From the Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom
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ABSTRACT |
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Dramatic changes in the patterns of transcription
are a common feature of early development. We have used F9 embryonal
carcinoma cells as a model system to study gene regulation during an
early stage of murine embryogenesis. We find that transcription by RNA polymerase I decreases when F9 cells differentiate into parietal endoderm. The reduced rate of transcription is associated with a
down-regulation of several components of the class I transcription apparatus. The most substantial change involves the essential factor
SL1, which is a multisubunit complex that contains the TATA-binding
protein and three TATA-binding protein-associated factors (TAFs). The
abundance of two of these TAFs, TAFI48 and TAFI95, decreases during F9 cell differentiation.
Developmental regulation of a specific class of genes may therefore be
achieved through changes in the availability of TAFs.
The early stages of mouse development involve rapidly changing
patterns of transcription, which play a key role in embryogenesis. These events are inaccessible to study at the biochemical level because
of the difficulty in obtaining homogeneous cell populations in
sufficient numbers. One way to circumvent this problem involves the use
of embryonal carcinoma (EC)1
cell lines, which mimic events within the early embryo. A well characterized example is the F9 EC cell line, which can be induced to
differentiate into parietal endoderm (PE) by treatment with retinoic
acid and cAMP (1). Differentiation of F9 cells is accompanied by a
dramatic decrease in the rate of transcription by RNA polymerase (pol)
III (2). This response appears to provide an accurate reflection of
regulatory phenomena that occur in early development because in
situ hybridization has demonstrated that the abundance of pol III
transcripts decreases substantially when PE forms in mouse embryos (3).
By using F9 cells as a model system, it has been possible to
demonstrate that the decrease in pol III transcription that accompanies
differentiation into PE reflects a specific decrease in the abundance
of a transcription factor called TFIIIB (2, 4).
Ribosome biogenesis requires equimolar amounts of each of the rRNAs.
Because 5 S rRNA is made by pol III, whereas the remaining rRNAs are
made by pol I, these polymerases are frequently regulated coordinately
(5, 6). We have therefore investigated whether the decrease in pol III
transcription that occurs during F9 cell differentiation is accompanied
by a similar change in pol I activity. Our results indicate that PE
cells contain less rRNA than F9 EC cells, and this reflects a reduced
rate of transcript initiation. Changes are observed in several
components of the pol I transcription machinery. PE cells display a
slight decrease in the catalytic activity of the polymerase itself.
They also show a slight reduction in the level of the basal factor UBF,
which binds to rRNA promoters and stimulates expression by folding the
DNA and excluding repressor proteins (7, 8). An additional role for UBF
is to interact with an essential initiation factor called SL1 or TIF-IB
and stabilize its association with the promoter (5, 8, 9). SL1/TIF-IB is a complex containing the TATA-binding protein (TBP) and three TBP-associated factors (TAFs) (5, 10). The abundance of two of these
TAFs, TAFI95 and TAFI48, is significantly
depressed following F9 cell differentiation. As a consequence, the
activity of SL1/TIF-IB is severely limiting for pol I transcription in
PE cell extracts. We conclude that the differentiation of F9 EC cells
is accompanied by changes in several components of the pol I
transcription machinery, which result in decreased production and
steady-state levels of the large rRNA.
Cell Culture--
F9 cells were cultured and differentiated as
previously described (2).
RNA Extraction and Analysis--
Total cellular RNA was
extracted using TRI reagent (Sigma), according to the manufacturer's
instructions. Agarose gel electrophoresis, Northern transfer, and
hybridization were conducted as previously described (2). Primer
extension analysis was carried out by the method of Carey et
al. (11). The primer was 5'-CAGGCACCGCGACAGACCCAAG-3', which is
complementary to bases +100 to +122 of the mouse rRNA gene.
Preparation of Extracts and Protein Fractions--
Whole cell
extracts were prepared according to the method of Manley et
al. (12) and also by a more direct freeze-thaw procedure (13).
Both approaches yielded similar results.
Phosphocellulose chromatography was carried out by the method of Segall
et al. (14), except that the final high salt elution was
made using 1.2 M KCl instead of 1.0 M.
SL1/TIF-IB was present in this 0.6-1.2 M KCl step fraction
(PC-D), which contained ~1% of recovered protein. UBF and TFIIB were
present in the 0.35-0.6 M KCl step fraction (PC-C).
Heparin gradient chromatography of SL1/TIF-IB was carried out as
previously described (15). Human UBF was fractionated by gradient
chromatography of a HeLa nuclear extract on Q-Sepharose, performed as
previously described (16), and it was found to elute at ~500
mM KCl, away from the bulk of protein. HeLa UBF was also
fractionated by heparin chromatography of a PC-C fraction to give the
CHep1.0 fraction (13).
Transcription Assays--
Transcription reactions were carried
out as previously described (2), except that pBR322 was not included,
the incubations were for 60 min, and Antibodies and Western Blotting--
Western immunoblot analysis
was performed as described previously (13). Antiserum against
recombinant UBF (18) was a generous gift from Brian McStay. Anti-TFIIB
antiserum C-18 was obtained from Santa Cruz Biotechnology. Monoclonal
antibody SL30 against TBP (19) was a generous gift from Nouria
Hernandez. Antisera raised against recombinant TAFI48,
TAFI68, and TAFI95 (20, 21) were generously
provided by Ingrid Grummt and Joost Zomerdijk.
The Level of Pol I Transcripts Decreases After F9 Cell
Differentiation--
Total RNA was extracted from undifferentiated F9
EC cells and from cells that had been induced to differentiate into PE
by culture in the presence of retinoic acid and dibutyryl cAMP. The abundance of the 28 S rRNA product of pol I was then compared in F9 EC
and PE cells by Northern blot analysis (Fig.
1A). After differentiating for
5 days (lane 2), the 28 S rRNA was found to be 2.7-fold less
abundant than in undifferentiated EC cells (lane 1), and
after 7 days of differentiation its level had decreased by 4.0-fold
(lane 3). This effect is specific, because the steady-state level of tRNA remains unchanged, as shown previously (2).
Large rRNA is synthesized as a 47 S precursor molecule, the 5' end of
which is degraded rapidly (22). Because the sequences at the start of
the primary transcript are highly unstable, their level in the cell
reflects the rate of ongoing initiation by pol I. Primer extension
analysis with various amounts of total RNA was used to quantitate the
5' end of the primary rRNA transcript and test whether its abundance is
altered by differentiation (Fig. 1B). PhosphorImager
quantitation revealed that the level of the pre-rRNA 5' sequences is
3-5-fold higher in EC cells relative to PE cells. We conclude that a
decrease in the rate of transcript initiation can account for the
reduced steady-state levels of large rRNA in differentiated PE cells.
Pol I Transcription Apparatus Becomes Less Active following F9 Cell
Differentiation--
To compare the activity of the pol I
transcription apparatus before and after differentiation, whole cell
extracts were prepared and assayed for their ability to initiate
transcription on a rRNA gene template. EC cell extracts were found to
support much higher levels of pre-rRNA synthesis than extracts prepared
in parallel from PE cells (Fig.
2A). This was the case with
several sets of extracts that had been prepared using either of two
different extraction protocols. EC cell extracts were more active than
PE cell extracts when these assays were carried out over a range of
protein/DNA ratios.2
Quantitation of these experiments indicated that the activity of the
pol I transcription initiation apparatus is 6-9-fold higher in EC
extracts than in PE extracts. It is unclear why the EC/PE differential
is greater in vitro than in vivo. As a control
for the specificity of these effects, we assayed the ability of F9 cell
extracts to support pol II initiation from an HPRT promoter. Previous
work has demonstrated that HPRT transcription is not decreased after F9
cell differentiation (2). Consistent with this finding, we found that
EC and PE extracts initiate transcription at the HPRT promoter with
comparable efficiencies (Fig. 2B). We conclude that the
observed decrease in initiation by pol I is a specific regulatory
event.
The Low Rate of Pol I Transcription Initiation in PE Cells Is Not
Caused by an Excess of Dominant Repressor--
The above-described
results suggest that the ability of pol I to initiate transcription is
greater in EC than in PE cells. To determine whether this reflects the
production of an excess of dominant repressor following
differentiation, we performed mixing experiments (Fig.
3). Titrating increasing amounts of PE extract into a constant amount of EC cell extract did not diminish the
level of transcription that was obtained using EC extract alone. This
mixing approach therefore provided no evidence for a dominant repressor
of pol I transcription that is present in PE extracts in stoichiometric
excess. We therefore addressed the possibility that the reduced rate of
rRNA synthesis following differentiation is the result of a decrease in
the activity of one or more components of the pol I basal transcription
apparatus.
Several Components of the Pol I Transcription Apparatus Are
Down-regulated during F9 Cell Differentiation--
To determine
whether differentiation is accompanied by a change in the catalytic
capacity of pol I, we carried out random polymerization assays using
poly(dA-dT) as template. EC extracts were found consistently to have
slightly higher pol I catalytic activity than PE extracts that were
prepared in parallel.2 However, the difference between
matched pairs of extracts was only 1.6-2.2-fold. Although this change
in pol I activity may contribute to the observed regulation of rRNA
synthesis, it seems insufficient to account fully for the reduced
levels of pol I transcription that are found in PE cell extracts.
To examine whether differentiation affects the abundance of UBF, we
carried out Western blots with EC and PE cell extracts (Fig.
4A). The amount of UBF was
found to decrease by 1.7-3.1-fold after differentiation, with an
average difference of ~2.5-fold. Similar changes in UBF levels were
also detected when EC- and PE-derived fractions were compared after
chromatography.2 To examine whether there is a generalized
reduction in the level of transcription factors following F9 cell
differentiation, the same extracts were probed for the presence of
TFIIB, and the amount of this basal pol II factor was found to remain
virtually constant when the EC and PE cell extracts were compared (Fig.
4B). We also found that the abundance of the pol III factor
TFIIIC2 does not decrease when F9 cells differentiate (2, 4). We
conclude from these data that the observed reduction in the level of
UBF is a specific phenomenon.
The Activity of SL1/TIF-IB Decreases When F9 Cells Differentiate
and Is Limiting for Pol I Transcription in PE Cell Extracts--
If
the reduced transcriptional activity of PE cell extracts is caused by a
lack of UBF, it should be possible to stimulate expression by adding
more UBF. However, we found that titrating in partially purified UBF
made little difference to the rate of pol I transcription in PE-derived
extracts (Fig. 5A). In
contrast, an SL1-containing fraction tested in parallel produced a
substantial increase in the rate of rRNA synthesis. Indeed, this
fraction was sufficient to raise the rate of transcription in a PE cell extract to levels obtained using EC cell extracts. These data suggest
that there is a relative excess of UBF activity following F9 cell
differentiation, despite its decrease in abundance. Instead, the
transcription of rRNA genes appears to be limited in PE extracts by a
lack of SL1/TIF-IB activity. A rather different response was obtained
when EC cell extracts were tested in the same way (Fig. 5B).
Although adding the SL1/TIF-IB fraction produced a slight increase in
transcription by the EC extract, the UBF fraction gave a much more
dramatic stimulation (lanes 1-7). In contrast, when tested
in parallel, PE extract again responded to SL1/TIF-IB but not to the
UBF fraction (lanes 8-14). These results provide evidence
for a shift in the balance of the pol I factors as F9 cells
differentiate; the UBF fraction stimulates transcription in EC extracts
but not in PE extracts, whereas the activity of SL1/TIF-IB becomes
severely limiting after differentiation.
We carried out complementation assays to test whether the activity of
SL1/TIF-IB is diminished in PE cell extracts. The assay system
exploited the fact that SL1 from humans is unable to function on a
mouse rRNA promoter, whereas the other components of the human pol I
transcription apparatus are fully active on a murine template (15, 17).
A HeLa cell extract is therefore unable to transcribe a mouse rRNA gene
unless a source of murine SL1/TIF-IB is provided. The addition of 2 µg of EC cell extract was sufficient to allow expression from the
murine promoter in this system. In contrast, 10 µg of PE cell extract
was required to give a comparable signal when tested in parallel (Fig.
6A). Because the HeLa extract used in this assay provides an excess of the other pol I factors, the
relative inactivity of the PE cell extract must be because of a
deficiency in the species-specific factor SL1/TIF-IB. As a further test
of this conclusion, we used phosphocellulose chromatography to purify
this factor approximately 100-fold from EC and PE cell extracts. These
fractions were then compared for their ability to reconstitute
transcription from the murine promoter in the complementation assay.
When we added the SL1/TIF-IB fraction from F9 EC cells, efficient
transcription of the mouse gene was obtained, whereas the corresponding
fraction from PE cells was significantly less active when tested in
parallel (Fig. 6B). Although 0.36 µg of the EC-derived
fraction was sufficient to give a signal in this assay (lane
2), approximately four times more of the PE-derived fraction was
required to cross the detection threshold (lane 7). We
conclude that F9 cell differentiation is accompanied by a significant decrease in the activity of SL1/TIF-IB.
The Abundance of TAFI95 and TAFI48
Decreases Specifically When F9 Cells Differentiate--
Western blots
were carried out to compare the abundance of each of the four
components of SL1/TIF-IB in EC and PE extracts. The overall level of
TBP was found to be lower following differentiation (Fig.
7A). However, previous
analyses have shown that this decrease in TBP is associated primarily
with the down-regulation of TFIIIB and that the TBP content of
fractions containing SL1/TIF-IB and TFIID remains relatively constant
(4). Nevertheless, the abundance of TAFI48 (Fig.
7B) and TAFI95 (Fig. 7C) was also
found to be lower in the PE cell extracts than in the EC cell extracts.
In contrast, the abundance of TAFI68 remains unchanged
following differentiation (Fig. 7D).
Western blot analysis was also conducted on phosphocellulose fractions
containing SL1/TIF-IB that had been partially purified from EC and PE
cells. As in the crude extracts, the fractions showed little change in
the levels of TAFI68 (Fig.
8A, upper). Furthermore the TBP content of these fractions changed little during
differentiation (Fig. 8A, lower), as reported previously (4). However, both TAFI95 (Fig. 8B) and
TAFI48 (Fig. 8C) were much less abundant in the
fraction derived from PE cells compared with the equivalent EC-derived
fraction, as seen in the crude extracts. For each subunit of
SL1/TIF-IB, these results were confirmed using a second antiserum
raised against an alternative epitope (for TBP and TAFI95,
three separate antisera were used). The results suggest strongly that
F9 cell differentiation is accompanied by a specific decrease in the
abundance of two TAF components of SL1/TIF-IB.
The steady-state level of 28 S rRNA is significantly lower in F9
PE cells than in the undifferentiated EC progenitors. This decrease
reflects a reduction in the activity of the pol I transcription apparatus and a diminished rate of transcript initiation. We have detected a down-regulation in three separate components of the basal
machinery, pol I itself and the initiation factors UBF and SL1/TIF-IB.
Despite the generality of these changes to the pol I apparatus, the
effects are nevertheless specific, because the abundance of TFIIB and
the rate of pol II initiation at the HPRT promoter show little or no
difference between EC and PE cells.
A previous study using rat L6 myoblasts has shown that the abundance of
UBF decreases during myogenic differentiation (23). It may therefore be
that a reduction in UBF levels is a common feature of differentiation
in rodent cells. UBF is also a target for regulation during
differentiation of the human monocytic cell line U937 (24). In U937
cells, however, UBF is regulated by interaction with the retinoblastoma
protein, rather than a change in abundance (24). Genetic analysis has
shown that ratinoblastoma protein is important for hematopoiesis but is
not required for the early differentiation events that are mimicked by
F9 cells (25).
Despite the changes in UBF, the reduced rate of pol I transcription
that is found in PE cells appears to be caused primarily by a decrease
in the availability of SL1/TIF-IB. UBF activity is in relative excess
after differentiation, as shown by the fact that adding more UBF to PE
extracts does not increase the level of expression. Instead, add-back
experiments suggest that SL1/TIF-IB is the limiting component of the
pol I transcription apparatus. We cannot be certain that SL1/TIF-IB is
sufficient to restore active transcription to PE cell extracts, as our
fractions contain traces of UBF.2 It is therefore possible
that both SL1/TIF-IB and UBF are necessary to raise transcription in
the PE cell extracts to levels obtained using EC cell extracts.
Nevertheless, SL1/TIF-IB appears to be critical for this effect,
because fractions that lack this factor but contain active UBF produce
no stimulation when added to the PE cell extract. The requirement for
SL1/TIF-IB in these add-back experiments is consistent with the
substantial down-regulation of this factor, which is detected both in
complementation assays and also by immunoblotting.
Complementation assays with crude extracts or phosphocellulose
fractions reproducibly show that the activity of SL1/TIF-IB decreases
by 4- or 5-fold following differentiation. Despite this finding, the
abundance of TAFI68 remains virtually constant, the maximal
change observed being 1.2-fold. In contrast, substantial decreases are
observed with TAFI95 and TAFI48. Accurate
quantitation of the changes in these TAFs is difficult because of their
low abundance following differentiation. When various extracts and fractions are compared, we find that the levels of TAFI95
and TAFI48 can decrease by anything from 3- to 13-fold
after differentiation. It is difficult to be confident in these
numbers, however, because of the very low signal obtained when Western
blotting some PE cell extracts and fractions. Nevertheless, we are able
to conclude that there is a specific and substantial reduction in the
abundance of these subunits. SL1/TIF-IB is an essential component of
the pol I machinery that is involved in promoter recognition and
polymerase recruitment (5). Although murine SL1/TIF-IB activity has yet to be reconstituted from isolated subunits (21), both
TAFI48 and TAFI110 (the human equivalent of
TAFI95) are essential for transcription in the human system
(26, 27). It therefore seems highly probable that the decreased
abundance of TAFI48 and TAFI95 in PE cells can
account for the reduction in SL1/TIF-IB activity that is detected by
complementation assays.
To our knowledge, this constitutes the first report that SL1/TIF-IB is
subject to developmental control. However, tissue-specific changes in
TAFs have been described previously. Differentiated human B cells
contain a TAF component of TFIID that is not found in other cell types
and may contribute to B cell-specific patterns of pol II transcription
(28). Furthermore, a set of TAFs becomes restricted to the developing
nervous system during Drosophila development (29). Previous
studies have also provided precedent for TAF regulation during
differentiation of F9 cells. PE extracts were shown to contain
substantially reduced levels of the pol III-specific TBP-containing
complex TFIIIB (2, 4). This reflects a decrease in the abundance of a
TFIIIB TAF called BRF (TFIIB-related factor) (4). As a result of this
change, the rate of pol III transcription is considerably less in PE
than in EC cells or extracts (2, 4). Because the pol III product 5 S
rRNA is required in equimolar ratios with the rRNAs made by pol I, it
is not surprising that the two systems are regulated in parallel during
F9 cell differentiation. It is interesting that this coregulation is
achieved by a similar mechanism in both cases, a reduction in the
availability of specific TAFs.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-amanitin was present at a
final concentration of 200 µg/ml. The pol I template was pMrWT, which
contains bases
168 to +155 of the mouse rRNA promoter (17).
pHPRTcat has been described (2).
RESULTS
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Fig. 1.
F9 cell differentiation is accompanied by a
specific decrease in the abundance of pol I transcripts.
A, Northern blot analysis of total RNA (10 µg) from EC
cells (lane 1), PE cells after 5 days of differentiation
(lane 2), or PE cells after 7 days of differentiation
(lane 3). The blot was hybridized with a probe against 28 S
rRNA (upper panel) and then reprobed with a tRNA gene
(lower panel). B, primer extension analysis of
the levels of primary pol I transcript in total RNA (1 µg in
lanes 1 and 2; 6 µg in lanes 3 and
4) from EC cells (lanes 1 and 3) or PE
cells after 7 days of differentiation (lanes 2 and
4).
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Fig. 2.
PE extracts display a specific deficiency in
pol I transcriptional activity. A, a pMrWT template (20 ng) was transcribed using 14 µg (lanes 1 and 2)
or 8 µg (lanes 3 and 4) of EC (lanes
1 and 3) or PE (lanes 2 and 4)
cell extract. Extracts were prepared either by the freeze-thaw method
(lanes 1 and 2) or the Manley protocol
(lanes 3 and 4). B,
pHPRTcat (1 µg) was transcribed using 30 µg of EC
(lane 1) or PE (lane 2) cell extract, prepared by
the Manley protocol.
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Fig. 3.
Mixing of extracts provides no evidence for a
dominant repressor activity in PE cell extracts. A pMrWT template
(20 ng) was transcribed using 2 µl of EC cell extract (lanes
2-7) and 1 (lane 3), 2 (lanes 1 and
4), 3 (lane 5), or 4 µl (lane 6) of
PE cell extract. Both extracts had a protein concentration of 1.3 mg/ml.
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Fig. 4.
UBF levels decrease during F9 cell
differentiation. A, an SDS-7.8% polyacrylamide gel was
used to resolve 9 µg of a HeLa cell CHep1.0 fraction (lane
1) and 40 µg of EC and PE cell extracts (lanes 2 and
3, respectively) and then analyzed by Western immunoblotting
with anti-UBF antibody. B, an SDS-7.8% polyacrylamide gel
was used to resolve 40 µg of EC and PE cell extracts (lanes
1 and 2, respectively) and 9 µg of a HeLa cell
CHep1.0 fraction (lane 3) and then analyzed by Western
immunoblotting with anti-TFIIB antibody C-18.
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Fig. 5.
The activity of SL1/TIF-IB is limiting for
pol I transcription in PE cell extracts. A, a pMrWT
template (20 ng) was transcribed using 9 µg of EC (lane 1)
or PE (lanes 2-7) cell extract. Reactions were supplemented
with 1.5 or 2.6 µg of Q-Sepharose-fractionated human UBF (lanes
3 and 4, respectively) or with 0.6, 1.1, or 2.0 µg
(lanes 5, 6, and 7, respectively) of the
EC-derived SL1 fraction (PC-D). B, a pMrWT template (20 ng)
was transcribed using 8 µg of EC (lanes 1-7) or PE
(lanes 8-14) cell extract. Reactions were supplemented with
0.7 (lanes 2 and 9), 1.5 (lanes 3 and
10), or 2.6 µg (lanes 4 and 11) of
Q-Sepharose-fractionated human UBF or with 0.5 (lanes 5 and
12), 0.9 (lanes 6 and 13), or 1.6 µg
(lanes 7 and 14) of the EC-derived SL1 fraction
(PC-D).
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Fig. 6.
The activity of SL1/TIF-IB is diminished
following F9 cell differentiation. A, a pMrWT template
(20 ng) was transcribed using 10 µg of HeLa nuclear extract
supplemented with 2, 4, 6, or 8 µg of EC cell extract (lanes
2-5, respectively) or with 2, 4, 6, 8, 10, or 12 µg
(lanes 7-12, respectively) of PE cell extract.
B, a pMrWT template (20 ng) was transcribed using 10 µg of
HeLa nuclear extract supplemented with 2, 4, or 6 µl (lanes
2-4, respectively) of an EC cell-derived SL1 fraction (PC-D) or
with 6, 8, 10, 12, 14, or 16 µl (lanes 6-11,
respectively) of the SL1 fraction (PC-D) derived from PE cells. Both
PC-D fractions had a protein concentration of 0.18 mg/ml.
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Fig. 7.
The abundance of specific SL1 subunits
decreases during differentiation. A, whole cell extract
(56 µg) of EC (lane 1) or PE (lane 2) cells was
resolved on an SDS-7.8% polyacrylamide gel and then analyzed by
Western immunoblotting with antibody SL30 against TBP. B,
whole cell extract (56 µg) of PE (lane 1) or EC
(lane 2) cells and the HeLa cell PC-D fraction (5.6 µg,
lane 3) were resolved on an SDS-7.8% polyacrylamide gel and
then analyzed by Western immunoblotting with antibody against
TAFI48. C, SL1 prepared from an EC extract by
gradient chromatography on heparin (24 µg; lane 1) and
whole cell extract (56 µg) of EC (lane 2) or PE
(lane 3) cells were resolved on an SDS-7.8% polyacrylamide
gel and then analyzed by Western immunoblotting with antibody against
TAFI95. D, SL1 prepared from an EC extract by
gradient chromatography on heparin (24 µg; lane 1) and
whole cell extract (56 µg) of EC (lane 2) or PE
(lane 3) cells were resolved on an SDS-7.8% polyacrylamide
gel and then analyzed by Western immunoblotting with antibody against
TAFI68.
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Fig. 8.
F9 cell differentiation is accompanied by
specific decreases in the abundance of TAFI95
and TAFI48. A, an SL1 fraction
(PC-D; 5.6 µg) derived from HeLa (lane 1), EC (lane
2), or PE (lane 3) cells was resolved on an SDS-7.8%
polyacrylamide gel and then analyzed by Western immunoblotting with
antibodies against TAFI63/68 (top half) and TBP
(bottom half). Mouse TAFI68 is 26 amino acids
longer than its human homologue TAFI63 (21) and
consequently migrates somewhat more slowly. Mouse TBP (mTBP)
is 23 amino acids shorter than its human homologue (hTBP)
and consequently migrates faster. B, an SL1 fraction derived
from PE (lane 1) or EC (lanes 2 and 3)
cells, was resolved on an SDS-7.8% polyacrylamide gel and then
analyzed by Western immunoblotting with antibodies against
TAFI95. Lanes 1 and 2 contain 5.6 µg of PC-D fraction, whereas lane 3 contains 24 µg of
SL1 that was prepared from an EC extract by gradient chromatography on
heparin. C, an SL1 fraction (PC-D; 5.6 µg) derived from
HeLa (lane 1), PE (lane 2), or EC (lane
3) cells was resolved on an SDS-7.8% polyacrylamide gel and then
analyzed by Western immunoblotting with antibodies against
TAFI48.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank David Stott for advice concerning the growth and differentiation of F9 cells and Carol Cairns, Ingrid Grummt, Brian McStay, and Joost Zomerdijk for antibodies and discussions.
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FOOTNOTES |
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* This work was funded by Project Grant G9437137MB (to R. J. W.) from the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Jenner Research Fellow of the Lister Institute of Preventive
Medicine. To whom correspondence should be addressed. Tel.:
44-141-330-4628; Fax: 44-141-330-4620; E-mail:
rwhite{at}udcf.gla.ac.uk.
The abbreviations used are: EC, embryonal carcinoma; HPRT, hypoxanthine-guanine phosphoribosyltransferase; PE, parietal endo- derm; pol, polymerase; TBP, TATA-binding protein; TAF, TBP-associated factor; TF, transcription factor; TIF-IB, transcription initiation factor IB; UBF, upstream binding factor; PC, phosphocellulose.
2 H. Alzuherri and R. J. White, unpublished data.
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REFERENCES |
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