From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B. P. 163-67404 Illkirch Cédex, Communauté Urbaine de Strasbourg, France
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ABSTRACT |
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We have investigated the expression levels of the
TATA-binding protein (TBP) and several TBP-associated factors
(TAFIIs) in differentiated adult mouse tissues.
Immunoblots performed using monoclonal antibodies show that there are
considerable variations in the levels of TBP and many TAFII
proteins present in various tissues. Consequently, the relative levels
of TAFIIs and TBP vary significantly from one tissue to
another. TBP and several TAFIIs are overexpressed in both
testis and small intestine, while in marked contrast, many of these
proteins, including TBP itself, were substantially down-regulated in
nervous tissues and in the heart. These tissues do, however, show a
high expression level of the TBP-like factor, which thus may represent
an alternative factor for the specialized transcription program in some
differentiated tissues. While there are significant variations in the
levels of TAFII28 protein, reverse transcription-coupled
polymerase chain reaction shows similar expression of the
TAFII28 mRNA in different tissues. The variations in
TAFII28 protein levels therefore result from
post-transcriptional regulatory events.
TFIID1 is a multiprotein
complex, which together with TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH
assists RNA polymerase II to correctly initiate transcription (1).
TFIID is composed of the TATA-binding protein (TBP), which specifically
binds the TATA element, and a series of evolutionary conserved
TBP-associated factors (TAFIIs). TAFIIs have
been shown to be involved in promoter recognition (2, 3) and to act as
specific transcriptional coactivators in vitro and in
transfected mammalian cells (Refs. 4-8 and references therein; for
review, see Ref. 9). Genetic experiments in yeast have shown a variable
requirement for TAFIIs, some of which are required for the
expression of only a subset of promoters involved for example in cell
cycle control, while others are more generally required (10-14).
Recently, a subset of TAFIIs have been found in other
complexes devoid of TBP, such as the PCAF·SAGA complex in humans and
in yeast (15-17), and the TBP-free
TAFII-containing complex (TFTC)
(18). Despite the fact that TFTC does not contain TBP it can replace
TFIID in both basal and activated transcription in vitro,
suggesting that TBP may not always be an essential transcription factor
in vivo.
While much has been learned about the function of TFIID in biochemical
assays and in yeast, little is known concerning the expression of its
constituent subunits in animal tissues. Previous studies on TBP (19)
have demonstrated an overexpression of TBP mRNA and to a lesser
extent of the TBP protein in testis. The mRNAs of several
TAFIIs have been shown to be equally expressed in several
rat tissues (20). However, the TAFII105 mRNA is widely expressed yet the corresponding protein shows cell specificity, being
much more abundant in mature lymphoid B cells (8).
The above observations prompted us to investigate the expression
of TBP and TAFII proteins rather than their mRNAs in a
variety of adult murine tissues. Immunoblots performed with a series of monoclonal antibodies show that the relative expression levels of these
TAFIIs, and TBP can vary extensively from tissue to tissue, suggesting that the transcription program in different tissues may have
differential requirements for TFIID components. Furthermore, the levels
of TBP and many TAFIIs is significantly reduced in extracts
from the nervous system (brain, cerebellum, eye, spinal cord), kidney,
and in the heart. Interestingly, several of these tissues show high
expression levels of the previously described TBP-like factor (TLF)
(18), raising the possibility that TLF may functionally substitute for
TBP in certain tissues. In the case of TAFII28, whose
mRNA is equivalently expressed in many tissues, the variations in
TAFII28 protein must result from post-transcriptional events.
Preparation of Murine Tissue Extracts--
Four individual
6-week-old Black 6 mice were sacrificed and the tissues extracted and
immediately frozen in liquid nitrogen. Protein extracts were made as
described (21) by shearing the tissues in 2 × boiling Laemmli
buffer containing 10 mM Preparation of Cell Line Extracts--
Cell extracts were
prepared as described previously (5) by three cycles of freeze-thaw in
100 µl of buffer A (50 mM Tris-HCl (pH 7.9), 20%
glycerol, 1 mM dithiothreitol, and 0.01% Nonidet P-40)
containing 0.5 M KCl and 2.5 µg/ml leupeptin, pepstatin, aprotinin, antipain, and chymostatin. The proteins were quantified by
Bradford test and the equivalent amounts were used for immunoblots.
Antibody Preparation--
Monoclonal antibodies (mAbs)
against TBP (3G3), TAFII55 (19TA), TAFII135
(20TA), TAFII100 (1TA), TAFII30 (4G2),
TAFII20 (22TA), and TAFII18 (16TA) and mouse
polyclonal sera against TLF were prepared as described previously (4,
18, 22-25). mAb 35TA was raised against purified Escherichia
coli expressed GST-mTAFII28.
RNA Preparation and RT-PCR--
RNA from tissue samples was
prepared as described previously (26). RT-PCR was performed on 1 µg
of total RNA using the following primers 5'-GGACAAGAAGGAGAAGAA-3' and
5'-CTTCTTGTGCTTTGAGTTGGGGAT-3' specific to different exons of
mTAFII28 generating a 360-base pair fragment. Samples were
denatured for 3 min at 94 °C and annealed for 10 min at 50 °C. A
mix of avian myeloblastosis virus reverse transcriptase and
Taq polymerase was added and incubated for another 20 min at
the same temperature. 30 cycles of PCR were then performed. After 15, 23, and 30 cycles an aliquot of each sample was removed and
electrophoresed, transferred to a hybond membrane, and hybridized with
a 32P-labeled TAFII28-specific oligonucleotide
probe. As a control a 200-base pair fragment of the hypoxanthine
guanine phosphoribosyltransferase (HPRT) gene was amplified in the same
reactions and detected by hybridization using an HPRT-specific
oligonucleotide probe. Amplification with no avian myeloblastosis virus
reverse transcriptase was also performed as a negative control.
Variations in TAFII Protein Content of Adult Murine
Tissues--
To investigate the levels of TFIID components in adult
murine tissues immunoblots were performed using mAbs against a
selection of TAFIIs, which are either TFIID-specific
(TAFII28, TAFII18) or are present in other
TAFII-containing complexes (TAFII135, TAFII100, TAFII55, TAFII30,
TAFII20). Six-week-old mice were sacrificed, dissected, and
their organs were immediately frozen in liquid nitrogen. Equivalent
amounts of the proteins extracted from each tissue (see "Materials
and Methods" and Fig. 1) were used to
make several replica immunoblots along with extracts from human HeLa and murine F9 cells as controls. All the antibodies used detected both
the human TAFIIs and their murine counterparts. Analogous results to those shown below were observed in blots from independently prepared extracts (data not shown).
TAFII135 and TAFII100 could be detected in all
tissues, with the exception of the spinal cord where
TAFII100 was seen only very weakly, while
TAFII135 was undetectable (Fig.
2, see lane 17). Both
TAFII135 and TAFII100 were strongly
overexpressed in the testis where, and for the sake of clarity, a
5-fold shorter exposure is shown (lane 15, Fig. 2). An
exposure time comparable with that shown in the other lanes resulted in
a saturated black signal (data not shown). Varying levels of
TAFII55 could also be detected in all tissues with
overexpression in the testis being less dramatic than for
TAFII135 and TAFII100 (note that for
TAFII55 the same exposure time is shown in all
tissues).
Although these TAFIIs are widely expressed, their
relative expression levels vary from tissue to tissue. For example,
equivalent signals for TAFII135 and TAFII100
are seen in the liver, lung, and adrenal gland (Fig. 2, lanes
9-11, respectively), while the signal for TAFII100 is
stronger than that for TAFII135 in the pituitary and the
small intestine (lanes 3 and 4, respectively). The opposite relationship is observed in the eye, tongue, and spleen
(lanes 2, 7, and 8, respectively).
Similarly, the ratio of TAFII55 and TAFII100
signals changes when one compares the pituitary or the liver, where the
signal for TAFII100 is the stronger, with the heart and
lung, where the opposite is seen (lanes 3, 9,
6, and 10, respectively). Therefore, not only do
the expression levels of a given TAFII vary from tissue to
tissue, but the relative abundance of TAFIIs also varies.
The presence of several other TAFIIs in these tissues was
also assayed. TAFII30 can be readily detected in most
tissues with the exception of the eye and the pituitary, where only low
levels of expression are seen (lanes 2 and 3,
respectively). In contrast, TAFII30 is below the limit of
detection in the heart and spinal cord (lanes 6 and
17, respectively). Like the other TAFIIs, it is
strongly overexpressed in the testis (lane 15).
The histone fold-containing TAFII28 can be clearly detected
only in the small intestine and the testis (lanes 4 and
15, respectively), while it is barely detectable in most
other tissues and undetectable in the heart, brain, kidney, and spinal
cord (lanes 6, 13, 16, and
17, respectively). A similar expression pattern was seen for its heterodimeric partner TAFII18 (27). The histone
fold-containing TAFII20 was also up-regulated in the testis
and small intestine and down-regulated in heart, brain, kidney, and
spinal cord.
These results again highlight some significant variations in the ratios
of TAFIIs present in different tissues. For example, equivalent amounts of TAFII30 are seen in small intestine
and spleen (lanes 4 and 8, respectively), while
TAFII28, TAFII20, and TAFII18 are
down-regulated in spleen, whereas TAFII55 is up-regulated. Similarly, equivalent amounts of TAFII30 are seen in the
brain and cerebellum (lanes 13 and 12,
respectively), while all the other TAFIIs are
down-regulated in brain compared with cerebellum. Furthermore, while
TAFII30, TAFII28, TAFII20, and
TAFII18 are down-regulated in the eye;
TAFII135, TAFII100, and TAFII55 are expressed at levels comparable with those of several other tissues.
These results also reveal a general pattern of TAFII
expression. Many TAFIIs are overexpressed in the testis.
This was most dramatic for TAFII135, TAFII100,
and TAFII30, while TAFII55 was only mildly
overexpressed. Overexpression of other RNA polymerase II transcription
factors, TBP (also confirmed by this study, see below), TFIIB, and the
largest subunit of RNA polymerase II, have been described previously in
testis (19). It is possible that the TAFIIs, like these
other factors, are overexpressed in the round haploid spermatids.
In addition to testis, most of the TAFIIs tested were
strongly expressed in the small intestine. This is particularly true for TAFII28, TAFII20, and TAFII18
which were as well expressed as in the testis. In contrast, many
TAFIIs were down-regulated to the point of being
undetectable in tissues such as brain, heart, kidney, and spinal cord.
This is also the case in the kidney with the exception of
TAFII30, which is as abundant as in intestine. Comparison
of the signals observed in the brain, kidney, and lung with those
obtained with serial dilutions of the small intestine extract showed
that the levels of TAFII135 and TAFII100 were
5-fold lower in the brain and kidney than in intestine, while those in the lung were around 3-fold lower (data not shown). Note that the
levels of these TAFIIs are even lower in the spinal cord
and heart than in the brain or kidney. Similar titrations showed that TAFII55 levels were 10-fold lower in the brain and kidney
than in the testis, while the levels in the lung were 2-3-fold lower (data not shown). This suggests that the distinct transcriptional programs of each tissue show differing requirements for a given TAFII.
Partially Complementary Expression of TBP and TLF in Mouse
Tissues--
The same extracts were also tested for the expression of
TBP. TBP is strongly expressed in the testis (Fig.
3A, lane 15) and in
the small intestine and the pituitary (lanes 8 and
9, respectively). It is interesting that one of the highest
levels of TBP is found in the pituitary, since many TAFIIs
are under expressed in this extract. Intermediate expression levels
were detected in the adrenal, lung, liver, spleen, and tongue
(lanes 2-6, respectively). Strikingly, only very low levels
of TBP could be detected in the brain and cerebellum (lanes
12 and 13, respectively), and TBP was virtually undetectable in the heart, eye, kidney, and spinal cord (lanes 7, 10, 16, and 17, respectively;
note that since comparable exposures of two different blots are
presented, the adrenal gland was included in both to allow comparison
of the left and right panels). In these experiments, TBP was detectable
in brain, cerebellum, heart, eye, kidney, and spinal cord only when
very long saturating exposures of the blots were made (data not shown),
while the nonsaturating exposures shown in Fig. 3A highlight
the differences in expression levels. Titration experiments using
serial dilutions of the testis and small intestine extracts showed that
TBP levels were 3-5-fold lower in the small intestine than in the
testis, 5-6-fold lower in the lung, and more than 10-fold lower in the
brain and kidney (data not shown). These results reveal a considerable
variation in TBP expression levels among the different tissues.
The above result is rather unexpected considering the important role
which TBP is thought to play in transcription. This prompted us to look
at the expression of TLF, a factor highly related to the TBP core
domain (18)2 and which
consequently may be able to functionally substitute for TBP.
The highest levels of TLF were detected in the adrenal, small
intestine, brain, and spinal cord (Fig. 3A, lanes
2, 8, 13, and 17, respectively).
TLF was also present in the liver, tongue, heart, pituitary, eye,
cerebellum, and kidney (lanes 4, 6, 7, 9, 10, 12, and 16,
respectively), but was undetectable in the lung and spleen (lanes
3 and 5, respectively). TLF was expressed in the
testis, but in contrast to the other factors examined, it was under,
rather than overexpressed, in this tissue (compare the contrasting
levels of TBP and TLF in testis, lane 15, with brain or
spinal cord in lanes 13 and 17, respectively, and
the expression of TBP and TLF in the pituitary and eye, lanes
9 and 10, respectively). The fact that TLF expression can be
readily detected in the eye, heart, spinal cord, and kidney (note also that TAFII30 is readily detectable in the kidney extract)
extracts shows that there is no intrinsic defect in these extracts
which would explain the observed low levels of TAFIIs and
TBP. The presence of TLF in these extracts rather underlines the real
differences which exist in the expression levels of TBP and
TAFIIs.
TLF was also present in the extracts from several cultured cell lines,
being readily detected in total cell extracts from pluripotent murine
F9 embryonal carcinoma cells (Fig. 3A, lane 1,
and Fig. 3B, lane 2) and embryonic stem cells
(Fig. 3B, lane 5) or from differentiated 3T3
fibroblasts and simian COS cells (lanes 3 and 4,
respectively), but much more weakly in HeLa cells (lane
1).
Previous studies on TBP protein expression have been limited to
only a few tissues and have employed polyclonal antisera. Here we have
used a very sensitive monoclonal antibody against TBP that reveals
unexpected and very significant variations in TBP expression. As
described previously (19), TBP is overexpressed in the testis. This,
however, is not unique since high expression was also observed in the
small intestine and the pituitary. In contrast, TBP like many
TAFIIs, was strongly down-regulated in the nervous tissues,
eye, kidney, and in the heart.
In many of the tissues with low TBP expression, especially those of the
nervous system, prominent levels of TLF were observed. Nevertheless,
TLF expression was not limited only to nervous tissues or to tissues
with low TBP levels, since it was also abundantly expressed in the
adrenal and the small intestine extracts. Immunohistochemistry will
help determine whether TBP and TLF are overexpressed in the same cell
populations in these organs. Similarly, it will be interesting to
determine which cells within the nervous system express TLF. The
available antibody does not yet permit such studies.
In yeast and in mammalian cells, TAFIIs are essential for
cell cycle progression and they regulate the expression of cell cycle
genes (14, 28-31). Thus, while there is a stringent requirement for
high levels of TBP and TAFIIs in proliferating cells,
nothing is known concerning the requirement for these proteins in
terminally differentiated tissues. Our finding that the levels of TBP
and several TAFII are very dramatically reduced in several
differentiated tissues suggests that there is a differential
requirement for TFIID in rapidly proliferating cells versus
differentiated tissues.
The low expression of TBP and many TAFIIs does not reflect
an absence of polymerase II transcription in these tissues. Previous measurements of polymerase II transcription rates in different organs
have shown only two to three-fold reductions in the kidney and the
brain compared with the liver (32). Moreover, transcription rates were
lower in the lung than in the brain and kidney. There is therefore no
correlation between global transcription rates and measured TBP levels.
Our results would rather suggest that in certain differentiated
tissues, TLF may play an important role in very specialized transcription program. While this manuscript was in preparation, Ohbayashi et al. (33) described the expression of TLF
(TBP-like protein) in a limited set of rat tissues. As observed here,
TLF levels were especially high in the brain and heart. However, these authors (33) showed that recombinant TLF does not support transcription in vitro and does not bind to adenovirus E4 and major late
promoter TATA boxes. Further experiments will be required to determine whether TLF is a transcription factor under more physiological conditions using extracts from TLF-expressing tissues and natural promoters. In addition, as suggested for TBP-related factor (34), TLF
may itself be associated with a specialized set of associated factors
programming it for the transcription of specific genes.
TAFII28 Protein Levels Do Not Correlate with That of
Its mRNA--
Among the TAFIIs examined here,
TAFII28 is typical of a TAFII, whose expression
varies considerably from tissue to tissue. We tested whether these
variations could result from differences in mRNA levels.
RT-PCR was performed with total RNA from each tissue and exon-specific
TAFII28 primers (see "Materials and Methods"). Aliquots of the reaction were analyzed after 15, 23 (shown in Fig.
4), and 30 cycles of PCR. Although the
TAFII28 protein was most abundant in testis and small
intestine, yet undetectable in the heart, kidney, and spinal cord, only
minor differences in the corresponding levels of mRNA were
observed. These variations closely mirrored those of the HPRT control
and thus are probably due to intrinsic variations in the RNA samples
rather than real significant differences in TAFII28
mRNA levels. The TAFII28 mRNA was, however,
expressed at significantly lower levels in the adrenal gland.
The above results (and Northern
blotting)3 indicate that
there is no direct correlation between the levels of
TAFII28 protein and those of its mRNA. Most of the
variations in TAFII28 protein expression therefore result
from post-transcriptional events. This is in keeping with previous
studies that showed rather homogenous expression of several
TAFIIs mRNAs in normal rat tissues (20). The increases
in several TAFII proteins seen in testis do not, as is the
case for TBP (19), require a strong up-regulation of their mRNAs.
Moreover, while TBP mRNA levels are increased in testis, correlated
with increased protein expression, TBP mRNA levels are not
down-regulated in brain and kidney (19), where there is a considerable
reduction in the corresponding protein. These results in conjunction
with the previous results concerning TAFII105 (8) would
indicate that the levels of many TAFII proteins are mainly
regulated post-transcriptionally.
The nature of this post-transcriptional regulation is at present
unknown. It is possible that the efficiency of translation of the
mRNAs varies in different tissues. Alternatively, it is interesting
to note that when a given TAFII is depleted in yeast, the
integrity of the TFIID complex is compromised and the levels of other
TAFIIs are also strongly reduced (12, 35, 36). This
suggests that many TAFII proteins accumulate only when they are stably associated in the TFIID complex, otherwise they are be
rapidly degraded. Therefore, the levels of one TAFII may
indirectly control those of others, if it becomes limiting for TFIID
complex assembly. In the nervous tissues, it may even be the low levels of TBP itself that are limiting for TFIID assembly. Further knowledge of how the different TAFII-containing complexes are
assembled inside cells and what the limiting factors in this process
are will help in understanding the mechanisms which regulate
TAFII expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-mercaptoethanol. The extracts
were analyzed by SDS-PAGE and staining with Coomassie Blue stain to
normalize each preparation.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Analysis of tissues extracts by SDS-PAGE and
staining with Coomassie Brilliant Blue. 3 µl of the extracts
whose tissue of origin is indicated above each lane were analyzed by
SDS-PAGE and staining with Coomassie Blue. The positions of migration
of molecular mass standards are shown on the left along with the
relative molecular mass in kilodaltons.
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Fig. 2.
Detection of TAFII proteins in
extracts from HeLa and F9 cells and mouse tissues. The tissues of
origin are indicated above each lane. The quantities of extract loaded
in each lane were adjusted to that of 3 µl of the kidney extract
shown in Fig. 1. The positions of migration of each TAFII
are indicated. As long exposures of the panels for TAFII30
and TAFII28 are shown, several other proteins are detected
nonspecifically by these antibodies. The dash indicates the
bone fide position of migration of TAFII30 and
TAFII28 to distinguish them from closely migrating species,
while "o" indicates the presence of an artifact seen
with both the anti-TAFII30 and anti-TAFII28
antibodies. The signals for TAFII135 and
TAFII100 in lanes 14 and 15 represent
a 5-fold shorter exposure than those in the other lanes.
TAFII135, TAFII100, and TAFII55
were detected on the same blots as were TAFII28,
TAFII20, and TAFII18, while
TAFII30, which closely comigrates with TAFII28,
was taken from another blot.
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Fig. 3.
Expression of TBP and TLF. A,
the origin of the extracts is shown above each lane. The positions of
mouse (m) TBP and TLF are indicated by the
arrows. "o" indicates a second protein
nonspecifically revealed by the anti-TLF antisera. B, the
origin of the cell lines is shown above each lane. Mouse (m)
and human (h) TBP are indicated along with TLF.
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Fig. 4.
Expression of TAFII28
mRNA. The origin of the RNA used is indicated above each lane.
RT-PCR products were hybridized with probes for TAFII28 and
HPRT. The positions of the corresponding fragments are indicated.
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ACKNOWLEDGEMENTS |
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We thank P. Chambon for support; Y. G. Gangloff and M. Abrink for critical reading of the manuscript; Y. Lutz and the monoclonal antibody facility; G. Cristina for help with the mice; the staff of cell culture, animal, and oligonucleotide facilities; G. Gangloff and M. Abrink for critical reading of the manuscript; and B. Boulay, J. M. Lafontaine, R. Buchert, and C. Werlé for illustrations.
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FOOTNOTES |
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* 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.
Supported by fellowships from the Association pour la Recherche
contre le Cancer and a TMR grant from the European Union.
§ To whom correspondence should be addressed. Tel.: 33-3-88-65-34-40 (ext. 45); Fax: 33-3-88-65-32-01.
2 J.-C. Dantonel, J.-M. Wurtz, O. Poch, D. Moras, L. Tora, submitted for publication.
3 L. Perletti and I. Davidson, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: TF, transcription factor; TBP, TATA-binding protein; TAFII, TBP-associated factor; TLF, TBP-like factor; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; RT-PCR, reverse transcription-polymerase chain reaction; HPRT, hypoxanthine guanine phosphoribosyltransferase.
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