2429
Journal of Cell Science 110, 2429-2440 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS3612
When rDNA transcription is arrested during mitosis, UBF is still associated
with non-condensed rDNA
Jeannine Gébrane-Younès*, Nathalie Fomproix and Danièle Hernandez-Verdun
Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France
*Author for correspondence (e-mail: [email protected])
SUMMARY
The mechanisms that control inactivation of ribosomal gene
(rDNA) transcription during mitosis is still an open
question. To investigate this fundamental question, the
precise timing of mitotic arrest was established. In PtK1
cells, rDNA transcription was still active in prophase,
stopped in prometaphase until early anaphase, and
activated in late anaphase. Because rDNA transcription can
still occur in prophase and late anaphase chromosomes, the
kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by
electron microscopy from the G2/M transition to late
anaphase in the secondary constriction, the chromosome
regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA
was observed in addition to axial condensed rDNA. Thus,
the persistence of this non-condensed rDNA during inactive
transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation.
Analysis of the three-dimensional distribution of the rDNA
transcription factor, UBF, revealed that it was similar at
each stage of mitosis in the secondary constriction. In
addition, the colocalization of UBF with non-condensed
rDNA was demonstrated. This is the first visual evidence of
the association of UBF with non-condensed rDNA. As we
previously reported that the rDNA transcription machinery
remained assembled during mitosis, the colocalization of
rDNA fibers with UBF argues in favor of the association of
the transcription machinery with certain rDNA copies even
in the absence of transcription. If this hypothesis is correct,
it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot
explain the arrest of rDNA transcription during mitosis. It
is proposed that modifications of the transcription
machinery occurring in prometaphase could explain the
arrest of transcription, while reverse modifications in late
anaphase could explain activation.
INTRODUCTION
polymerase I (RNA pol I) transcription, i.e. to be specific for
rDNA transcription (Masson et al., 1996).
During mitosis the rDNAs are clustered in the secondary
constriction of chromosome regions designated nucleolar
organizer regions (NORs). It has been reported that the
upstream binding factor (UBF), a transcription factor specific
for rDNAs, remains associated with mitotic NORs (Rendon et
al., 1992; Roussel et al., 1993, 1996; Zatsepina et al., 1993).
Similarly the SL1 complex that functions cooperatively with
UBF to activate RNA pol I transcription (Bell et al., 1988) is
localized in 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.
The persistence of the assembled rDNA transcription
machinery in the NORs during mitosis does not prove that the
machinery is still associated with rDNAs. Indeed, inactivation
In mitosis of higher eukaryotes, when chromatin is condensed
into chromosomes, transcription is inhibited (Prescott, 1964).
The mechanisms that regulate gene inactivation during mitosis
are not completely understood. It is reasonable to predict at
least two main levels of inactivation that are not necessarily
exclusive. Inactivation could be controlled either at the level of
the transcription machinery by dissociation or modification, or
at the level of the chromatin by condensation or modification.
Ribosomal genes (rDNAs) are a good model to investigate
such fundamental processes, because they are actively transcribed during the cell cycle with optimal transcription in G2
and arrest during mitosis (Hadjiolov, 1985). However, the
precise mitotic stage during which arrest of rDNA transcription takes place remains to be determined to correlate this event
with other major mitotic changes such as the condensation of
chromatin into chromosomes. It is now possible to determine
the transition between active and inactive rDNA transcription
in correlation with precise mitotic phases by run-on in situ
assays (Wansink et al., 1993). Indeed this method has been
found to be very sensitive and can be adapted to favor RNA
Key words: Mitosis, rDNA, rRNA transcription, UBF transcription
factor, Secondary constriction, Cell cycle, DNA conformation,
Electron microscopy
2430 J. Gébrane-Younès, N. Fomproix and D. Hernandez-Verdun
of transcription could be due to the dissociation of both
partners even if the machinery is stored near by. Such association or dissociation can be investigated at high resolution
revealing the relative distribution of the rDNA chromatin fibers
and the proteins of the transcription machinery. To date, only
light and confocal microscopy were used to visualize the colocalization of proteins of the rDNA machinery in mitotic chromosomes (Roussel et al., 1993, 1996; Suja et al., 1997; Weisenberger and Scheer, 1995; Zatsepina et al., 1993). Even though
the presence of condensed chromatin in the axis of the
secondary constriction is well documented (Ghosh and
Paweletz, 1990; Hernandez-Verdun and Derenzini, 1983; Hsu
et al., 1967; Ploton et al., 1987b; Thiry et al., 1988), the
existence of rDNA surrounding the axis is still controversial
(Hernandez-Verdun and Derenzini, 1983; Thiry et al., 1988).
Using PtK1 cells, we first determined the mitotic stages
during which transition between active and inactive transcription takes place and vice-versa. By comparing active and
inactive mitotic stages we looked for differences in rDNA configuration or differences in association of the rDNAs with the
transcription machinery. UBF was chosen to test such association because recognition and binding to the rDNA promoter
is promoted by UBF (Bell et al., 1989; Jantzen et al., 1990;
Learned et al., 1986) and binding of UBF is the first step of
the assembly of the RNA pol I transcription machinery (for a
review see Moss and Stefanovsky, 1995).
DNA was revealed in situ by specific contrast with uranyl
acetate after extraction of RNA and phosphate groups from
phosphoproteins and blockage of protein contrast, as shown by
Testillano et al. (1991). As this method is presently the only one
that can be combined with immunogold labeling, it was used to
visualize DNA and localize UBF in the same chromosomes.
We conclude that the transcription machinery is still colocalized with non-condensed rDNA copies even in the absence
of transcription.
MATERIALS AND METHODS
Cell culture
PtK1 cells (Potorous tridactylis kidney cells) were grown on microgrid
coverslips (CELLocate, Eppendorf) in Eagle’s minimum essential
medium (EMEM) containing 0.85 g/l NaHCO3 (Gibco BRL) supplemented with 10% (v/v) fetal calf serum (ICN Flow) and 2% glutamine
(Gibco BRL), in 5% CO2 at 37°C. Alphanumeric labeling of the grids
facilitated the location of individual mitotic cells by light microscopy.
The cells were treated in situ.
Run-on transcription in permeabilized cells
Run-on transcription was performed as previously described (Wansink
et al., 1993) except that glycerol was omitted from the medium and the
reactions were performed at room temperature (RT). Briefly, cultured
cells were washed rapidly in PBS, pH 7.4, and with the permeabilization buffer (20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA,
0.5 mM PMSF). The cells were permeabilized in the same buffer containing 0.05% Triton X-100 (IBI, USA) for 5 minutes at RT and permeabilization was stopped by extensive washing in the same buffer
without Triton X-100. The cells were then incubated in run-on buffer
(Wansink et al., 1993) containing 0.5 mM of ATP, CTP, GTP and 0.2
mM Br-UTP (Sigma) for 20 minutes. The cultured cells were then
washed twice with PBS containing 5 units/ml of RNase inhibitor, fixed
immediately in 2% paraformaldehyde for 40 minutes at 4°C, and permeabilized with 0.1% Triton X-100 (PBS/Triton). For control experiments, 10 µM dideoxyTTP (Boehringer) and 5 µg/ml aphidicolin
(Sigma), or actinomycin D (1 µg/ml), or α-amanitin (100 µg/ml), were
added in the run-on buffer. Enzymatic digestion with RNase A (50
µg/ml in PBS) was performed for 10 minutes after run-on transcription.
Run-on transcription in ‘weakly fixed’ cells
Run-on transcription was performed as previously described by
Moore and Ringertz (1973). The cells were fixed for exactly 5 minutes
in absolute ethanol/acetone (1:1 v/v), at 4°C, washed 3 minutes in
reaction mixture containing 100 mM Tris-HCl, pH 7.9, 12 mM 2-mercaptoethanol, 150 mM sucrose, 12 mM MgCl2 and incubated in runon buffer containing 0.5 mM of ATP, CTP, GTP and 0.2 mM Br-UTP
for 15 minutes. They were then washed 3 minutes in the reaction
mixture, postfixed immediately in 2% paraformaldehyde for 40
minutes at 4°C and permeabilized with 0.1% Triton X-100 in PBS for
5 minutes at RT.
To detect transcription, the cells were incubated for 60 minutes with
a monoclonal anti-Br-deoxyuridine antibody (Sigma) diluted 1/100 in
PBS, then for 30 minutes with goat anti-mouse FITC-conjugated
antibody (Jackson Immunoresearch Laboratories) and for 5 minutes in
DAPI (1/10,000). Control of the anti-Br-dU antibody specificity was
performed by inclusion of UTP instead of Br-UTP in the run-on buffer.
All samples were mounted with Citifluor and photographed using
TMAX 400 Kodak film in a Leica microscope or directly observed
on a video microscope control. The scanned images and the video
microscope images were assembled on a Macintosh computer
equipped with an Adobe Photoshop 3.0 software program. Images
were printed directly from the computer on colour paper using a dye
sublimation printer (Colorease, Eastman-Kodak).
Pre-embedding UBF-immunogold labeling for electron
microscopy
The human autoimmune serum (B15) containing high titers of antibodies against the RNA pol I transcription factor UBF and recently
characterized (Roussel et al., 1993) was used for the present study.
After brief rinsing in serum-free EMEM, the cells were fixed in 4%
(w/v) paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, for 60
minutes at 4°C, rinsed in the same buffer and processed for the
immunocytochemical reaction. All operations were carried out in PBS
containing 0.1% saponine (PBS/Sap) at RT, with shaking. The cells
were preincubated with 5% normal goat serum and 1% BSA in
PBS/Sap for 30 minutes, before incubation with B15 serum diluted
1/100 in PBS/Sap plus 0.1% BSA for 60 minutes. After rinsing in
PBS/Sap, the cells were incubated with goat anti-human IgG conjugated to 5 nm colloidal gold particles (GAH G5, BioCell, Cardiff, UK),
diluted 1/10 in PBS/Sap plus 0.1% BSA for 60 minutes.
After labeling, the cells were rinsed with PBS/Sap, fixed in 2%
(v/v) glutaraldehyde in 0.1 M cacodylate, pH 7.4, for 60 minutes at
4°C, rinsed, postfixed in 1% (w/v) OsO4 in the same buffer for 30
minutes, rinsed in distilled water, dehydrated through an ethanol series
and immersed in an Epon/ethanol mixture (1:1, v/v) followed by pure
Epon 812. Finally, an Epon-filled gelatin capsule was inverted over
the microgrid coverslip.
Control for labeling was carried out by omitting the first antibodies.
NAMA-Ur method for DNA performed ‘en bloc’
The cells were fixed either in 3% (v/v) glutaraldehyde or in 4% (w/v)
paraformaldehyde in 0.025 M cacodylate buffer, pH 6.9, for 60 minutes
at RT, washed in 0.1 M cacodylate buffer, pH 6.9, and then immersed
in 0.5 N NaOH (NA) in 4% (w/v) paraformaldehyde overnight (Testillano et al., 1991). After rinsing in distilled water, treating in 1% acetic
acid, rinsing again in distilled water and dehydrating in ethanol, the
cells were subjected to the methylation and acetylation (MA)
procedure (Tandler and Solari, 1982). They were immersed in a freshly
prepared mixture of methanol and acetic anhydride (5:1, v/v)
overnight. They were then washed in pure methanol, in ethanol, and
finally embedded in Epon as described above. After these treatments
only DNA was highly contrasted, whatever the fixation method used.
rDNA and UBF during mitosis 2431
To better examine the relationship between UBF labeling and DNA
distribution in the secondary constriction, the two techniques
described above were performed on the same cells: the cells were first
immunogold labeled for UBF and thereafter the NAMA-Ur method
applied, before embedding in Epon.
set of chromosomes, very close to the centromere in PtK1 chromosomes (see below). At the onset of transcription, two beads
were observed at each secondary constriction. In all cases, the
two NORs were expressed or repressed simultaneously.
Serial ultrathin sections and contrast
After resin polymerization, the embedded cells were separated from
the microgrid coverslips by brief immersion of the Epon-filled capsule
in liquid nitrogen. As a consequence, the surface of the block carried
the imprint of the alphanumeric labeling. The mitotic cells previously
located by light microscopy were serially sectioned parallel to the
plane of the growing cells. Ultrathin sections (110 nm) were mounted
onto single slot Formvar-coated copper grids.
The immunolabeled sections were conventionally contrasted with
uranyl acetate (10 minutes at RT) followed by lead citrate (5 minutes
at RT).
When the NAMA method was used, sections were contrasted with
5% (w/v) aqueous uranyl acetate (Ur) for 2 hours at 37°C.
RESULTS
Kinetics of arrest and onset of RNA pol I
transcription during mitosis
To determine the kinetics of transcription, in situ run-on was
used. This technique was based on the incorporation of Br-UTP
into newly synthesized RNA, followed by the detection of the
modified precursors using specific antibodies (Wansink et al.,
1993). Depending on the run-on conditions, the activity of
RNA pol II or RNA pol I could be favored. In the present study,
we used different approaches to label rDNA transcription (see
Materials and Methods). The labeling obtained in permeablized or ‘weakly fixed’ cells was similar. Nevertheless,
observation of mitosis was optimized using ‘weakly fixed’
cells. In these conditions, run-on labeling was visible as intense
fluorescent beads forming an alignment (Fig. 1a). Based on
phase contrast, the labeling was only observed in nucleoli.
Double immunostaining of RNA transcription and nucleolar
fibrillarin was performed to ascertain that the signal was
localized in nucleoli (data not shown). Moreover, the labeling
was abolished by low doses of actinomycin D (1 µg/ml). Consequently, the run-on labeling was merely due to rRNA transcription. The number of beads varied during interphase but
was always greater than ten.
When the cell entered into mitosis, transcription was not
completely abolished. In prophase when the compaction of the
chromatin into chromosomes was visible, beads corresponding
to RNA pol I transcripts were detected in the residual nucleolar
structures (Fig. 1b). The main modification of labeling at this
stage was assembly of the beads. ‘Weakly fixed’ cells upon
which run-on was performed did not progress into the cell
cycle during the time of precursor incorporation. Therefore it
was reasonable to exclude labeling of RNAs transcribed at the
beginning of run-on before entry into mitosis.
In prometaphase, while the nuclear envelope was disrupted and
the chromosomes individualized, transcription was no longer
observed (Fig. 1c). This inhibition of transcription (Fig. 1d) was
maintained in metaphase and in early anaphase (anaphase A). On
the contrary, transcription could be detected (Fig. 1e) in late
anaphase (anaphase B). Beads corresponding to transcription
were visible in the two NORs located at the external sites in each
Fig. 1. Ribosomal transcription during the PtK1 cell cycle. The runon assay was performed on ‘weakly fixed’ cells. Images (a-e) show
ribosomal transcription revealed by immunofluorescence. Images (a′e′) present DNA staining with DAPI and allow identification of the
mitotic stage. (a) Interphase cell; several beads of transcription are
visible in the area that corresponds to the nucleolus. (b) Prophase
cell; transcription is still visible. (c and d) Prometaphase and
anaphase A, respectively; no labeling is observed. (e) Anaphase B;
transcription starts as small dots. In each set of chromosomes, two
doublets (each of two beads) of transcription are observed, i.e. one
doublet at each secondary constriction. Depending on the focus,
either one or two beads (arrows) are detected. Bar, 5 µm.
2432 J. Gébrane-Younès, N. Fomproix and D. Hernandez-Verdun
The three-dimensional approach
We took advantage of the kinetics of RNA pol I activity during
mitosis to examine the changes occurring at the activity-repression transition and vice-versa in the secondary constriction. At
preselected stages of mitosis, electron microscopic observations were performed. Because three-dimensional information
could be crucial for interpretation, at least three or four serially
sectioned secondary constrictions of each mitotic stage were
examined for each experiment (i.e. DNA staining as well as
UBF immunolocalization alone or with DNA staining).
Fig. 2. Early PtK1 prophase after ‘en bloc’ NAMA-Ur
staining for DNA. (A) General view showing the
chromosomes in the process of condensation. The X
chromosome associated with nucleolar material
distinct from the rest of the nucleus, is clearly
distinguishable with the ‘in building’ secondary
constriction (between arrowheads). Bar, 1 µm.
(B) Detail of the same secondary constriction at two
sections below the level shown in A. This mid section
in the series shows an axis of condensed chromatin
forming a bridge between the two parts of the long
arm of the X chromosome. Fine DNA fibers emanate
from this axis and occupy the width of the
chromosome (arrows). Note the longitudinal direction
of some fibers (arrowheads). This non-condensed
rDNA appears as long fibers in the amorphous
material. Such organization as well as the specificity
of the staining (see Materials and Methods) reasonably
exclude that other components than DNA form these
fibers. Bar, 0.1 µm.
PtK1 cells have two NORs located at the secondary constriction on the long arms of the two X chromosomes. In this
species, the primary and the secondary constrictions are very
near each other (Hsu et al., 1967). The discrimination between
the primary (centromere) and the secondary constriction
(NORs) is facilitated by the presence exclusively in the primary
constriction of the kinetochore with a typical organization (for
a review see Rattner, 1991) and by the interaction of the kinetochore with microtubules. Therefore recognition of the
secondary constriction by electron microscopy can be estab-
rDNA and UBF during mitosis 2433
lished without ambiguity especially by observing serial
sections as in the present work. Indeed in serial sections the
relative position of the kinetochore and secondary constriction
can be ascertained in folded chromosomes as is the case in situ.
The serial sections made in prophase, prometaphase and
metaphase cells, showed that the secondary constriction could
occur on eight successive ultrathin sections (110 nm),
depending on the plane of section. On the other hand, at
anaphase when there is only one chromatid, the extent of the
secondary constriction was visible on four sections. This threedimensional approach clearly demonstrated that the size of the
secondary constriction is not modified throughout mitosis.
Conformation of the DNA visualized in the
secondary constriction
The conformation of the DNA in the secondary constriction
was investigated by the NAMA-Ur method, that specifically
reveals the DNA by electron micoscopy.
At early prophase, the nuclear envelope was still present and
well delineated by the NAMA-Ur DNA staining method, due
to the small patches of aligned, condensed chromatin. The condensing X chromosome could be easily identified among the
other chromosomes in the nucleus by its association with
homogeneous material of low contrast (Fig. 2a). This material
which probably results from nucleolar proteins still grouped at
Fig. 3. Metaphase from a PtK1
cell after ‘en bloc’ NAMA-Ur
staining for DNA. (A) General
view showing the chromosomes
gathered at the metaphase plate.
Only the DNA is well contrasted
and an X chromosome is
identified with its secondary
constriction (arrow). Bar, 1 µm.
(B) Detail of the secondary
constriction shown in A. Fine
DNA fibers are still noncondensed and some fibers run
parallel to the axis of the
condensed chromatin (arrows).
Bar, 0.1 µm.
2434 J. Gébrane-Younès, N. Fomproix and D. Hernandez-Verdun
At metaphase (Fig. 3a) the distribution of the chromosomes
did not seem to be random, since the NOR-bearing chromosomes were always situated at the periphery of the metaphase
plate and continued to occupy the same external position in the
two chromosomal sets at anaphase. Even in metaphase when
the chromosomes are known to exist in their maximum condensation state, the successive sections of NORs always
showed non-condensed rDNA fibers (Fig. 3b). They formed a
network in which the fibers were mainly longitudinally
oriented, i.e. parallel to the axis of the condensed chromatin.
It is interesting to note that the non-condensed rDNA fibers did
not completely surround the two condensed chromatin axes as
seen when all the sections of several secondary constrictions
were examined. Nevertheless the presence of non-condensed
rDNA fibers was also detected between the two axes of the two
chromatids. These observations reveal a polarized chromatid
organization of the rDNA in the secondary constriction for
which there is presently no explanation.
At anaphase, when there is only one chromatid per chromosome, the NOR appeared either as a large band containing
the non-condensed rDNA fibers, or as an axis of condensed
chromatin incompletely surrounded by the non-condensed
rDNA fibers, depending on the plane of the section.
Therefore the rDNA in the secondary constriction exhibits
the same conformation at each stage of mitosis, an axially
located condensed chromatin incompletely surrounded by noncondensed DNA fibers.
Fig. 4. Immunoelectron microscopic localization of UBF in a
prometaphase PtK1 cell. Longitudinal section of the long arm of the
X chromosome with a NOR heavily labeled with gold particles
(some indicated by thin arrows) in its fibrillar part tangentially
sectioned. A kinetochore at the primary constriction (between thick
arrows) is visible. Bar, 0.1 µm.
early prophase, disappears at prometaphase. When examining
serial sections of the identified X chromosome, a level was
reached where the secondary constriction appeared detached
from the sheathing material. An axis of condensed chromatin
in continuity with the adjacent arms of the X chromosome was
seen, around which fine DNA fibers emanated and occupied
the width of the chromosome. With this DNA staining method
it was clear that some of the distal fibers run in a longitudinally
curved direction parallel to the axis of the condensed chromatin
(Fig. 2b). These DNA fibers exhibited a non-condensed configuration as opposed to the rDNA in the axis of the chromosome, which appeared more intensely stained.
At prometaphase, the nucleolar material previously associated with the X chromosome was no longer seen. Even with
more condensed chromosomes, good longitudinal profiles with
two chromatids were infrequent. The secondary constriction
was observed on one or two chromatids, depending on the
plane of the section. As in the previous stage, the noncondensed ribosomal chromatin was composed of fine fibers
extending around the two axes of condensed chromatin of both
chromatids. By examining several serial sections of the entire
secondary constriction, it appeared that the quantity of noncondensed DNA fibers may vary from an X chromosome to its
homologue in the same cell or between sister chromatids.
Three-dimensional distribution of the RNA pol I
transcription factor UBF
The ultrastructure of the secondary constriction after the preembedding immunogold labeling exhibits the characteristic
architecture observed by standard methods, a condensed
chromatin axis surrounded by low contrasted fibrillar material
resembling the fibrillar center of interphase nucleoli. The threedimensional observation revealed that the low contrasted fibrillar
material failed to appear on all the serial sections of the secondary
constriction. This is compatible with a crescent-shaped organization for the fibrillar material of the NORs as already proposed
(Ploton et al., 1987a; Robert-Fortel et al., 1993; Suja et al., 1997).
The presence of UBF was detected in this low contrasted
fibrillar material and was never detected in the condensed
chromatin axis, using immunogold labeling (Fig. 4). UBF was
invariably associated with the fibrillar material, whatever the
mitotic stage from prophase to anaphase B. Rare were the
sections of fibrillar material devoid of labeling. Serial ultrathin
sections of labeled cells in prophase, prometaphase as well as
in metaphase showed the same pattern of distribution of UBF
throughout the NORs. Five nm gold particles could be seen in
the fibrillar material mostly in the external region of the
chromatid but also close to the axis of the condensed chromatin
(Figs 4, 5) and running between the two axes of condensed
chromatin. Similarly, the distribution of UBF in anaphase A
and anaphase B predominated in the external region of the low
contrasted fibrillar material (Fig. 6). The labeling density on
serial sections, indicated that the amount of UBF can vary from
one X chromosome to another independently of the mitotic
phase (compare Figs 4, 5 and 6) and in the same chromosome
between sister chromatids. However quantification at each
mitotic stage was not possible because only three or four
complete series of immunolabeling were made.
rDNA and UBF during mitosis 2435
Fig. 5. Immunoelectron microscopic localization of UBF in a metaphase PtK1 cell. Six adjacent serial longitudinal sections of the secondary
constriction. (A) The two axes (stars) of condensed chromatin appear fused and only the fibrillar material of the NOR located to the left of
the left-hand chromatid is decorated with gold particles (some indicated by arrows). This labeled NOR extended to E, whereas the righthand chromatid shows fibrillar material ending in F. A signal could be seen running between the sister chromatids in C, D and E (arrows).
Bar, 0.1 µm.
2436 J. Gébrane-Younès, N. Fomproix and D. Hernandez-Verdun
Fig. 6. Immunoelectron
microscopic localization of UBF
in a late anaphase PtK1 cell.
(A) Overview of a mid section
in the series showing one NOR
in each set of chromosomes
(arrows). Note the short distance
between the primary constriction
(thin arrow) and the secondary
constriction (thick arrow). Bar, 2
µm. (B,C) Two other successive
sections of the NOR 1 shown in
A. (B) Tangentially sectioned
fibrillar material of the NOR is
labeled all over its surface,
whereas in C, when the portion
of the X chromosome situated
between the two constrictions
appears (between arrowheads),
UBF is mostly present at the
edges of the fibrillar material.
Some gold particles are
indicated by arrows. The star
is placed in the direction of
the primary constriction. Bar,
0.1 µm.
The three-dimensional localization of UBF at each stage of
mitosis indicates a similar distribution in prophase and
anaphase B as well as in prometaphase and metaphase, that is
independent of the transcriptional activity of the rDNA.
Colocalization of UBF with non-condensed rDNA in
the NOR-bearing chromosomes
Even though immunogold electron microscopy on serial
sections allowed us to localize UBF in the volume of the
NORs, except in the axial condensed chromatin, and even
though the NAMA-Ur cytochemical technique showed the
presence of fine DNA fibers in the same region, we could not
anticipate the relationship between UBF and rDNA in the
secondary constriction. To address this question, we performed
immunogold labeling of UBF followed by the NAMA-Ur cytochemical technique to reveal DNA. Using these two methods
on the same cells and examining successive serial sections,
whatever the mitotic phase, the gold particles appeared only on
the non-condensed DNA fibers in the secondary constriction,
the condensed chromatin axis being devoid of labeling. UBF
remained colocalized with certain rDNAs even when transcription was impaired as can be seen in Fig. 7 at anaphase A.
Not all DNA fibers were covered by gold particles. Again, the
density of labeling was higher on DNA fibers located at the
edges of the secondary constriction and close to the axis of the
condensed chromatin (Fig. 7A-D).
To summarize the present results, a model of the organization of rDNA during mitosis is proposed (Fig. 8).
DISCUSSION
Bipartite organization of the secondary constriction
The fine structure of the secondary constriction of PtK1 chromosomes examined by electron microscopy was described
earlier (Hsu et al., 1967). It was reported to be composed of an
axis of condensed chromatin surrounded by clear fibrillar structures (Ghosh and Paweletz, 1990; Hsu et al., 1967; Suja et al.,
1997). This organization appears to be a common feature of
rDNA and UBF during mitosis 2437
Fig. 7. Early anaphase from a
PtK1 cell immunolabeled with
UBF and then treated with the
NAMA-Ur technique before
embedding. (A) In this set of
chromosomes, the X chromosome
is identified by its secondary
constriction (thick arrow), very
near the primary constriction (thin
arrow). Star indicates the direction
of the pole. Bar, 1 µm. (B) Detail
of the NOR shown in A. The gold
particles mostly colocalize with
the fine DNA fibers situated at the
external limit of the NOR (arrows)
or near the axis of condensed
chromatin (arrowheads). Bar,
0.1 µm. (C) High magnification
of the same secondary constriction
at three sections above the level
shown in A. Labeling of UBF is
intense but not uniformly
distributed. Most of the 5 nm gold
particles (some indicated by
arrowheads) lie over the peripheral
well-contrasted DNA fibers. The
arrows indicate the axis of
condensed chromatin tangentially
sectioned. Bar, 0.1 µm. (D) Same
early anaphase, but the NOR
belongs to another X
chromosome. Again, UBF is
preferentially associated with the
fine DNA fibers at the external
side of the NOR. Bar, 0.1 µm.
the secondary constriction in different species (Goessens,
1984; Goessens et al., 1987; Ploton et al., 1987a).
These ultrastructural features indicate a bipartite organization of the secondary constriction, arguing in favor of two
distinct domains with possible segregation of the components.
The condensed chromatin is only visible in the chromosome
axis and clear fibrillar structures have been proposed to be
mainly composed of non-histone nucleolar proteins. However,
the actual proof of the presence of nucleolar proteins in the
clear fibrillar structures except for the AgNOR proteins, is
almost exclusively based on light and confocal microscopy
data (for a review see Thiry and Goessens, 1996), i.e. using
approaches that do not permit the discrimination of structures
around the axis of the condensed chromatin.
Conformation and distribution of the rDNAs in the
secondary constriction
In PtK1 cells, all the rDNA copies are clustered in the
secondary constriction (Robert-Fortel et al., 1993). Therefore
the study of the DNA in the secondary constriction makes it
possible to study the configuration of the rDNAs. Using
hybridization with specific probes, rDNAs were detected in the
chromosome axis and were suspected in lateral expansions
around the axis (Robert-Fortel et al., 1993). This observation
performed by confocal microscopy as well as the visualization
of DNA in the width of the chromosome by very sensitive DNA
staining (Saitoh and Laemmli, 1994; Suja et al., 1997) suggest
that the clear fibrillar structure around the chromosome axis
visualized by electron microscopy could also contain rDNAs.
However, the relative proportion of DNA and proteins should
be in favor of the proteins which would explain the appearance
of a constriction when DNA is revealed by a non-sensitive
method in the standard caryotype.
In electron microscopy, the chromatin in the axis of the
secondary constriction is similar to the condensed chromatin
in other parts of the chromosome, allowing us to conclude that
2438 J. Gébrane-Younès, N. Fomproix and D. Hernandez-Verdun
Fig. 8. Schematic representation of the rDNA configuration during
mitosis. In the secondary constriction, the rDNA copies (in purple)
are either condensed as the adjacent parts of the chromosome (in
mauve), or non-condensed and associated with the transcription
factor UBF (yellow circles).
some rDNA copies are condensed during mitosis. In the clear
fibrillar structure around the axis, condensed chromatin is not
visible, but non-condensed and non-nucleosomal DNA fibers
were detected using osmium ammine (Hernandez-Verdun and
Derenzini, 1983), a specific and sensitive DNA stain for
electron microscopy (reviewed by Derenzini, 1995). However,
the presence of DNA fibers in the clear fibrillar part of the
secondary constriction is still controversial (for a review see
Thiry and Goessens, 1996), since DNA was not found outside
the chromosome axis using the immunodetection approach
(Thiry et al., 1988). In the present study, non-condensed
chromatin fibers, probably non-nucleosomal, were visualized
around the axis of condensed chromatin using the NAMA-Ur
method. Because this nucleic acid staining method has the
advantage of being compatible with immunogold detection of
proteins (Testillano et al., 1995), it makes it possible to localize
the DNA fibers in the same sites as UBF (as discussed below).
Our results demonstrate the presence in the secondary constriction of non-condensed chromatin fibers corresponding to
rDNA copies, in addition to the condensed rDNA copies in the
chromosome axis. We conclude that the bipartite organization
of the secondary constriction also concerns the conformation
of the rDNA with condensed and non-condensed genes.
Distribution of UBF in the secondary constriction
The presence of UBF in the clear fibrillar structure and its
exclusion from the chromosome axis is demonstrated here by
immunogold labeling on serial sections at each stage of the
mitosis.
However, the distribution of UBF was not homogeneous in
the volume of the clear fibrillar structure of the NORs, whatever
the mitotic stage. Since the distribution pattern of UBF remains
the same during mitosis, and since UBF remains colocalized
with the DNA (see below), this may indicate a stable organization of rDNA in the secondary constriction. The use of fluorescence in situ hybridization on extracted metaphase chromosomes (Bickmore and Oghene, 1996), presented visual
evidence that within rDNAs, non-transcribed spacer sequences
and DNA fragments including the promoter regions are more
closely apposed to the chromosome axis than are rDNAs. This
is in agreement with our observation of UBF labeling near the
axis of the condensed chromatin, but cannot actually explain the
UBF labeling at the external edges of the fibrillar material.
In light microscopy, most of the proteins of the rDNA transcription machinery (RNA pol I, UBF, TBP TAF1 and topoisomerase I) are localized in the secondary constriction (Jordan
et al., 1996; Roussel et al., 1996; Roussel et al., 1993; Weisenberger and Scheer, 1995; Zatsepina et al., 1993). The colocalization of these complexes was described using confocal
microscopy, but not yet established at high resolution.
However, as the transcription factors varied in the same proportion, a stoichiometric association of these complexes has
been proposed (Roussel et al., 1996). If this hypothesis is
correct, it can be anticipated that the other components of the
transcription machinery associated with UBF should follow the
same distribution in the clear fibrillar structure of the NORs.
Colocalization of UBF and rDNAs in the secondary
constriction
The visualization of non-condensed DNA fibers with UBF
labeling strongly supports the hypothesis that these components remain associated even during mitosis. It is well known
that UBF binding on rDNAs is due to several HMG-Box
sequences (Bazett-Jones et al., 1994; Moss and Stefanovsky,
1995) that bend, wrap and supercoil the enhancer DNA of the
rDNA promoter (Putnam et al., 1994). Presently we cannot
predict if the conformation of the non-condensed rDNA is the
cause or the consequence of such association, but obviously it
is not modified by chromosome condensation during mitosis.
In yeast, it has been proposed that open chromatin structures
result from specific protein-DNA interactions occurring before
the onset of transcription in the nucleosome-free enhancers
(Dammann et al., 1995).
Non-condensed rDNA copies appeared to be associated with
UBF but not the condensed rDNA. This indicates a heterogeneous distribution of UBF in all the copies of the same chromosome. It is tempting to propose that this heterogeneous distribution reflects a state already established during interphase
since only part of the rDNA copies are actually associated with
UBF in functional nucleoli (Junéra et al., 1997). In the case of
mitosis, we predict that the same mechanism would preserve
the two kinds of rDNAs even in the absence of transcription.
This fits with the observation on isolated ribosomal chromatin
that the two states of chromatin, containing nucleosomes, or
lacking a repeating nucleosome structure, coexist in rDNAs
throughout the cell cycle (Conconi et al., 1989).
Arrest of rDNA transcription during mitosis
In the past, autoradiography staining was used to study the
variation of the transcription during mitosis (Baserga, 1962;
Prescott, 1964). However, this approach was not efficient for
the detection of weak signals, whereas the run-on method is a
very sensitive approach that allows the visualization of weak
modulation of transcription. In human cells, ongoing rDNA
transcription was visualized by electron microscope autoradiography (Hernandez-Verdun et al., 1980) and by the run-on
approach (Roussel et al., 1996) and was observed in telophase.
In the present study, run-on transcription was performed in
PtK1 cells in which all mitotic stages are easy to identify and
allow the precise determination of the mitotic stages during
which arrest or onset of rDNA transcription takes place.
rDNA and UBF during mitosis 2439
The run-on assay performed in mitotic PtK1 cells demonstrates that rDNA transcription is active in prophase and late
anaphase. At these mitotic stages, chromatin condensation into
chromosomes is visible, suggesting that this mechanism can be
disconnected from rDNA transcription at least at these stages.
However, this does not exclude the possibility that modification of rDNA compaction exists between prometaphase and
early anaphase, i.e. the period of rDNA transcription inactivation.
Two kinds of rDNA conformation, nucleosomal and nonnucleosomal have been described, the transcriptionally active
genes being devoid of nucleosomes and randomly distributed
along the rDNA copies (Dammann et al., 1995). Here two types
of rDNA conformation are also found, condensed and noncondensed conformations, for each mitotic stage including
periods of active and inactive genes. Global condensation of
chromatin into chromosomes cannot be directly responsible for
inactivation of rDNA transcription during mitosis. Indeed,
remodeling of chromatin as well as modification(s) of nucleosomes cannot be excluded.
The mechanism(s) that controls arrest of rDNA transcription
during mitosis is still an open question. It has been found that
the disassembly of the rDNA transcription machinery is not the
cause of the arrest of rDNA transcription (Jordan et al., 1996;
Roussel et al., 1996) and regulation at the level of transcription elongation has been proposed, since most transcripts are
released from the NORs at mitosis (Weisenberger and Scheer,
1995). As suggested here, arrest of rDNA transcription is
probably not the consequence of the dissociation of the transcription factor UBF from rDNA. However, further investigations will be needed to determine whether modification of the
molecular association of UBF with rDNA after prophase is
responsible for the arrest of transcription. The presence of
specific repressor(s) could be hypothesized or inactivation of
the transcription machinery at prometaphase, i.e. the time of
nuclear envelope breakdown. Similarly, modifications should
take place between early and late anaphase to explain activation of transcription at the end of mitosis.
The authors are grateful to A. Lepage for help in cell culture and
to M. Barre and R. Schwartzmann for photographic work. We are particularly grateful to A.-L. Haenni 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 (contract no. 6703). N. Fomproix was
recipient of a Fellowship from ‘la Ligue Nationale contre le Cancer’.
REFERENCES
Baserga, R. (1962). A study of nucleic acid synthesis in ascites tumor cells by
two-emulsion autoradiography. J. Cell Biol. 12, 633-637.
Bazett-Jones, D., Leblanc, B., Herfort, M. and Moss, T. (1994). Short-range
DNA looping by Xenopus HMG-box transcription factor, xUBF. Nature 264,
1134-1137.
Bell, S. P., Learned, R. M., Jantzen, H.-M. and Tjian, R. (1988). Functional
cooperativity between transcription factors UBF1 and SL1 mediates human
ribosomal RNA synthesis. Science 241, 1192-1197.
Bell, S. P., Pikaard, C. S., Reeder, R. H. and Tjian, R. (1989). Molecular
mechanisms governing species-specific transcription of ribosomal RNA.
Cell 59, 489-497.
Bickmore, W. and Oghene, K. (1996). Visualizing the spatial relationships
between defined DNA sequences and the axial region of extracted metaphase
chromosomes. Cell 84, 95-104.
Conconi, A., Widmer, R. M., Koller, T. and Sogo, J. M. (1989). Two different
chromatin structures coexist in ribosomal RNA genes throughout the cell
cycle. Cell 57, 753-761.
Dammann, R., Lucchini, R., Koller, T. and Sogo, J. M. (1995). Transcription
in the yeast rRNA gene locus: distribution of the active gene copies and
chromatin structure of their flanking regulatory sequences. Mol. Cell. Biol.
15, 5294-5303.
Derenzini, M. (1995). Ultrastructural Cytochemistry (ed. G. Morel), pp. 69-93.
CRC Press, Boca Raton.
Ghosh, S. and Paweletz, N. (1990). The nucleolar chromatin and the
secondary constriction. Cell Biol. Intern. Rep. 14, 681-687.
Goessens, G. (1984). Nucleolar structure. Int. Rev. Cytol. 87, 107-158.
Goessens, G., Thiry, M. and Lepoint, A. (1987). Relations between nucleoli
and nucleolus-organizing regions during the cell cycle. Chrom. Today 9, 261271.
Hadjiolov, A. A. (1985). The Nucleolus and Ribosome Biogenesis, vol. 12 (ed.
M. Alfert, W. Beermann, L. Goldstein, K. R. Porter and P. Sitte), pp. 1-268.
Springer-Verlag, Wien, New-York.
Hernandez-Verdun, D., Bourgeois, C. A. and Bouteille, M. (1980).
Simultaneous nucleologenesis in daughter cells during late telophase. Biol.
Cell 37, 1-4.
Hernandez-Verdun, D. and Derenzini, M. (1983). Non-nucleosomal
configuration of chromatin in nucleolar organizer regions of metaphase
chromosomes in situ. Eur. J. Cell Biol. 31, 360-365.
Hsu, T. C., Brinkley, B. R. and Arrighi, F. E. (1967). The structure and
behavior of the nucleolus organizers in mammalian cells. Chromosoma 23,
137-153.
Jantzen, H.-M., Admon, A., Bell, S. P. and Tjian, R. (1990). Nucleolar
transcription factor hUBF contains a DNA-binding motif with homology to
HMG proteins. Nature 344, 830-836.
Jordan, P., Mannervik, M., Tora, L. and Carmofonseca, M. (1996). 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, 225-234.
Junéra, H. R., Masson, C., Géraud, G., Suja, J. and Hernandez-Verdun, D.
(1997). Involvement of in situ conformation of ribosomal genes and selective
distribution of UBF in rRNA transcription. Mol. Biol. Cell 8, 145-156.
Learned, R. M., Learned, T. K., Haltiner, M. M. and Tjian, R. T. (1986).
Human rRNA transcription is modulated by the coordinate binding of two
factors to an upstream control element. Cell 45, 847-857.
Masson, C., Bouniol, C., Fomproix, N., Szöllösi, M. S., Debey, P. and
Hernandez-Verdun (1996). Conditions favoring the RNA polymerase I
transcription in permeabilized cells. Exp. Cell Res. 226, 114-125.
Moore, G. P. M. and Ringertz, N. R. (1973). Localization of DNA-dependent
RNA polymerase activities in fixed human fibroblasts by autoradiography.
Exp. Cell Res. 76, 223-228.
Moss, T. and Stefanovsky, V. Y. (1995). Promotion and regulation of
ribosomal transcription in eukaryotes by RNA polymerase I. Prog. Nucl.
Acid Res. Mol. Biol. 50, 25-65.
Ploton, D., Beorchia, A., Menager, M., Jeannesson, P. and Adnet, J. J.
(1987a). The three-dimensional ultrastructure of interphasic and metaphasic
nucleolar argyrophilic components studied with high-voltage electron
microscopy in thick sections. Biol. Cell 59, 113-120.
Ploton, D., Thiry, M., Menager, M., Lepoint, A., Adnet, J. J. and Goessens,
G. (1987b). Behaviour of nucleolus during mitosis. A comparative
ultrastructural study of various cancerous cell lines using the Ag-NOR
staining procedure. Chromosoma 95, 95-107.
Prescott, D. M. (1964). Cellular sites of RNA synthesis. Prog. Nucl. Acid Res.
Mol. Biol. 3, 33-57.
Putnam, C. D., Copenhaver, G. P., Denton, M. L. and Pikaard, C. S. (1994).
The RNA polymerase I transactivator upstream binding factor requires its
dimerization domain and high-mobility-group (HMG) box 1 to bend, wrap,
and positively supercoil enhancer DNA. Mol. Cell. Biol. 14, 6476-6488.
Rattner, J. B. (1991). The structure of the mammalian centromere. BioEssays
13, 51-56.
Rendon, M. C., Rodrigo, R. M., Goenechea, L. G., Garcia-Herdugo, G.,
Valdivia, M. M. and Moreno, F. J. (1992). Characterization and
immunolocalization of nucleolar antigen with anti-NOR serum in HeLa
cells. Exp. Cell Res. 200, 393-403.
Robert-Fortel, I., Junéra, H. R., Géraud, G. and Hernandez-Verdun, D.
(1993). Three-dimensional organization of the ribosomal genes and Ag-NOR
proteins during interphase and mitosis in PtK1 cells studied by confocal
microscopy. Chromosoma 102, 146 -157.
Roussel, P., André, C., Masson, C., Géraud, G. and Hernandez-Verdun, D.
2440 J. Gébrane-Younès, N. Fomproix and D. Hernandez-Verdun
(1993). Localization of the RNA polymerase I transcription factor hUBF
during the cell cycle. J. Cell Sci. 104, 327-337.
Roussel, P., André, C., Comai, L. and Hernandez-Verdun, D. (1996). The
rDNA transcription machinery is assembled during mitosis in active NORs
and absent in inactive NORs. J. Cell Biol. 133, 235-246.
Saitoh, Y. and Laemmli, U. K. (1994). Metaphase chromosome structure:
bands arise from a differential folding path of the highly AT-rich scaffold.
Cell 76, 609-622.
Suja, J. A., Gébrane-Younès, J., Géraud, G. and Hernandez-Verdun, D.
(1997). Relative distribution of rDNA and proteins of the RNA Polymerase
I transcription machinery at chromosomal NORs. Chromosoma 105, 459469.
Tandler, C. J. and Solari, A. J. (1982). Methanol-acetic anhydride: an efficient
blocking agent for electron microscope cytochemistry. Its application to
mouse testis and other tissues. Histochemistry 76, 351-361.
Testillano, P. S., Sánchez-Pina, M. A., Olmedilla, A., Ollacarizqueta, M. A.,
Tandler, C. J. and Risueño, M. C. (1991). A specific ultrastructural method
to reveal DNA: The NAMA-Ur. J. Histochem. Cytochem. 39, 1427-1438.
Testillano, P. S., Gonzalez-Melendi, P., Ahmadian, P. and Risueno, M. C.
(1995). The methylation-acetylation method: an ultrastructural
cytochemistry for nucleic acids compatible with immunogold studies. J.
Struct. Biol. 114, 123-139.
Thiry, M., Sheer, U. and Goessens, G. (1988). Immunoelectron microscopic
study of nucleolar DNA during mitosis in Ehrlich tumor cells. Eur. J. Cell
Biol. 47, 346-357.
Thiry, M. and Goessens, G. (1996). The Nucleolus during the Cell Cycle.
Springer-Verlag, Heidelberg.
Wansink, D. G., Schul, W., van der Kraan, I., van Steensel, B., van Driel, R.
and de Jong, L. (1993). Fluorescent labeling of nascent RNA reveals
transcription by RNA polymerase II in domains scattered throughout the
nucleus. J. Cell Biol. 122, 283-293.
Weisenberger, D. and Scheer, U. (1995). A possible mechanism for the
inhibition of ribosomal RNA gene transcription during mitosis. J. Cell Biol.
129, 561-575.
Zatsepina, O. V., Voit, R., Grummt, I., Spring, H., Semenov, M. V. and
Trendelenburg, M. F. (1993). The RNA polymerase I-specific transcription
initiation factor UBF is associated with transcriptionally active and inactive
ribosomal genes. Chromosoma 102, 599-611.
(Received 21 May 1997 – Accepted 28 July 1997)