anti-ICP4 antibodies, as indicated. (b) Synchronized infected cells were harvested at 6 h postinfection, fixed, and subjected to in situ hybridization using a biotin-labeled HSV-1 DNA probe and streptavidin-fluorescein. Cells were then stained with anti-ICP4 antibody and DAPI. Bars, 10 µm.

To extend previous reports that HSV-1 DNA is nucleosome free, we examined infected cells using two cellular imaging methods. First, we immunostained interphase and mitotic infected cells with antibodies that recognize histone H4 and a mitosis-specific epitope on histone H3. Viral replication compartments in both interphase and mitotic cells showed no detectable staining with antibodies that recognize histones H4 (Fig. 3 a) or H3 (Fig. 3 b). This absence of staining is in sharp contrast to the pronounced replication compartment staining seen for other host cell DNA-binding proteins such as RNAP II, basal transcription factors, p53, Rb, and DNA ligase, as well as for viral replication and regulatory proteins such as ICP4, ICP8, ICP27, and viral replication proteins (Wilcock and Lane 1991; Rice et al. 1994; Zhong and Hayward 1997; de Bruyn Kops et al. 1998). These data suggest that histones H3 and H4 do not contribute significantly to the protein complement of HSV nucleoprotein fibers.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Viral replication compartments contain little if any histone H4 or H3. Cycling HeLa S3 cells were infected with HSV-1 strain KOS1.1 for 6 h, fixed, and immunostained with antibody that recognizes histone H4 (a) or a mitosis-specific phospho-epitope on histone H3 (b). Cells were also stained with anti-ICP4 conjugated to Texas red and DAPI, to visualize DNA. Anti-histone H3 recognizes histone H3 predominantly in mitotic cells. Bars, 10 µm.

HeLa S3 cells were infected, stained with anti-ICP4 antibody, and embedded for electron microscopy. Thin sections were prepared by ultramicrotomy and deposited onto lettered EM specimen grids. The sections were first imaged in the fluorescence microscope to find replication compartments and host chromatin (Fig. 4 a). The regions of interest were then found in the electron microscope (with the aid of the lettered grids) and imaged by ESI to obtain high resolution structure and analytical information. The light and EM images were then superimposed in order to identify the regions of interest in the electron images (Fig. 4 b). The viral replication compartment in this cell occupied a large volume of the nucleus, with host chromatin (arrows in Fig. 4, c–f) forced to the nuclear periphery, as seen previously (Besse et al. 1995). Fig. 4c and Fig. d, show the boxed region in Fig. 4 b imaged at higher magnification. Similarly, Fig. 4e and Fig. f, show the boxed region in Fig. 4 d imaged at an even higher magnification. At these higher magnifications, the replication compartments appeared to contain both fibers and granules composed of both nitrogen and phosphorus. The phosphorus signal in these fibers appeared to be qualitatively close to the detection limit, significantly lower than that of 10-nm nucleosomal DNA fibers (Boisvert et al. 2000). On the other hand, the nitrogen content of these fibers was significantly higher, indicating that the DNA fibers associate with protein. These data confirm previous conclusions derived from biochemical and electron microscopic assays that show HSV DNA in a non-nucleosomal extended conformation in interphase infected cells (Muller et al. 1980; Puvion-Dutilleul et al. 1985).

View larger version (97K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Viral replication compartment DNA is non-nucleosomal and noncondensed during interphase. HeLa S3 cells were blocked in S-phase, released from the block for 3 h and infected with wild-type virus. At 7 h postinfection, mitotic and interphase infected cells were harvested, fixed, and immunostained with anti-ICP4 conjugated to Texas red. Cells were then fixed and sectioned as described in Materials and Methods. Individual cells were first imaged by standard immunofluorescence microscopy, to locate viral replication compartments (a). The same cell as seen in a was then visualized by electron microscopy. The immunofluorescence image was rescaled and superimposed, in this instance, on the phosphorus map EM image (b). The boxed area in b was imaged at a higher magnification for net phosphorus (c) and net nitrogen (d). Arrows indicate host chromatin, and the viral replication compartments are indicated by RC. The boxed area in d was imaged at an even higher magnification for net phosphorus (e) and net nitrogen (f). Replication compartment material appears to contain diffuse noncondensed granules and fibers containing both phosphorus and nitrogen, characteristic of nucleic acids and protein. Bar, 5 µm.

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Viral replication compartment DNA is non-nucleosomal and noncondensed during mitosis. HeLa S3 cells were infected, harvested, and processed for immunofluorescence and ESI, as described in the legend to Fig. 4. A viral replication compartment in a mitotic cell was visualized by ICP4 staining and immunofluorescence (a). The same cell as seen in a was imaged by electron microscopy and the two images were superimposed (b). In this instance the immunofluorescence image was superimposed on the nitrogen map EM image. The box in b, encompassing part of a replication compartment, was imaged for net phosphorus (c) and net nitrogen (d). Arrows indicate host chromosomes and RC indicates replication compartment. The box in c was imaged for net phosphorus (e) and net nitrogen (f). The boxed area in d was imaged at higher magnification (50 k) for net phosphorus (g) and net nitrogen (h). The phosphorus and nitrogen maps (c–h) show mitotic replication compartments as diffuse noncondensed areas containing both phosphorus- and nitrogen-containing fibers of <10 nm, similar to the pattern seen in interphase replication compartments (Fig. 4, c–f). Bars: (b) 5 µm; (h) 50 nm.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Mitotic replication compartments do not stain with anti-RNAP II antibody, 8WG16. Cycling HeLa cells were either uninfected (a and b) or infected with HSV-1 for 6 h (c–f), fixed, and stained with 8WG16 and anti–ICP4-Texas red conjugate. DNA was stained with DAPI. (a and b), RNAP II staining was nuclear with exclusion from nucleoli in uninfected cells. (c–f), RNAP II localized to viral replication compartments in interphase nuclei, as seen previously (Rice et al. 1994, Rice et al. 1995). Mitotic replication compartments stained weakly with 8WG16. Bar, 10 µm.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Mitotic replication compartments stain with anti-RNAP II antibody, ARNA-3. Cycling infected HeLa cells were fixed and stained with ARNA-3 and anti–ICP4-Oregon green conjugate. DNA was stained with DAPI. ARNA-3 antibody stained viral replication compartments and cytoplasm in mitotic cells. Bar, 10 µm.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 10 In vivo cross-linking of RNAP II large subunit and ICP4 to uninfected and infected DNA. Uninfected or HSV-1–infected synchronized U2OS cells were separated into mitotic and interphase populations and treated with 0.1% formaldehyde to cross-link proteins to DNA. Cross-linked proteins were fractionated from non–cross-linked proteins, treated with nucleases and analyzed by Western blotting. (a) Blot probed with ARNA-3 antibody, which recognizes all phosphorylation variants of the large subunit of RNAP II. IIo is the hyperphosphorylated variant and IIa is nonphosphorylated. X-link denotes proteins cross-linked to DNA; Protein denotes proteins not cross-linked. (b) The same blot as in a, reprobed with anti-ICP4 antibody. Lane 1, protein cross-linked to DNA from 1.1 x 107 mitotic uninfected cells. Lane 2, free protein from 1.3 x 105 mitotic uninfected cells. Lane 3, protein cross-linked to DNA from 1.2 x 107 interphase uninfected cells. Lane 4, protein cross-linked to DNA from 1.0 x 107 mitotic HSV-1–infected cells. Lane 5, free protein from 1.3 x 105 mitotic HSV-1–infected cells. Lane 6, protein cross-linked to DNA from 1.1 x 107 interphase HSV-1–infected cells.

In uninfected cells (Fig. 6 a), transcription of cellular genes was repressed during mitosis, as reported previously (Parsons and Spencer 1997). Mitotic transcription levels declined from 17% to 0% of interphase (G2) levels, as assessed by PhosphorImager analysis. In infected interphase cells (Fig. 6 b, left panels), host gene transcription levels were lower than those in uninfected cells, as seen previously (Spencer et al. 1997). In contrast, viral gene transcription levels were high in infected interphase (G2) cells. When infected cells entered mitosis (Fig. 6 b, right), transcription of both host and viral genes was repressed to less than 10% of interphase (G2) levels. Data from these assays indicate that transcription of both host and viral genes is repressed in mitotic cells.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Transcription is repressed on both viral and cellular genes during mitosis. Whole cell run-on transcription assays were performed as described in Materials and Methods, on synchronized U2OS cells, either uninfected (a) or infected with HSV-1 for 7 h (b). Cells from the same flasks were separated into interphase (G2) and mitotic populations and assays were done using the same number of cells per sample. Single-stranded run-on transcription probes detect sense (S) or antisense (AS) transcription from cellular genes c-myc, -actin, GAPDH, and histone H2B, and viral genes ICP27, ICP8, gC, and UL36.

View larger version (63K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Fluorouridine incorporation in infected interphase and mitotic cells. Cycling SK-N-SH cells were infected with HSV-1, pulse labeled with fluorouridine, fixed, and stained with anti–BrdU- and Alexa 488–conjugated secondary antibody, and anti–ICP4-Texas red conjugate. DNA is stained with DAPI. (a), Infected interphase nucleus before formation of replication compartments. (b) Infected interphase nucleus containing replication compartments. (c) Infected mitotic cell containing viral replication compartment. Bar, 5 µm.

As the 8WG16 antibody does not recognize hyperphosphorylated forms of the RNAP II large subunit, we also stained infected cells with ARNA-3, an antibody that recognizes all phosphorylation variants of the large subunit (Rice et al. 1994, Rice et al. 1995). In contrast to the staining patterns observed with 8WG16, ARNA-3 stained replication compartments in both interphase and mitotic cells (Fig. 9).

These immunostaining data suggested that RNAP II may remain engaged on viral DNA during mitosis, but may be hyperphosphorylated and hence unreactive with antibody 8WG16. Therefore, it was important to confirm whether RNAP II was engaged on viral DNA or was indirectly associated with viral replication compartments. To do so, we analyzed RNAP II using in vivo cross-linking assays. We have used formaldehyde cross-linking previously to show that RNAP II is not transcriptionally engaged on mitotic chromatin in uninfected cells (Parsons and Spencer 1997). At low formaldehyde concentrations, cross-linking is selective for proteins that are in direct contact with DNA (Wrenn and Katzenellenbogen 1990). Infected or uninfected U2OS cells were separated into mitotic and interphase populations, treated with 0.1% formaldehyde for 10 min, and the cross-linked and non–cross-linked proteins fractionated on cesium chloride gradients. Cross-linked and non–cross-linked proteins were treated with nucleases, formaldehyde cross-links were reversed and proteins analyzed by Western blotting. Fig. 10 (top) shows cross-linked and non–cross-linked RNAP II large subunits in infected and uninfected cells. As seen previously (Parsons and Spencer 1997), the non–cross-linked large subunit of RNAP II in both interphase and mitotic uninfected cells displayed two phosphorylation variants: IIo, which is hyperphosphorylated, and IIa, which is nonphosphorylated (Fig. 10, lane 2; Dahmus 1994, Dahmus 1996). In interphase cells, the transcriptionally engaged form of the RNAP II large subunit is hyperphosphorylated and the nonengaged form is nonphosphorylated. In mitotic cells, the IIo form may result from mitosis-specific phosphorylations and may be distinct from the IIo form present in interphase cells (Kim et al. 1997; Parsons and Spencer 1997). In interphase uninfected cells, the IIo form of the RNAP II large subunit cross-linked to DNA (Fig. 10, lane 3), consistent with IIo being transcriptionally engaged on DNA. In mitotic uninfected cells, little if any RNAP II large subunit cross-linked to DNA (lane 1), as seen previously (Parsons and Spencer 1997). In HSV-1–infected interphase cells, a hyperphosphorylated form of the RNAP II large subunit also cross-linked to DNA (lane 6), but in mitotic infected cells little if any RNAP II large subunit cross-linked to DNA (lane 4). In contrast, the viral immediate-early protein ICP4 cross-linked to DNA in both interphase and mitotic infected cells (Fig. 10, lanes 4 and 6).

Taken together, the immunofluorescence localization and in vivo cross-linking assays suggest that RNAP II is not transcriptionally engaged in either infected or uninfected mitotic cells. It is possible however, that RNAP II interacts indirectly with replication compartments during mitosis, perhaps by associating with other proteins that bind to viral DNA, such as ICP4.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A remarkable feature of mitosis is the cessation of all nuclear RNA synthesis and the loss of RNAP II and transcription factors from condensing mitotic chromatin. Mitotic transcription repression and loss of DNA-binding factors may have important cell cycle regulatory consequences. It is possible that removal of large protein complexes such as RNAP II elongation complexes may be necessary in order to condense DNA into mitotic chromosomes. In addition, the cyclic disengagement of transcription factors from DNA at each cell cycle could have consequences for gene regulation. It has been shown that the rapid mitotic cycles of early Drosophila embryos causes premature termination of transcription on long transcription units, thereby preventing expression of full-length mRNA until cell cycles lengthen (Shermoen and O'Farrell 1991; O'Farrell 1992). There is also evidence that exit from mitosis may be a cell cycle checkpoint. Growth-related genes such as c-fos, c-jun, c-myc, and p53 are sequentially induced after exit from mitosis in patterns similar to those in cells entering the cell cycle from quiescence (Cosenza et al. 1991, Cosenza et al. 1994). Cells require the presence of serum growth factors as they exit mitosis in order to proceed to the next cell cycle (Zetterberg and Larsson 1985). It is possible that mitotic transcription repression could facilitate changes in gene expression as cells exit mitosis.

In this study, we have exploited HSV-1 infection of human cells in order to assess the requirement for nucleosomal chromatin condensation in bringing about mitotic transcription repression and loss of transcription complexes from mitotic DNA. Data from this study indicate that during lytic infection, HSV-1 DNA is nucleosome free, is devoid of 10–30-nm chromatin structure, and does not undergo mitotic chromosome condensation. The reasons why HSV-1 DNA remains free of histones and nucleosomes are unknown. It is possible that the cellular pool of histones is not sufficient to interact with the large amount of newly synthesized viral DNA after infection. It is also possible that viral DNA is maintained in a nucleosome-free state due to its strong binding to viral regulatory proteins such as ICP4 and ICP8 (de Bruyn Kops and Knipe 1988; Allen and Everett 1997). We have not examined whether DNA within viral replication compartments binds condensins or other SMC proteins. However, as the presence of nucleosomes appears to be a prerequisite for chromatin condensation, HSV-1 DNA may remain noncondensed simply due to the absence of nucleosomes (Newport 1987).

Our data show that transcription is repressed on both condensed and noncondensed genomes during mitosis. Although this suggests that mitotic transcription repression can occur in the absence of chromosome condensation, it is possible that the interaction of transcriptionally active DNA with template modifying factors could contribute to cessation of transcription, even in the absence of chromosome condensation. Such template modifying factors could include condensins, SMC proteins, or topoisomerases. Similarly, mitotic loss of chromatin remodeling factors such as SWI/SNF (Sif et al. 1998) or interaction of chromatin with negative regulatory proteins (Knoepfler and Eisenman 1999; Maldonado et al. 1999) could be involved in repressing transcription from either condensed or noncondensed templates. In addition, mitotic modifications to viral DNA-binding proteins such as ICP4 or ICP8 could contribute to mitotic transcription repression of the HSV-1 genome. Our conclusions support earlier in vitro studies that show repression of RNAP III transcription in mitotic Xenopus extracts in the presence of topoisomerase inhibitors or in the absence of normal nucleosome structure (Hartl et al. 1993).

Our experiments do not address the question of what biochemical mechanisms lead to mitotic transcription repression in the absence of nucleosomal chromatin condensation. However, our data are consistent with the hypothesis that mitosis-specific modifications to transcription proteins may be sufficient to bring about mitotic transcription repression. Biochemical studies show that mitosis-specific phosphorylations on members of the RNAP II basal transcription machinery inhibit transcription initiation in vitro. Similarly, mitotic phosphorylation of RNAP I and RNAP III transcription factors mediate transcription repression in vitro (Hartl et al. 1993; Gottesfeld et al. 1994; Heix et al. 1998; Kuhn et al. 1998; Klein and Grummt 1999). These in vitro studies suggest that mitotic phosphorylation alone could repress transcription of all three nuclear RNA polymerases during mitosis. Similarly, phosphorylation of transcription proteins after their removal from DNA early in mitosis may keep these proteins from reassociating with promoters, leading to loss of transcription factors from mitotic chromatin (Segil et al. 1991; Martinez-Balbas et al. 1995; Gottesfeld and Forbes 1997).

Our data show that RNAP II remains disengaged from both condensed and noncondensed DNA during mitosis. This suggests that chromosome condensation is not required for removal of transcription complexes from DNA or for keeping these complexes off mitotic chromosomes. However, it is possible that interactions of mitotic chromatin with condensation proteins such as condensins, SMC proteins and topoisomerases could be involved. Similarly, mitotic modifications to viral or host chromatin templates could make significant contributions to either establishing or maintaining transcription factor loss during mitosis, even in the absence of chromosome condensation. Our study does not address the question of how transcription complexes and activator proteins are lost from mitotic chromatin. It is possible that RNAP II elongation complexes may simply complete a round of transcription and terminate within the 15–30 min of prophase. Thereafter, RNAP II may be rendered incapable of reinitiating due to either mitosis-specific phosphorylations or to template modifications that occur after normal transcription termination. In this case, transcription complexes are not removed from mitotic chromatin, but are prevented from reinitiating a new round of transcription. Although this may be the case for RNAP II elongation complexes, it seems likely that other mechanisms operate to remove DNA binding factors such as TBP, Oct-1, and HSF1, as well as nascent RNA.

Our findings on HSV-1 mitotic transcription repression have interesting parallels with recent studies of mitotic repression of RNAP I transcription (Sirri et al. 2000). Although RNAP I transcription is repressed during mitosis, RNAP I and its transcription regulators remain localized to some nucleolar organizing regions during mitosis (Zatsepina et al. 1993; Weisenberger and Scheer 1995; Jordan et al. 1996; Roussel et al. 1996; Gebrane-Younes et al. 1997; Sirri et al. 1999). Nucleolar organizing regions that remain associated with the RNAP I transcription machinery during mitosis also appear to contain noncondensed chromatin. Recent studies (Sirri et al. 2000) show that rRNA transcription can be restored in mitotic cells after treatment with the cdc2-cyclin B kinase inhibitor roscovitine, although RNAP II transcription is not restored. It will be of interest to determine whether specific cdc2-cyclin B inhibitors can re-establish HSV-1 transcription in mitotic cells, on viral templates that remain noncondensed during mitosis.

In summary, our study suggests that nucleosomal chromatin condensation is not required for mitotic repression of RNAP II transcription or for dissociation of transcription factors from mitotic DNA. These conclusions are consistent with previously hypothesized roles for mitotic modifications to the RNAP II transcription machinery in effecting mitotic repression, but do not exclude the possibility that template modifications may contribute to mitotic repression in vivo.

	Acknowledgments

 
We are grateful to Michael Hendzel for helpful advice and discussions and to Dean Jackson for helpful discussion in establishing the protocol for labeling nascent RNA with halogenated nucleotides. We thank Maryse Fillion for preparation of stained cells for ESI analysis and Wei-Xiang Dong for excellent technical assistance during EM imaging. We also thank Priscilla Schaffer for providing the n212 virus and Henry Parker for review of the manuscript.

This work was supported by grants to C.A. Spencer from the Medical Research Council of Canada and the Alberta Cancer Board, and to D.P. Bazett-Jones from the Medical Research Council of Canada and the National Sciences and Engineering Research Council of Canada. C.A. Spencer is a Senior Scholar of the Alberta Heritage Foundation for Medical Research. M.J. Kruhlak is supported by a studentship award from University Technologies International (University of Calgary).

Submitted: 7 February 2000

Revised: 4 May 2000

Accepted: 25 May 2000

The online version of this article contains supplemental material.

Abbreviations used in this paper: 3-D, three-dimensional; DAPI, 4',6-diamidine-2-phenylindole; ESI, electron spectroscopic imaging; HSV-1, herpes simplex virus type 1; MOI, multiplicity of infection; RNAP, RNA polymerase.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

Akoulitchev S. Reinberg D. The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7, Genes Dev 12, 1998 3541–3550.[Abstract/Free Full Text]

Allen K.E. Everett R.D. Mutations which alter the DNA binding properties of the herpes simplex virus type 1 transactivating protein Vmw175 also affect its ability to support virus replication, J. Gen. Virol., 78, 1997, 2913–2922.[Abstract]

Bazett-Jones D.P. Hendzel M.J.. Electron spectroscopic imaging of chromatin, Methods, 17, 1999, 188–200.[Medline]

Bazett-Jones D.P. Hendzel M.J. Kruhlak M.J.. Stoichiometric analysis of protein- and nucleic acid-based structures in the cell nucleus, Micron, 30, 1999, 151–157.[Medline]

Besse S. Vigneron M. Pichard E. Puvion-Dutilleul F.. Synthesis and maturation of viral transcripts in herpes simplex virus type 1 infected HeLa cellsthe role of interchromatin granules, Gene Expr, 4, 1995, 143–161.[Medline]

Boisvert F.-M. Hendzel M.J. Bazett-Jones D.P.. Promyelocytic leukemia (PML) nuclear bodies are protein structures that do not accumulate RNA, J. Cell Biol., 148, 2000, 283–292.[Abstract/Free Full Text]

Cai W. Astor T.L. Liptak L.M. Cho C. Coen D.M. Schaffer P.A.. The herpes simplex virus type 1 regulatory protein ICP0 enhances virus replication during acute infection and reactivation from latency, J. Virol., 67, 1993, 7501–7512.[Abstract/Free Full Text]

Cosenza S.C. Carter R. Pena A. Donigan A. Borrelli M. Soprano D.R. Soprano K.J.. Growth-associated gene expression is not constant in cells traversing G- 1 after exiting mitosis, J. Cell Physiol., 147, 1991, 231–241.[Medline]

Cosenza S.C. Yumet G. Soprano D.R. Soprano K.J.. Induction of c-fos and c-jun mRNA at the M/G1 border is required for cell cycle progression, J. Cell Biochem., 55, 1994, 503–512.[Medline]

Dahmus M.E.. The role of multisite phosphorylation in the regulation of RNA polymerase II activity, Prog. Nucl. Acid Res. Mol. Biol., 48, 1994, 143–179.[Medline]

Dahmus M.E.. Reversible phosphorylation of the C-terminal domain of RNA polymerase II, J. Biol. Chem., 271, 1996, 19009–19012.[Free Full Text]

de Bruyn Kops A. Knipe D.M.. Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein, Cell, 55, 1988, 857–868.[Medline]

de Bruyn Kops A. Knipe D.M.. Preexisting nuclear architecture defines the intranuclear location of herpesvirus DNA replication structures, J. Virol., 68, 1994, 3512–3526.[Abstract/Free Full Text]

de Bruyn Kops A. Uprichard S.L. Chen M. Knipe D.M.. Comparison of the intranuclear distributions of herpes simplex virus proteins involved in various viral functions, Virology, 252, 1998, 162–178.[Medline]

Everett R.D. Earnshaw W.C. Findlay J. Lomonte P.. Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110, EMBO (Eur. Mol. Biol. Organ.) J., 18, 1999, 1526–1538.[Medline]

Gebara M.M. Sayre M.H. Corden J.L.. Phosphorylation of the carboxy-terminal repeat domain in RNA polymerase II by cyclin-dependent kinases is sufficient to inhibit transcription, J. Cell Biochem., 64, 1997, 390–402.[Medline]

Gebrane-Younes J. Fomproix N. Hernandez-Verdun D.. When rDNA transcription is arrested during mitosis, UBF is still associated with non-condensed rDNA, J. Cell Sci., 110, 1997, 2429–2440.[Abstract]

Godowski P.J. Knipe D.M.. Mutations in the major DNA-binding protein gene of herpes simplex virus type 1 result in increased levels of viral gene expression, J. Virol., 47, 1983, 478–486.[Abstract/Free Full Text]

Godowski P.J. Knipe D.M.. Transcriptional control of herpesvirus gene expressiongene functions required for positive and negative regulation, Proc. Natl. Acad. Sci. USA., 83, 1986, 256–260.[Abstract/Free Full Text]

Gottesfeld J.M. Forbes D.J.. Mitotic repression of the transcriptional machinery, Trends Biochem. Sci., 22, 1997, 197–202.[Medline]

Gottesfeld J.M. Wolf V.J. Dang T. Forbes D.J. Hartl P.. Mitotic repression of RNA polymerase III transcription in vitro mediated by phosphorylation of a TFIIIB component, Science, 263, 1994, 81–84.[Abstract/Free Full Text]

Gourves A.S. Le Gac N.T. Villani G. Boehmer P.E. Johnson N.P.. Equilibrium binding of single-stranded DNA with herpes simplex virus type I-coded single-stranded DNA-binding protein, ICP8, J. Biol. Chem., 275, 2000, 10864–10869.[Abstract/Free Full Text]

Haider S.R. Juan G. Traganos F. Darzynkiewicz Z.. Immunoseparation and immunodetection of nucleic acids labeled with halogenated nucleotides, Exp. Cell Res., 234, 1997, 498–506.[Medline]

Hartl P. Gottesfeld J. Forbes D.J.. Mitotic repression of transcription in vitro, J. Cell Biol., 120, 1993, 613–624.[Abstract/Free Full Text]

Heix J. Vente A. Voit R. Budde A. Michaelidis T.M. Grummt I.. Mitotic silencing of human rRNA synthesisinactivation of the promoter selectivity factor SL1 by cdc2/cyclin B-mediated phosphorylation, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 7373–7381.[Medline]

Hendzel M.J. Bazett-Jones D.P.. Probing nuclear ultrastructure by electron spectroscopic imaging, J. Microsc, 182, 1996, 1–14.[Medline]

Hendzel M.J. Kruhlak M.J. Bazett-Jones D.P.. Electron spectroscopic imaging and correlative fluorescence microscopy of the cell nucleus, Scanning Microsc, 12, 1998, 306–316.

Hendzel M.J. Boisvert F. Bazett-Jones D.P.. Direct visualization of a protein nuclear architecture, Mol. Biol. Cell., 10, 1999, 2051–2062.[Abstract/Free Full Text]

Jordan P. Mannervik M. Tora L. Carmo-Fonseca M.. In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive, J. Cell Biol., 133, 1996, 225–234.[Abstract/Free Full Text]

Kattar-Cooley P. Wilcox K.W.. Characterization of the DNA-binding properties of herpes simplex virus regulatory protein ICP4, J. Virol., 63, 1989, 696–704.[Abstract/Free Full Text]

Kim E. Du L. Bregman D.B. Warren S.L.. Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA, J. Cell Biol., 136, 1997, 19–28.[Abstract/Free Full Text]

Klein J. Grummt I.. Cell cycle-dependent regulation of RNA polymerase I transcriptionthe nucleolar transcription factor UBF is inactive in mitosis and early G1, Proc. Natl. Acad. Sci. USA., 96, 1999, 6096–6101.[Abstract/Free Full Text]

Knipe D.M.. Virus–host cell interactions, Fields B. Knipe D., Virology, 1990, 293–316 Raven Press New York.

Knipe D.M. Spang A.E. Definition of a series of stages in the association of two herpesviral proteins with the cell nucleus, J. Virol., 43, 1982, 314–324.[Abstract/Free Full Text]

Knipe D.M. Senechek D. Rice S.A. Smith J.L.. Stages in the nuclear association of the herpes simplex virus transcriptional activator protein ICP4, J. Virol., 61, 1987, 276–284.[Abstract/Free Full Text]

Knoepfler P.S. Eisenman R.N.. Sin meets NuRD and other tails of repression, Cell, 99, 1999, 447–450.[Medline]

Kuhn A. Vente A. Doree M. Grummt I.. Mitotic phosphorylation of the TBP-containing factor SL1 represses ribosomal gene transcription, J. Mol. Biol., 284, 1998, 1–5.[Medline]

Lee C.K. Knipe D.M.. An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8, J. Virol., 54, 1985, 731–738.[Abstract/Free Full Text]

Leinbach S.S. Summers W.C.. The structure of herpes simplex virus type 1 DNA as probed by micrococcal nuclease digestion, J. Gen. Virol., 51, 1980, 45–59.[Abstract/Free Full Text]

Leinbach S.S. Casto J.F.. Identification and characterization of deoxyribonucleoprotein complexes containing the major DNA-binding protein of herpes simplex virus type 1, Virology, 131, 1983, 274–286.[Medline]

Lentine A.F. Bachenheimer S.L.. Intracellular organization of herpes simplex virus type 1 DNA assayed by staphylococcal nuclease sensitivity, Virus Res, 16, 1990, 275–292.[Medline]

Leresche A. Wolf V.J. Gottesfeld J.M.. Repression of RNA polymerase II and III transcription during M phase of the cell cycle, Exp. Cell Res., 229, 1996, 282–288.[Medline]

Locklear L. Jr. Ridsdale J.A. Bazett-Jones D.P. Davie J.R.. Ultrastructure of transcriptionally competent chromatin, Nucleic Acids Res, 18, 1990, 7015–7024.[Abstract/Free Full Text]

Lomonte P. Everett R.D.. Herpes simplex virus type 1 immediate-early protein Vmw110 inhibits progression of cells through mitosis and from G(1) into S phase of the cell cycle, J. Virol., 73, 1999, 9456–9467.[Abstract/Free Full Text]

Long J.J. Leresche A. Kriwacki R.W. Gottesfeld J.M.. Repression of TFIIH transcriptional activity and TFIIH-associated cdk7 kinase activity at mitosis, Mol. Cell. Biol., 18, 1998, 1467–1476.[Abstract/Free Full Text]

Lukonis C.J. Weller S.K.. Formation of herpes simplex virus type 1 replication compartments by transfectionrequirements and localization to nuclear domain 10, J. Virol., 71, 1997, 2390–2399.[Abstract]

Maldonado E. Hampsey M. Reinberg D.. Repressiontargeting the heart of the matter, Cell, 99, 1999, 455–458.[Medline]

Martinez-Balbas M.A. Dey A. Rabindran S.K. Ozato K. Wu C.. Displacement of sequence-specific transcription factors from mitotic chromatin, Cell, 83, 1995, 29–38.[Medline]

Michael N. Roizman B.. Binding of the herpes simplex virus major regulatory protein to viral DNA, Proc. Natl. Acad. Sci. USA., 86, 1989, 9808–9812.[Abstract/Free Full Text]

Mouttet M.E. Guetard D. Bechet J.M.. Random cleavage of intranuclear herpes simplex virus DNA by micrococcal nuclease, FEBS Lett, 100, 1979, 107–109.[Medline]

Muggeridge M.I. Fraser N.W.. Chromosomal organization of the herpes simplex virus genome during acute infection of the mouse central nervous system, J. Virol., 59, 1986, 764–767.[Abstract/Free Full Text]

Muller U. Schroder C.H. Zentgraf H. Franke W.W.. Coexistence of nucleosomal and various non-nucleosomal chromatin configurations in cells infected with herpes simplex virus, Eur. J. Cell Biol., 23, 1980, 197–203.[Medline]

Newport J.. Nuclear reconstitution in vitrostages of assembly around protein-free DNA, Cell, 48, 1987, 205–217.[Medline]

O'Farrell P.H.. Developmental biology. Big genes and little genes and deadlines for transcription, Nature, 359, 1992, 366–367.[Medline]

Parsons G.G. Spencer C.A.. Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes, Mol. Cell. Biol., 17, 1997, 5791–5802.[Abstract]

Phelan A. Dunlop J. Patel A.H. Stow N.D. Clements J.B.. Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors, J. Virol., 71, 1997, 1124–1132.[Abstract]

Pignatti P.F. Cassai E.. Analysis of herpes simplex virus nucleoprotein complexes extracted from infected cells, J. Virol., 36, 1980, 816–828.[Abstract/Free Full Text]

Prescott D.M. Bender M.A.. Synthesis of RNA and protein during mitosis in mammalian tissue culture cells, Exp. Cell. Res., 26, 1962, 260–268.[Medline]

Puvion-Dutilleul F. Laithier M. Sheldrick P.. Ultrastructural localization of the herpes simplex virus major DNA-binding protein in the nucleus of infected cells, J. Gen. Virol., 66, 1985, 15–30.[Abstract/Free Full Text]

Quinlan M.P. Chen L.B. Knipe D.M.. The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication, Cell, 36, 1984, 857–868.[Medline]

Randall R.E. Dinwoodie N.. Intranuclear localization of herpes simplex virus immediate-early and delayed-early proteinsevidence that ICP 4 is associated with progeny virus DNA, J. Gen. Virol., 67, 1986, 2163–2177.[Abstract/Free Full Text]

Rice S.A. Long M.C. Lam V. Spencer C.A.. RNA polymerase II is aberrantly phosphorylated and localized to viral replication compartments following herpes simplex virus infection, J. Virol., 68, 1994, 988–1001.[Abstract/Free Full Text]

Rice S.A. Long M.C. Lam V. Schaffer P.A. Spencer C.A.. Herpes simplex virus immediate-early protein ICP22 is required for viral modification of host RNA polymerase II and establishment of the normal viral transcription program, J. Virol., 69, 1995, 5550–5559.[Abstract]

Rixon F.J. Atkinson M.A. Hay J.. Intranuclear distribution of herpes simplex virus type 2 DNA synthesisexamination by light and electron microscopy, J. Gen. Virol., 64, 1983, 2087–2092.[Abstract/Free Full Text]

Roussel P. Andre C. Comai L. Hernandez-Verdun D.. The rDNA transcription machinery is assembled during mitosis in active NORs and absent in inactive NORs, J. Cell Biol., 133, 1996, 235–246.[Abstract/Free Full Text]

Ruyechan W.T.. The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration, J. Virol., 46, 1983, 661–666.[Abstract/Free Full Text]

Sacks W.R. Schaffer P.A.. Deletion mutants in the gene encoding the herpes simplex virus type 1 immediate-early protein ICP0 exhibit impaired growth in cell culture, J. Virol., 61, 1987, 829–839.[Abstract/Free Full Text]

Sahasrabuddhe C.G. Adlakha R.C. Rao P.N.. Phosphorylation of non-histone proteins associated with mitosis in HeLa cells, Exp. Cell Res., 153, 1984, 439–450.[Medline]

Segil N. Roberts S.B. Heintz N.. Mitotic phosphorylation of the Oct-1 homeodomain and regulation of Oct-1 DNA binding activity, Science, 254, 1991, 1814–1816.[Abstract/Free Full Text]

Segil N. Guermah M. Hoffmann A. Roeder R.G. Heintz N.. Mitotic regulation of TFIIDinhibition of activator-dependent transcription and changes in subcellular localization, Genes Dev, 10, 1996, 2389–2400.[Abstract/Free Full Text]

Shermoen A.W. O'Farrell P.H.. Progression of the cell cycle through mitosis leads to abortion of nascent transcripts, Cell, 67, 1991, 303–310.[Medline]

Sif S. Stukenberg P.T. Kirschner M.W. Kingston R.E.. Mitotic inactivation of a human SWI/SNF chromatin remodeling complex, Genes Dev, 12, 1998, 2842–2851.[Abstract/Free Full Text]

Sinden R.R. Pettijohn D.E. Francke B.. Organization of herpes simplex virus type 1 deoxyribonucleic acid during replication probed in living cells with 4,5',8-trimethylpsoralen, Biochemistry, 21, 1982, 4484–4490.[Medline]

Sirri V. Roussel P. Hernandez-Verdun D.. The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery, J. Cell Sci., 112, 1999, 3259–3268.[Abstract]

Sirri V. Roussel P. Hernandez-Verdun D.. In vivo release of mitotic silencing of ribosomal gene transcription does not give rise to precursor ribosomal RNA processing, J. Cell Biol., 148, 2000, 259–270.[Abstract/Free Full Text]

Smiley J.R. Smibert C. Everett R.D.. Regulation of cellular genes by HSV products, Wagner E.. Herpesvirus Transcription and Its Regulation, 1991, 151–179 CRC Press, Inc. Boca Raton, FL.

Spencer C.A. Dahmus M.E. Rice S.A. Repression of host RNA polymerase II transcription by herpes simplex virus type 1, J. Virol., 71, 1997, 2031–2040.[Abstract]

Taylor J.H.. Nucleic acid synthesis in relation to the cell division cycle, Annu. NY Acad. Sci., 90, 1960, 409–421.[Medline]

Thompson N.E. Steinberg T.H. Aronson D.B. Burgess R.R.. Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II, J. Biol. Chem., 264, 1989, 11511–11520.[Abstract/Free Full Text]

Uprichard S.L. Knipe D.M.. Assembly of herpes simplex virus replication proteins at two distinct intranuclear sites, Virology, 229, 1997, 113–125.[Medline]

Weisenberger D. Scheer U.. A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis, J. Cell Biol., 129, 1995, 561–575.[Abstract/Free Full Text]

White R.J. Gottlieb T.M. Downes C.S. Jackson S.P.. Cell cycle regulation of RNA polymerase III transcription, Mol. Cell. Biol., 15, 1995, 6653–6662.[Abstract]

Wilcock D. Lane D.P.. Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells, Nature, 349, 1991, 429–431.[Medline]

Wrenn C.K. Katzenellenbogen B.S.. Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cellsoptimization and effect of ligand, Mol. Endocrinol., 4, 1990, 1647–1654.[Abstract/Free Full Text]

Yao F. Schaffer P.A.. An activity specified by the osteosarcoma line U2OS can substitute functionally for ICP0, a major regulatory protein of herpes simplex virus type 1, J. Virol., 69, 1995, 6249–6258.[Abstract]

Zatsepina O.V. Voit R. Grummt I. Spring H. Semenov M.V. Trendelenburg M.F.. The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes, Chromosoma, 102, 1993, 599–611.[Medline]

Zetterberg A. Larsson O.. Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells, Proc. Natl. Acad. Sci. USA., 82, 1985, 5365–5369.[Abstract/Free Full Text]

Zhong L. Hayward G.S.. Assembly of complete, functionally active herpes simplex virus DNA replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays, J. Virol., 71, 1997, 3146–3160.[Abstract]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Facebook

Reddit

Technorati

Twitter

This Article Abstract Full Text (PDF, 684K) PPT slides of all figures Supplememtal Material Index Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Spencer, C. A. Articles by Bazett-Jones, D. P. Search for Related Content PubMed PubMed Citation Articles by Spencer, C. A. Articles by Bazett-Jones, D. P. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Social Bookmarking                            What's this?