3259
Journal of Cell Science 112, 3259-3268 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0557
The mitotically phosphorylated form of the transcription termination factor
TTF-1 is associated with the repressed rDNA transcription machinery
Valentina Sirri, Pascal Roussel and Danièle Hernandez-Verdun*
Institut Jacques Monod, UMR 7592, Paris, France
*Author for correspondence at Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France (e-mail: [email protected])
Accepted 15 July; published on WWW 22 September 1999
SUMMARY
The transcription termination factor TTF-1 exerts two
functions in ribosomal gene (rDNA) transcription:
facilitating initiation and mediating termination of
transcription. Using HeLa cells, we show that TTF-1
protein is colocalized with the active transcription
machinery in the nucleolus and also with the inactive
machinery present in certain mitotic nucleolar organizer
regions (NORs) when rDNA transcription is repressed. We
also show that TTF-1 is specifically phosphorylated during
mitosis in a manner dependent on the cdc2-cyclin B kinase
pathway and on an okadaic acid-sensitive phosphatase.
Interestingly, the mitotically phosphorylated form of
TTF-1 appearing at the G2/M transition phase was more
easily solubilized than was the interphase form. This
INTRODUCTION
Global inhibition of transcription occurs during mitosis in
higher eukaryotes. Recently several mechanisms for mitotic
repression of transcription have been proposed for RNA
polymerase (RNA pol) II and III (for a review, see Gottesfeld
and Forbes, 1997). Amongst these mechanisms, the best
substantiated mechanism in vitro is mitotic phosphorylation
leading to inhibition of transcription factor activity (Gottesfeld
et al., 1994; Hartl et al., 1993; Leresche et al., 1996; Segil et
al., 1991; White et al., 1995). In vivo, mitotic displacement or
release of transcription factors and ejection of RNA pol II
elongation complexes have also been observed (MartinezBalbàs et al., 1995; Parsons and Spencer, 1997; Segil et al.,
1996). However, other mechanisms can also participate in
silencing of mitotic transcription, such as chromatin
condensation, the action of general repressor proteins and
premature termination (Gottesfeld and Forbes, 1997).
Concerning inhibition of RNA pol I transcription during
mitosis, there are indications that the ribosomal gene (rDNA)
transcription machinery, as defined in vitro, remains associated
in the secondary constriction of certain chromosomes. These
are the chromosome regions designated nucleolar organizer
regions (NORs), where rDNAs are clustered. The fact that
RNA pol I complexes remain associated with rDNAs during
mitosis is supported by the detection of certain RNA pol I
indicates that the chromatin-binding affinity of TTF-1
appears to be different in mitotic chromosomes compared
to the interphase nucleolus. Correlated with this, the other
DNA-binding factor, UBF, which interferes with chromatin
conformation in the rDNA promoter, was more strongly
bound to rDNA during mitosis than at interphase. The
reorganization of the mitotic rDNA promoter might be
induced by phosphorylation of certain components of the
rDNA transcription machinery and participate in silencing
of rDNA during mitosis.
Key words: Ribosomal gene, UBF, TTF-1, Cell cycle, cdc2-cyclin B
kinase
subunits (Jordan et al., 1996; Matsui and Sandberg, 1985;
Matsui et al., 1979; Roussel et al., 1996; Scheer et al., 1993;
Weisenberger and Scheer, 1995) in the mitotic NORs and by
immunoprecipitation of the RNA pol I complexes that are still
assembled during mitosis (Roussel et al., 1996). In addition,
the upstream binding factor (UBF), a transcription factor
specific for rDNAs, was detected in mitotic NORs (Rendon et
al., 1992; Roussel et al., 1996, 1993; Zatsepina et al., 1993).
Similarly the promoter selectivity factor (SL1) that functions
cooperatively with UBF to activate RNA pol I transcription
(Bell et al., 1988) was localized to the same sites as the RNA
pol I complex (Roussel et al., 1996). Consequently, the three
components (RNA pol I complex, UBF and SL1) of the rDNA
transcription machinery that are sufficient to promote rDNA
transcription in vitro (for a review, see Moss and Stefanovsky,
1995), are found in the NORs during mitosis (Bell et al., 1989;
Jordan et al., 1996; Roussel et al., 1996; Weisenberger and
Scheer, 1995). Therefore, dissociation of the transcription
machinery cannot explain mitotic arrest of rDNA transcription.
Moreover, it was recently reported that mitotic phosphorylation
of SL1 by the cdc2-cyclin B kinase impairs the association of
SL1 with UBF and consequently represses rDNA transcription
during mitosis (Heix et al., 1998; Kuhn et al., 1998).
In the integrated structure of the mitotic cells, several
parameters, in addition to the transcription machinery per se,
must interact and be coordinated to achieve mitotic inhibition
3260 V. Sirri and others
of rDNA transcription. In active interphase cells, the
transcription termination factor (TTF-1) (for a review, see
Reeder and Lang, 1994) that binds terminator elements present
both upstream and downstream of rDNAs, seems to play a
crucial role in the global structural organization of the rDNA
transcription units (Sander and Grummt, 1997) as well as in
the position of the nucleosome close to the transcription start
site (Längst et al., 1998, 1997b). In addition, in agreement with
the in vivo effect of the promoter-proximal terminator element
(Mitchelson and Moss, 1987; Moss et al., 1992), TTF-1 was
reported to be an activator of RNA pol I transcription (Längst
et al., 1997a). However, there is presently only sparse
information concerning TTF-1 in vivo when the rDNA
transcription machinery and chromatin are integrated in a
highly ordered organization. Furthermore, nothing is known
concerning the fate of TTF-1 during mitosis when the rDNA
transcription machinery is repressed. Cloning of the human and
mouse TTF-1 revealed homology between the DNA-binding
domains of TTF-1 and Reb1p, the RNA pol I termination factor
in Saccharomyces cerevisiae (Evers and Grummt, 1995) and
similar mechanisms are used by yeast and mammals for
termination of RNA pol I transcription (Mason et al., 1997).
Moreover, as for TTF-1, Reb1p performs other functions in the
cell in addition to its role in RNA pol I termination. In
particular, Reb1p can rearrange nucleosomes (Fedor et al.,
1988) and this activity could remodel chromatin over the yeast
promoter in a manner similar to the way TTF-1 appears to act
at the level of the mouse promoter (Längst et al., 1997b).
In the present study, we have characterized the first antibody
directed against human TTF-1. Using this probe, TTF-1 was
found colocalized with the rDNA transcription machinery in
the nucleolus during interphase and in certain NORs during
mitosis. Consequently, as was previously established for RNA
pol I, UBF and SL1, TTF-1 is not released from the rDNAs
during mitotic repression of rDNA transcription. Moreover,
TTF-1 was shown to be specifically phosphorylated during
mitosis in a manner dependent on the cdc2-cyclin B kinase
pathway. Mitotic phosphorylation of TTF-1, as well as the
lower DNA-binding affinity of the phosphorylated TTF-1,
might participate in mitotic silencing of the rDNA transcription
machinery.
MATERIALS AND METHODS
Antisera and antibodies
The rabbit polyclonal anti-mouse TTF-1 antibodies were obtained
from I. Grummt (German Cancer research Center, Heidelberg,
Germany). The human autoimmune sera with specificity against UBF
(A17) and an RNA pol I subunit (V11) have been described (Roussel
et al., 1996, 1993). A third autoimmune serum, P21, was characterized
in the present study. Except for P21, the other autoimmune sera
contained only autoantibodies characterized as IgGs. The autoimmune
serum P21 contained autoantibodies characterized as IgGs and IgAs.
Protein A covalently linked to agarose was used to deplete IgG
molecules of P21. The absence of contaminating IgGs in the
remaining IgA fraction was checked by immunoblotting and
immunofluorescent labeling. The IgA fraction prepared from P21 was
designated IgA-P21. The rabbit polyclonal anti-phosphohistone H1
antibodies were from Upstate Biotechnology (Lake Placid, NY).
FITC- and Texas Red-conjugated secondary antibodies with
specificity for human IgAs and human IgGs, respectively, and
peroxidase-conjugated secondary antibodies with specificity for
human IgAs were obtained from Jackson ImmunoResearch
Laboratories, Inc. (West Grove, PA). Peroxidase-conjugated
secondary antibodies with specificity for human or rabbit IgGs were
obtained from Amersham (France).
Purified and enriched nucleolar proteins
Human RNA pol I and SL1-depleted extracts were kindly provided
by L. Comai (School of Medicine, Department of Molecular
Microbiology and Immunology, CA). Nucleolin was from P.
Belenguer and F. Amalric (Toulouse, France). Recombinant human
TATA-binding protein (TBP)-associated factor (hTAFI-110), a gift of
H. Beckmann, was purified as previously described (Zomerdijk et al.,
1994). Calf thymus topoisomerase I was from Bethesda Research
Laboratories (France). GU/RNA helicase II was provided by B. C.
Valdez (Baylor College of Medicine, Huston).
Cell culture and synchronization
HeLa cells were cultured in Eagle’s minimum essential medium
(Sigma, France) supplemented with 10% (v/v) fetal calf serum. Cells
were seeded 3 times a week, and extracts were prepared 24 hours
after seeding from exponentially growing cells. For M phase
synchronization, HeLa cells were blocked in mitosis by colchicine
treatment (0.02 µg/ml) for 14 hours. Mitotic cells were harvested by
mechanical shock. For early G1 phase synchronization, the cells
blocked in mitosis were grown in fresh medium without colchicine
for 5 hours. For S phase synchronization, a double thymidine block
was applied. Exponentially growing cells were exposed to 2 mM
thymidine for 16 hours and then resuspended in fresh medium
without thymidine and supplemented with 0.024 mM 2′deoxycytidine and allowed to grow for 9 hours. Thymidine (2 mM)
was then added again for 16 hours, causing cells to accumulate near
the G1/S boundary. The cells were harvested at different times (30
minutes to 6 hours) after release from the second block, yielding a
population highly enriched in S phase. The G2 population was
reached at 8 hours after release from the double thymidine block.
For protein analyses, the cells were resuspended in SDS-PAGE
sample buffer (Laemmli, 1970), sonicated, boiled for 5 minutes and
centrifuged. The supernatant corresponding to the same number of
cells was loaded into the gel. For flow cytometry analyses, the cells
were washed twice in PBS, and fixed in 80% ethanol at −20°C. The
cells were washed in PBS and stained with 0.5 µg/ml of Hoechst
33342 to label the DNA. The fluorescence of the DNA was measured
using an EPICS ELITE Flow Cytometer (Coultronic France, Coulter
Corporations Margency, France).
Preparation of chromosome, nuclear and nucleolar protein
extracts for 1-D and 2-D gel electrophoresis
The procedure used to isolate chromosomes was described previously
(Roussel et al., 1996). For 1-D electrophoresis, the pellet containing
the chromosomes was resuspended in SDS-PAGE sample buffer,
sonicated, boiled for 5 minutes and centrifuged. For 2-D
electrophoresis the pellet was resuspended in lysis buffer containing
9.5 M urea, 2% Nonidet P-40 (NP-40), 2% ampholytes (1 part BioLyte 3/10 and 2 parts Bio-Lyte 5/7) and 5% β-mercaptoethanol.
Samples were lysed for 1 hour at room temperature and centrifuged
at 100,000 g for 30 minutes. The proteins in the supernatant
corresponding to the cytoplasmic fraction were precipitated by 5 vol
of cold acetone and kept at −20°C for 1 hour. The precipitated proteins
were collected by centrifugation and solubilized in SDS-PAGE
sample buffer. The cytoplasmic extracts were sonicated, boiled for 5
minutes and centrifuged.
Nuclear and nucleolar protein extracts were prepared from
exponentially growing HeLa cells as previously described (Roussel et
al., 1996). All steps were performed at 4°C and all the solutions
contained a protease inhibitor cocktail (Boehringer Mannheim,
France). The nuclear and nucleolar proteins were solubilized in SDS-
TTF-1 during mitosis 3261
PAGE sample buffer for 1-D electrophoresis. For 2-D electrophoresis
the proteins were solubilized as described above.
Salt solubilization from interphase and mitotic cells
Salt solubilization of nuclei, chromosomes, and permeabilized mitotic
cells was performed as follows. Nuclei and chromosomes were
purified from HeLa cells as described above. The mitotic cells were
permeabilized in PBS containing 0.1% NP-40 for 2 minutes. All steps
were performed at 4°C using a protease inhibitor cocktail. Samples
were resuspended in the extraction buffer (10 mM Hepes-KOH, pH
7.5 and 1 mM EDTA), containing increasing concentrations of NaCl
(0.1-1 M), incubated on ice for 10 minutes, and centrifuged at 6,000
g for 10 minutes (Yan et al., 1993). Another technique for the
extraction of DNA-binding proteins (Andrews and Faller, 1991) was
also used. In brief, interphase and mitotic cells were resuspended in
lysis buffer (10 mM Hepes-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM
KCl and 0.5 mM DTT) and centrifuged. The pellets were incubated
on ice for 20 minutes in extraction buffer (10 mM Hepes-KOH, pH
7.9, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT and 25% glycerol)
containing increasing concentrations of NaCl (0.1-1 M). With both
methods, after centrifugation the supernatant and the pellet were
normalized to an equal volume of SDS-PAGE sample buffer for
quantitative comparison.
Phosphatase treatment and analysis by 1-D and 2-D gel
electrophoresis
The chromosome pellets prepared as described above, were
resuspended in lambda phosphatase (λ-PPase) buffer (50 mM TrisHCl, 0.1 mM Na2EDTA, 5 mM DTT and 0.01% Brij 35)
supplemented with 2 mM MnCl2 and a protease inhibitor cocktail and
incubated with 1,000 units of λ-PPase (Biolabs, New England) for 30
minutes at 30°C. Controls were treated in the same way but without
λ-PPase. The reaction was stopped by adding SDS-PAGE sample
buffer for 1-D electrophoresis, or lysis buffer for 2-D electrophoresis.
To visualize a migration shift, a 7.5% SDS-polyacrylamide gel was
used for 1-D analysis.
1-D and 2-D gel electrophoresis and electrotransfer
Proteins were separated by 1-D gel electrophoresis using a Protean
II cell (Bio-Rad Laboratories, Richmond, CA) according to
established methods (Laemmli, 1970). For 2-D gel electrophoresis,
the first dimension was isoelectrofocalization in 3.5%
polyacrylamide gels crosslinked with 0.3% piperazine diacrylamide
(Bio-Rad Laboratories, Richmond, CA) and contained 9 M urea, 2%
ampholytes (1 part Bio-Lyte 3/10 and 2 parts Bio-Lyte 5/7) and 2%
NP-40. The anode and the cathode solutions were 0.74 M
phosphoric acid and 0.75 M ethylenediamine, respectively. The rod
gels were run for 10 minutes at 500 V followed by 3.5 hours at 750
V and were then equilibrated (Beis and Lazou, 1990). For the second
dimension, electrophoresis was performed in 8% SDSpolyacrylamide gels. After 1-D and 2-D gel electrophoresis, the
polypeptides were electrotransferred to reinforced nitrocellulose
membranes (BA-S 85, Schleicher & Schuell, Dassel). To visualize
the total proteins, the membranes were stained using 0.1% Ponceau
Red in 0.3% TCA (Sigma, France) and Auro-dye total protein
staining methods (Amersham). For immunoblotting, the strips were
blocked by incubation for 1 hour in PBS containing 5% (w/v) dried
milk and 0.05% (v/v) Tween 20, and incubated with the sera for 2
hours in the same buffer. They were then washed three times with
PBS containing 5% (w/v) dried milk and 0.5% (v/v) Tween 20 and
incubated for 1 hour in the presence of HRP-labeled second
antibodies. After several rounds of washing, the HRP activity was
detected using the enhanced chemiluminescence kit (Super-Signal,
Pierce Chem. Co. Rockford, IL) and recorded on a Fuji X-Ray
film. For reprobing, the blots were incubated at room temperature
for 5 minutes in 3 M KSCN to remove antibodies and washed with
PBS.
Kinase inhibitor treatments
Two highly selective inhibitors, roscovitine (Biomol, Tebu, France)
and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB, Sigma)
were used to inhibit cdc2-cyclin B kinase and casein kinase II,
respectively. HeLa cells at late S phase (6 hours after release from the
double thymidine block), were treated or not with 25 µM roscovitine
or 60 µM DRB for 2 hours. Colchicine (0.02 µg/ml) was then added
and treatment pursued for 14 hours. Non-adhering mitotic and
adhering G2 phase cells were harvested by mechanical shock and
trypsin treatment, respectively. Colchicine-arrested mitotic HeLa cells
were treated with 75 µM roscovitine for 15, 45 and 120 minutes. For
okadaic acid treatment, colchicine-arrested mitotic HeLa cells were
treated with 0.5 µM okadaic acid for 60 minutes, roscovitine (75 µM)
was then added and treatment continued for 30 minutes. Cells were
lysed in SDS-PAGE sample buffer as described above. The same
amount of proteins was run on a 10% polyacrylamide gel (ratio
acrylamide/bis-acrylamide, 30/0.2) and transferred to a nitrocellulose
membrane for western blot analysis.
Immunofluorescence labeling
For immunolocalization, HeLa cells were grown as monolayers on
glass slides. Cell monolayers were rinsed in PBS and fixed with 80%
ethanol at −20°C for 8 minutes. HeLa chromosome spreading for
immunolocalization was carried out as previously described (Roussel
et al., 1996) and DNA visualized with 4,6-diamidino-2-phenylindole
dihydrochloride (DAPI). Fluorescence microscopy was performed
using a Leitz DMRB microscope. The superimposition of images was
obtained by scanning micrographs. Images were then assembled using
Canvas and Adobe Photoshop and printed directly from the computer
on a printer (ColorEase PS Printer, Kodak). Confocal laser scanning
microscopy was performed using a BioRad MRC-600, mounted on
an Optiphot II Nikon microscope equipped with a 60× objective (plan
apo; NA 1.4). An Argon ion laser adjusted to 488 nm was used for
the fluorescein signal, and a Helium-Neon ion laser adjusted to 543
nm for Texas Red, as already described (Roussel et al., 1996).
RESULTS
The autoimmune serum IgA-P21 is directed against
human TTF-1
Using nucleolar extracts prepared from actively growing HeLa
cells, the IgA-fraction prepared from P21 (IgA-P21)
recognized only a 105 kDa polypeptide, as shown by
immunoblotting (Fig. 1A, lane 1). This protein was also present
in chromosome-associated protein extracts prepared from
mitotic cells (Fig. 1A, lane 2) but not in cytoplasmic extracts
(not shown). We first established that IgA-P21 did not
recognize nucleolar proteins described as possessing a similar
molecular mass. No positive reaction was observed using IgAP21 on nucleolin purified from CHO cells, topoisomerase I
purified from calf thymus, GU/RNA helicase II, or purified
recombinant hTAFI-110 (not shown). Analysis of SL1depleted extracts and of purified RNA pol I confirmed that the
nucleolar 105 kDa protein is not the hTAFI-110 moiety of the
SL1 complex (Fig. 1A, lane 3) nor a component of the RNA
pol I complex (Fig. 1A, lane 4). By 2-D western blot analysis
we demonstrated that the 105 kDa polypeptide is a basic (pI
9.5) nucleolar protein (Fig. 1B). Because in human cells,
TTF-1 (Evers and Grummt, 1995) is to date the only nucleolar
protein described as possessing a similar apparent molecular
mass and pI, IgA-P21 was compared with a polyclonal serum
(α-mTTF-1) directed against the murine TTF-1. In
preparations of nuclear proteins, both antibodies recognized a
3262 V. Sirri and others
Fig. 1. Identification of IgA-P21 as anti-TTF-1
antibodies. (A) Nucleolar, chromosomeassociated protein, SL1-depleted and purified
human RNA pol I (hRNA pol I) extracts
revealed by western blot (SDS-8% PAGE) with
IgA-P21 serum. The 105 kDa polypeptide
revealed by IgA-P21 was present in nucleolar,
chromosome-associated and SL1-depleted
protein extracts, and absent from purified
hRNA pol I. (B) HeLa nuclear extracts were
separated by 2-D electrophoresis and
transferred to a nitrocellulose filter. Nuclear
proteins separated in basic conditions were
revealed by total gold staining and probed with
IgA-P21. IgA-P21 serum directed against the
105 kDa polypeptide recognized a basic spot
(pI 9.5) by 2-D electrophoresis. (C-D) HeLa
nuclear proteins separated by 1-D (C) and 2-D
gel electrophoresis (D) were transferred to
membranes and probed with IgA-P21 and antimouse TTF-1 antibodies (α-mTTF-1). Both
antibodies revealed the same band at 105 kDa
on a 1-D gel and the same spot at pI 9.5 on a 2D gel.
band at 105 kDa on a 1-D gel (Fig. 1C) and the same spot (pI
9.5) on a 2-D gel (Fig. 1D). The IgA-P21 is the first serum to
be characterized that is directed against human TTF-1.
Colocalization of RNA Pol I, UBF and TTF-1 during
the cell cycle
The IgA-P21 was used for colocalization, taking advantage of
the discrimination between IgG and IgA antibodies. During
interphase RNA pol I and TTF-1 were mainly colocalized,
being detected in the same optical section by confocal
microscopy (not shown). Similar results were obtained using
the IgA-P21 and the anti-UBF antibodies (not shown). Taken
together these results demonstrate that UBF, RNA pol I and
TTF-1 are mainly colocalized in nucleoli during interphase.
During mitosis a comparison of the distribution of TTF-1,
UBF and RNA pol I was performed at all mitotic stages and
on chromosome spreads prepared from HeLa cells. Labeling
corresponding to RNA pol I and TTF-1 appeared in discrete
spots in association with chromosomes. The fluorescent
patterns observed for RNA pol I and for TTF-1 in the same
mitotic HeLa cells were similar (not shown). The same number
of similar spots, and colocalization of all three proteins, was
observed in the extended focus images obtained after 3-D
reconstruction of the serial optical sections of mitotic cells (not
shown).
Chromosome spreads prepared from HeLa cells blocked in
mitosis made it possible to recognize acrocentric chromosomes
that were previously shown (Henderson et al., 1972) to be the
NOR-bearing chromosomes in human cells. Immunolabeling
of UBF (Fig. 2B) and TTF-1 (Fig. 2C) obtained on HeLa
chromosome spreads (Fig. 2A) was similar. In both cases, six
spots or double-spots were visible. UBF and TTF-1 were
associated with the same acrocentric chromosomes upon
merging with the image of stained DNA (Fig. 2D). These two
proteins were not equally distributed between NORs. Some
NOR-bearing chromosomes scored negative and the intensity
of the labeling of the six positive NORs differed (Fig. 2D).
Interestingly, UBF and TTF-1 varied in the same proportions
in the six different positive NORs.
We obtained similar results for RNA pol I and TTF-1 using
V11 and IgA-P21 (not shown). Consequently, UBF, RNA pol
I and TTF-1 were associated with the same NORs in HeLa cells
and they varied in the same proportions in the different positive
NORs.
Variations of TTF-1 during the cell cycle
To analyze the variations of TTF-1 during the cell cycle, we
compared total cell extracts prepared from HeLa cells
synchronized in each phase of the cell cycle. An analysis by
flow cytometry was performed to verify the synchronization of
the cell cycle (Fig. 3A). Immunoblot analysis revealed a
decrease of TTF-1 in early G1 cells compared to exponentially
growing cells (Fig. 3B, lanes 1, 2). The amount of TTF-1
increased progressively during S phase with a maximum in G2
phase (Fig. 3B, lanes 4-9). Interestingly when cells were in the
G2/M transition phase, a form of TTF-1 that migrates slightly
more slowly appeared (Fig. 3B, lane 9). In mitotic cells, the
band corresponding to TTF-1 migrated more slowly than in
interphase cells (Fig. 3B, lane 10). The nature of the posttranslational modifications of TTF-1 in mitotic cells is
characterized and discussed below. We also analyzed the
variations of UBF, the other factor that binds to the rDNA
promoter, by probing the same blots with A17 serum. As
shown in Fig. 3B, UBF increased from the S phase up to the
G2 phase. No difference in UBF migration was detectable in
these conditions. Taken together, these results show that TTF-1
and UBF exhibit similar quantitative variations during cell
cycle progression.
TTF-1 during mitosis 3263
Fig. 2. Colocalization of UBF and TTF-1 by
immunofluorescence on the same HeLa chromosome
spread. (A) HeLa chromosome spread stained by
DAPI. (B) Labeling using anti-UBF antibodies
(A17). (C) Labeling using anti-TTF-1 antibodies
(IgA-P21). (D) Superposition of a chromosome
spread stained by DAPI and both antibodies. The
superposition shows colocalization of both antibody
markers. The same acrocentric chromosomes are
labeled. Both antibody markers vary in the same
proportion in the six positive chromosomes. Bars,
5 µm.
Phosphorylation of TTF-1 during the cell cycle
TTF-1 is present in NORs when ribosomal transcription is
repressed. To verify if post-translational modifications of
TTF-1 occur during mitosis, interphase and mitotic cell
extracts were compared using 1-D and 2-D gel
electrophoresis. The single band visible in 1-D gel by western
blots migrated slightly more slowly with mitotic than
interphase cell extracts (Fig. 4A). This mitotic shift was
associated with a modification of the pI observed by 2-D gel
electrophoresis. As shown in Fig. 4A, the mitotic TTF-1 form
was less basic (pI 8.5) than the interphasic form (pI 9.5). The
modified mitotic pI was observed using both human and
murine antibodies (Fig. 4A). This could indicate that the
mitotic form of TTF-1 is phosphorylated as opposed to the
interphasic form. To verify this hypothesis, chromosomeassociated protein extracts were treated with λ-PPase. The
mitotic shift was abolished and the interphasic form of TTF-1
appeared (Fig. 4B, lanes 1-3). These data confirmed that
TTF-1 is phosphorylated in mitotic cells and its
phosphorylation takes place at the G2/M transition, as shown
during cell cycle synchronization (Fig. 3B, lane 9).
The mitotic phosphorylated form of TTF-1 is
dephosphorylated by roscovitine treatment
The G2/M transition is triggered by activation of a protein
kinase cascade, at the head of which is the cdc2-cyclin B kinase
(Dunphy et al., 1988; Gautier et al., 1988; Lohka et al., 1988).
To investigate whether mitotic phosphorylation of TTF-1 is
mediated by cdc2-cyclin B kinase in vivo, roscovitine, a highly
selective inhibitor of cyclin-dependent kinases (De Azevedo et
al., 1997; Meijer et al., 1997), was used. Several cyclindependent kinases, including cdc2-cyclin B and cdk2-cyclin A
or E, are very sensitive to roscovitine (Ic50<7 µM) when tested
in vitro. However, because cdk2-cyclin E kinase and cdk2cyclin A kinase are active at the G1/S transition and during S
phase, respectively, the cdc2-cyclin B kinase is the only known
kinase inhibited by roscovitine when G2/M and mitotically
synchronized cells are treated with this inhibitor. Because the
3264 V. Sirri and others
Fig. 3. Levels of TTF-1 and UBF during the cell cycle. (A) Analysis
by flow cytometry of the DNA content of HeLa cells unsynchronized
(Unsyn) and synchronized in different cell cycle phases (early-G1, S
(4 hours) and G2/M) by the thymidine double-block method.
(B) Analysis by immunoblotting of the amount of TTF-1 and UBF
during the cell cycle. Exponentially growing cells (Unsyn) and
synchronized HeLa cells obtained as described in Materials and
Methods were analyzed. The proteins corresponding to the same
number of cells were separated by SDS-7.5% PAGE and
immunoblotted with anti-TTF-1 (IgA-P21) and anti-UBF (A17)
antibodies.
analysis by the PROSITE program of the TTF-1 sequence
showed several potential casein kinase II phosphorylation sites,
we also checked the possibility that casein kinase II is
responsible for TTF-1 phosphorylation by using DRB as
inhibitor (Zandomeni and Weimman, 1984).
To test the possible implication of cdc2-cyclin B kinase or
casein kinase II in phosphorylation of TTF-1, HeLa cells
synchronized at late S phase were treated or not with 25 µM
roscovitine or 60 µM DRB for 2 hours prior to exposure to
colchicine. The inhibitor concentrations were chosen so as to
maintain viability of the cells and not induce irreversible arrest
in G2 phase (Meijer et al., 1997). After exposure to colchicine
for 14 hours, 98% of untreated cells or cells treated with DRB
were arrested in mitosis. In contrast, cells treated with
roscovitine presented two populations: 90% of the cells were
arrested in G2 phase (interpreted as arrested in G2 phase by
inhibition of the cdc2-cyclin B kinase activity) and 10% in
mitosis (interpreted as not affected by roscovitine before
entering into mitosis). These two populations, i.e. nonadhering mitotic cells and adhering G2 phase cells, were
harvested by mechanical shock and trypsin treatment,
respectively, and analyzed separately. For kinase inhibitortreated and untreated cells, total proteins were extracted and
analyzed by immunoblotting with anti-hTTF-1 and antiphosphohistone H1 antibodies. Control mitotic cells
accumulated the phosphorylated form of TTF-1 contrary to
Fig. 4. Mitotic phosphorylation of TTF-1. (A) Interphase (I) and
mitotic (M) cell extracts prepared from HeLa cells were analyzed on
SDS-7.5% PAGE immunoblots using IgA-P21 (α-hTTF-1) and on 2D immunoblots with IgA-P21 (α-hTTF-1) or anti-mouse TTF-1
antibodies (α-mTTF-1). (B) Chromosome extracts treated or not with
λ-PPase were analyzed on 1-D and 2-D immunoblots with IgA-P21
(α-hTTF-1). Interphase extracts (I) were used as a control for λPPase treatment (lane 3).
unsynchronized and G2 synchronized cells (8 hours after
double thymidine block release) as expected (Fig. 5A, lanes 13). The same results were obtained in DRB-treated cells (Fig.
5A, lane 4) showing that casein kinase II is most probably not
implicated in mitotic TTF-1 phosphorylation. On the contrary,
in roscovitine-treated cells the non-phosphorylated form of
TTF-1 was observed in mitotic cells when compared to G2
phase cells (Fig. 5A, lanes 5, 6). Neither the cells blocked at
G2 phase nor the minor percentage of cells blocked in mitosis
possessed the mitotically phosphorylated form of TTF-1. This
result, expected for cells blocked in G2 phase, suggests that the
phosphorylation of TTF-1 in mitosis can be reversed upon
inhibition of the cdc2-cyclin B kinase. As a control for
inhibition of cdc2-cyclin B kinase activity by roscovitine, we
analyzed the phosphorylation state of histone H1, which is a
well-characterized substrate for cdc2-cyclin B kinase and is
routinely used for in vitro measurements of kinase activity
using anti-phosphohistone H1 antibodies. As expected, only
the control mitotic cells and the DRB-treated cells presented
the phosphorylated form of H1 (Fig. 5A, lanes 3, 4). When
cdc2-cyclin B kinase was inhibited by roscovitine treatment,
no signal was observed in mitotic cells (Fig. 5A, lane 6),
indicating that histone H1 was dephosphorylated.
To definitively establish that phosphorylation of TTF-1 in
mitosis can be reversed upon inhibition of the cdc2-cyclin B
TTF-1 during mitosis 3265
non-phosphorylated form of TTF-1 increased with the duration
of treatment and became the only detectable form after
120 minutes (Fig. 5B, compare lanes 4 and 5). The inhibition
of cdc2-cyclin B kinase activity in colchicine-arrested mitotic
HeLa cells treated with roscovitine was confirmed by
analyzing the phosphorylation state of histone H1 (Fig. 5B).
Indeed the phosphorylated form of histone H1 decreased with
the duration of the treatment (Fig. 5B, lanes 1-4) and
disappeared after 120 minutes as did the phosphorylated form
of TTF-1 (Fig. 5B, lane 4). These data indicate that in vivo the
cdc2-cyclin B kinase activity is indispensable for maintaining
phosphorylation of TTF-1 in colchicine-arrested mitotic HeLa
cells, as is also the case for histone H1. As illustrated in Fig.
5C, dephosphorylation of TTF-1 induced by inhibition of the
cdc2-cyclin B kinase involved an okadaic acid-sensitive
phosphatase. Indeed when colchicine-arrested mitotic HeLa
cells were treated or not with okadaic acid (0.5 µM) a potent
inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A),
for 60 minutes and then roscovitine-treated (75 µM) for 30
minutes, the mitotically phosphorylated form of TTF-1 (Fig.
5C, lane 2) was partially dephosphorylated by this roscovitine
treatment (Fig. 5C, lane 3) only in the absence of okadaic acid
(Fig. 5C, compare lanes 3 and 4). Taken together, these results
indicate that in vivo the cdc2-cyclin B kinase may be
implicated in phosphorylation of TTF-1 and its activity is
indispensable for maintaining phosphorylation of TTF-1
during mitosis, probably by inhibiting an okadaic acidsensitive phosphatase.
Fig. 5. Dephosphorylation of TTF-1 by inhibiting cdc2-cyclin B
kinase. (A) Protein extracts prepared from unsynchronized (lane 1)
and G2 synchronized (lane 2) HeLa cells, and from late S phase
synchronized HeLa cells (lanes 3-6) treated as follows, were
subjected to SDS-PAGE and immunoblotted with anti-TTF-1 (IgAP21) and anti-phosphohistone H1 antibodies. The late S phase
synchronized HeLa cells were treated for 2 hours with DRB (+DRB)
or roscovitine (+Rosc) or not treated with a kinase inhibitor (−).
Colchicine was then added (+col) and treatment pursued for
14 hours. After exposure to colchicine, 98% of the untreated cells or
cells treated with DRB were arrested in mitosis (M). They were
collected and analyzed respectively in lanes 3 and 4. After exposure
to colchicine, cells treated with roscovitine presented two
populations: 90% of the cells were arrested in G2 phase (G2) and
10% in mitosis (M). They were harvested and analyzed respectively
in lanes 5 and 6. (B) Protein extracts prepared from colchicinearrested mitotic HeLa cells treated with roscovitine for 0, 15, 45 and
120 minutes (lanes 1-4) and from unsynchronized HeLa cells (lane
5) were electrophoresed and immunoblotted with anti-TTF-1 (IgAP21) and anti-phosphohistone H1 antibodies. (C) Analysis of TTF-1
in protein extracts prepared from unsynchronized HeLa cells (lane 1)
and from colchicine-arrested mitotic HeLa cells (lanes 2-4) treated
with 0.5 µM okadaic acid for 60 minutes (lane 4) or not treated
(lane 3) prior to addition of roscovitine for 30 minutes (lanes 3 and
4). TTF-1 and the phosphorylated form of histone H1 are indicated
as TTF-1 and H1- P , respectively.
kinase, we analyzed the effect of a higher roscovitine
concentration (75 µM) over shorter periods of time (0, 15, 45
or 120 minutes) in mitotic HeLa cells previously blocked by
colchicine treatment (0.02 µg/ml for 14 hours). As shown in
Fig. 5B, the mitotically phosphorylated form of TTF-1 (Fig.
5B, lane 1) disappeared and the non-phosphorylated form
appeared after roscovitine treatment (Fig. 5B, lanes 1-4). The
Differences between interphase and mitotic TTF-1rDNA association
During active transcription, TTF-1 was described as a DNAbinding factor in vitro and in vivo (Grummt et al., 1985, 1986).
In mitosis, TTF-1 colocalized with the repressed transcription
machinery in certain NORs. However, nothing is known
regarding the binding of TTF-1 to rDNAs in mitotic
chromosomes. With this in mind, the solubilization of TTF-1
from interphase and colchicine-arrested mitotic HeLa cells was
compared and UBF was used as an internal reference for rDNA
binding.
The extraction procedures were controlled by checking
histone solubilization from isolated nuclei. H1 and H3 were
not solubilized at salt concentrations up to 0.25 M NaCl, but
became completely soluble at 0.5 M NaCl for H1 and at 2 M
NaCl for H3 (not shown), as already established for these
DNA-binding proteins (Hoffmann and Chalkley, 1978). In the
same conditions, TTF-1 was not solubilized at low salt
concentrations, became partially soluble at 0.5 M and nearly
entirely soluble at 1 M NaCl (Fig. 6A). The same blots
reprobed with A17 serum, showed that UBF was solubilized at
the same salt concentrations in interphase cells (Fig. 6A).
Therefore, in transcriptionally active conditions, such as
interphase nuclei, TTF-1 and UBF exhibit similar DNAbinding affinities.
TTF-1 was more easily solubilized from mitotic cells than
from interphase cells; it was partially soluble at 0.25 M NaCl
and nearly completely soluble at 0.5 M NaCl (Fig. 6B). On the
other hand, as previously reported (Imai et al., 1994), UBF was
more difficult to extract from mitotic cells than from interphase
cells. A larger proportion of UBF was present in pellet
fractions at 0.5 and 1 M NaCl in mitotic extracts (Fig. 6B) than
3266 V. Sirri and others
Fig. 6. Salt solubilization of TTF-1 and UBF. Proteins were extracted
at different NaCl concentrations from isolated nuclei (A),
permeabilized mitotic cells (B) and isolated chromosomes (C)
prepared from HeLa cells. Equal volumes of pellet (P) and
supernatant (S) fractions separated by SDS-8% PAGE were probed
on western blots with anti-TTF-1 (IgA-P21) and anti-UBF (A17)
antibodies.
in interphase cells (Fig. 6A). It is interesting to note that similar
results were obtained with permeabilized mitotic cells (Fig.
6B) or isolated chromosomes (Fig. 6C). These results,
reproduced using two extraction buffers (see Materials and
Methods; Andrews and Faller, 1991) for both interphase and
mitotic cells (not shown), demonstrate that, although the rDNA
transcription machinery remains colocalized in certain NORs
during mitosis, its interaction with rDNAs is most probably
modified.
DISCUSSION
TTF-1 distribution
TTF-1 has been almost exclusively studied in vitro and little is
known about its cellular localization. The present study
indicates that TTF-1 colocalizes with the rDNA transcription
machinery, namely RNA pol I and UBF, throughout the cell
cycle including mitosis. This colocalization was expected
during interphase because TTF-1 binds transcription
terminator elements present immediately upstream of the
promoter and in the 3′-external transcribed spacer of rDNAs,
and because it acts in activation and termination of rDNA
transcription. It was proposed that TTF-1 activates
transcription favouring slippage of the RNA pol I complex and
remodeling of the chromatin at the site of transcription
initiation (Längst et al., 1998). However, the localization of
TTF-1 in mitotic cells in which rDNA transcription is repressed
was not known. Here, we demonstrate that TTF-1 remains
colocalized with the inactive RNA pol I machinery during
mitosis. It was previously established that the RNA pol I, UBF
and SL1 remain assembled and localized on certain rDNAs
during mitosis, i.e. during repression of rDNA transcription
(Roussel et al., 1996). Consequently, all the partners defined as
composing the basal RNA pol I-dependent transcription
machinery remain associated with some rDNA genes, whether
or not the rDNA is being transcribed. It is remarkable that,
contrary to the basal RNA pol II transcription machinery, as
demonstrated for the majority of TFIID (Segil et al., 1996) and
for the transcription factors (Martinez-Balbàs et al., 1995; Segil
et al., 1991) that are released from mitotic chromatin, the rDNA
transcription machinery remains associated with certain NORbearing chromosomes, i.e. with some rDNAs during mitosis.
Interestingly, TTF-1 and the other factors presented a
stoichiometric distribution in the six positive NOR-bearing
chromosomes with similar amounts associated with both
chromatids of the same chromosome. This distribution may
ensure an equal partition of preassembled complexes in the two
daughter cells when the separation of chromatids occurs at
anaphase. rDNA transcription is repressed at mitotic prophase
and restored at telophase in the future daughter cells without
redistribution of the rDNA transcription machinery (Roussel et
al., 1996). The setting of the rDNA transcription machinery
occurs during the preceding interphase and determines rDNA
transcriptional activity at the following interphase. The setting
period could be the S phase in which UBF and TTF-1
accumulate and are located in the nucleoli. Further
investigations are required to determine whether these newly
synthesized proteins are actually associated with the rDNAs.
However the results obtained by salt solubilization are
compatible with this hypothesis since the majority of TTF-1 and
UBF require 0.5 M NaCl to be solubilized from interphase cells.
Phosphorylation of TTF-1
Human TTF-1 seems most probably not to be phosphorylated
in interphase despite the presence of potential protein kinase C
and casein kinase II sites (Evers and Grummt, 1995).
Conversely, we show that TTF-1 is undoubtedly
phosphorylated during mitosis. Mitotic phosphorylation of
TTF-1 might be under the direct or indirect control of the cdc2cyclin B kinase. Indeed the phosphorylated form of TTF-1
appears at the G2/M transition phase when cdc2-cyclin B
kinase is activated. Moreover the in vivo inhibition of cdc2cyclin B kinase activity induces dephosphorylation of the
mitotic form of TTF-1. At the present time, we cannot
conclude whether cdc2-cyclin B kinase directly phosphorylates
TTF-1 or whether it activates another kinase responsible for
TTF-1 phosphorylation. On the other hand, cdc2-cyclin B
kinase activity is indispensable to maintain the phosphorylated
form of TTF-1 during mitosis, probably by inhibiting an
okadaic acid-sensitive phosphatase (PP1 or PP2A). Indeed the
in vivo inhibition of cdc2-cyclin B kinase induces
dephosphorylation of the mitotic form of TTF-1 only in the
absence of okadaic acid.
rDNA-binding of TTF-1 during the cell cycle
TTF-1 has DNA-binding affinity (for a review see Reeder and
TTF-1 during mitosis 3267
Lang, 1994) and seems to be involved in the termination
process as well as in transcriptional activation of rDNAs (Evers
and Grummt, 1995; Längst et al., 1998). It has been proposed
that TTF-1 determines the chromatin organization of the active
rDNA promoter (Längst et al., 1998). TTF-1 can render
chromatin competent for transcription (Längst et al., 1997a).
In the present work, we compared TTF-1 DNA-binding affinity
in interphase cells actively transcribing the rDNA and in
mitotic cells with repressed transcription. Our data do not
directly address the binding of TTF-1 on rDNA sequences.
However, using well-defined conditions to characterize DNAbinding proteins and comparing them with H1 solubilization
(Hoffmann and Chalkley, 1978) in the same extracts, the DNAbinding affinity of TTF-1 can be predicted. It appears that this
DNA-binding affinity was lower during mitosis than during
interphase. This difference of TTF-1 solubilization using
mitotic cells compared to interphase cells most probably
reflects modifications of interactions between TTF-1 and
rDNAs. Because TTF-1 has been proposed to determine the
chromatin organization of the active rDNA promoter (Längst
et al., 1998), it seems reasonable to suggest that the mitotically
phosphorylated form of TTF-1 is involved in the repression of
rDNA transcription. Interestingly, the other DNA-binding
factor UBF (Bell et al., 1989; Jantzen et al., 1990; Learned et
al., 1986) has a higher DNA-binding affinity during mitosis
compared to interphase (Imai et al., 1994; this study).
Moreover, it was recently shown that phosphorylation by cdc2cyclin B kinase inactivates the TBP-containing factor SL1
(Kuhn et al., 1998) and impairs the capability of SL1 to interact
with UBF (Heix et al., 1998). Therefore, it seems established
that even though the rDNA transcription machinery remains
associated with rDNAs during mitosis, phosphorylation of
some components induces modifications in protein-protein and
protein-rDNA interactions that might repress rDNA
transcription.
The authors are grateful to the following laboratories for generously
providing reagents used in this study: I. Grummt for anti-mTTF-1
antibody, F. Amalric and P. Belenguer for nucleolin, B. C. Valdez for
GU/RNA helicase II and L. Comai for purified RNA pol I and SL1depleted extracts. We also thank M-C. Gendron for flow cytometry,
G. Géraud for help with confocal microscopy, and M. Barre and R.
Schwartzmann for photographic work. We are particularly grateful to
A. L. Haenni and C. Mann for critical reading of the manuscript. This
work was supported in part by grants from the Centre National de la
Recherche Scientifique and the Association pour la Recherche sur le
Cancer (Contrat 9143). V. S. is the recipient of a grant from the Centre
National de la Recherche Scientifique.
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