1 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, CNRS/INSERM/ULP, 1 Rue Laurent Fries, 67404 Illkirch Cédex,
France
2 Department of Anatomy, University of Turku, 20520 Turku, Finland
* Author for correspondence (e-mail: irwin{at}titus.u-strasbg.fr)
Accepted 27 January 2003
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Summary |
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Key words: X chromosome, Spermatogonia, TBP, Spermatogenesis, Haploid cells, Meiosis
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Introduction |
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Genetic and biochemical investigations in several organisms have shown that
TAFs play important roles in transcriptional regulation. Mutation of TAFs in
yeast and in mammalian cells leads to cell cycle arrest and gene-specific
transcriptional effects. TAFs have also been shown to play a role in promoter
recognition and selectivity and to interact directly with transcriptional
activators (for reviews, see Albright and
Tjian, 2000; Green,
2000
). These interactions are thought to contribute to recruitment
of TFIID to specific promoters.
While the majority of TAFs are `ubiquitously' expressed in all tissues,
albeit at different levels (Perletti et
al., 1999), two examples of TAFs with tissue-restricted expression
and function have been more recently described. In Drosophila
melanogaster, Cannonball (can, dTAF5L) is a testis-specific paralogue of
the somatic TAF5 protein. Mutation of this gene in Drosophila leads
to arrest of germ-cell development and male sterility
(Hiller et al., 2001
). In
mouse, the TAF4b protein, a paralogue of somatic TAF4, is expressed in a
tissue-restricted fashion with high expression in the granulosa cells of the
ovary and the testis (Dikstein et al.,
1996
; Freiman et al.,
2001
). Female TAF4b-/- mice show defective oocyte
maturation leading to sterility, while males are fertile and show no
spermatogenesis abnormalities. These two examples indicate that, in addition
to the somatically expressed TAFs, there are tissue-specific paralogues that
have much more specific functions.
Spermatogenesis is a cyclic process in which diploid spermatogonia differentiate into mature haploid spermatozoa. Spermatogonia committed for differentiation give rise to spermatocytes that undergo two meiotic divisions, to generate haploid round spermatids. During the process of spermiogenesis the haploid round spermatids undergo an elongation phase, during which they are sculptured into the shape of mature spermatozoa. This entails a major biochemical and morphological restructuring of the germ cell in which the majority of the somatic histones are replaced by protamines to pack the DNA into the sperm cell nucleus.
Several lines of evidence point to specialised transcriptional regulatory
mechanisms in spermatogenesis (for a review, see
Sassone-Corsi, 2002).
Components of the general transcription machinery such as TBP and
TAFIIs are strongly upregulated in testis
(Perletti et al., 1999
;
Schmidt and Schibler, 1995
).
In the case of TBP, upregulation takes place in late pachytene spermatocytes
and in haploid round spermatids (Martianov
et al., 2002
). In addition, male germ cells express paralogues of
the general transcription factors such as TFIIA
/ALF, a paralogue of
TFIIA, and TLF/TRF2, a paralogue of TBP
(Ozer et al., 2000
;
Upadhyaya et al., 1999
).
TLF-/- male mice are sterile as a result of a complete arrest of
spermatogenesis resulting from germ-cell apoptosis at the transition from
round to elongating spermatids (Martianov
et al., 2001
; Zhang et al.,
2001
). Hence, spermatogenesis is characterised by overexpression
of somatically expressed transcription factors and in some cases expression of
germ-cell-specific paralogues.
To add to this list, murine TAF7L (TAF2Q) is a testis-specific protein with
sequence similarity to somatic TAF7 [formerly TAFII55
(Lavigne et al., 1996;
Tora, 2002
)] encoded by the X
chromosome (Wang et al.,
2001
). RT-PCR experiments have indicated that TAF7L expression is
restricted to the germ-cell lineage, where its mRNA is strongly expressed in
spermatogonia.
While the TAF7L mRNA has been shown to be expressed in spermatogonia, nothing is known about its expression at other stages of spermatogenesis and whether it is associated with TBP as a bona fide TAF. We have investigated expression of the TAF7L protein during spermatogenesis and show that it is expressed in primary spermatocytes and haploid cells, but that its intracellular localisation is regulated during differentiation. A fraction of the TAF7L present in pachytene and haploid cells is tightly associated with TBP and in two hybrid screens TAF7L interacts with the TFIID subunit TAF1. These results are consistent with the idea that TAF7L is a bona fide TFIID subunit that replaces TAF7 as TFIID composition is remodelled during spermatogenesis. Analysis of the expression of other TAFs during spermatogenesis unexpectedly revealed that they are expressed differently both from each other and from TBP. Therefore, while TBP and TAFs are overexpressed in testis, they are overexpressed at distinct stages of the differentiation process indicating a dramatic remodelling of the composition of TFIID and other TAF-containing complexes during male germ-cell differentiation.
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Materials and Methods |
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Immunoblots and immunohistochemistry
Mouse seminiferous tubule segments at defined stages were isolated using
the transillumination-assisted microdissection method
(Parvinen and Hecht, 1981).
Immunohistochemistry was performed on fixed sectioned seminiferous tubules or
from staged squash preparations of microdissected tubules from wild-type C57
BL/6 mice as previously described
(Martianov et al., 2001
;
Nantel et al., 1996
).
Antibodies against TBP, TAF3, TAF4, TAF6, TAF7, TAF10, TAF12, and TAF13 are as
previously described (Bell et al.,
2001
; Brou et al.,
1993
; Gangloff et al.,
2001b
; Lavigne et al.,
1996
; Mengus et al.,
1997
; Mengus et al.,
1995
; Metzger et al.,
1999
; Perletti et al.,
1999
). Monoclonal (46TA) and polyclonal antibodies were generated
against the indicated TAF7L peptide coupled to ovalbumin as previously
described (Mengus et al.,
1997
).
Extract preparation and immunoprecipitation
Extracts were prepared from frozen mouse testis ground in a mortar in the
presence of liquid nitrogen. Cytoplasmic and nuclear extracts were then
prepared essentially as previously described
(Mengus et al., 1995).
Briefly, the powdered material obtained was resuspended in buffer A (50 mM
Tris-HCl, pH 7.9; 20% glycerol; 1 mM dithiothreitol; 0.5 mM EDTA) in the
presence of 0.01 M KCl. The resuspended material was dounced and the nuclei
collected by low speed centrifugation. The supernatant fraction (cytoplasmic
extract) was recovered and the nuclei resuspended in buffer A with 0.5 M NaCl.
After douncing and a 30 minute incubation in ice the nuclei were spun out (20
minutes at 30,000 g) and the supernatant fraction (nuclear
extract) recovered. The nuclear extract was precipitated by addition of 0.33
g/ml ammonium sulphate and the protein recovered by centrifugation. The
protein was resuspended in buffer A with 0.05 M KCl and dialysed overnight
against the same buffer. Typically 10 ml of cytoplasmic extract (around 100 mg
of protein) and 4 ml (around 6 mg) of nuclear extract were recovered from 20
mouse testes. Purified populations of pachytene spermatocytes and haploid
spermatids were prepared by centrifugal elutriation as described
(Fimia et al., 1999
;
Meistrich et al., 1981
). Cell
extracts were prepared from each population by several cycles of the freeze
thaw in buffer A with 0.5 M NaCl and 0.1% NP40 followed by incubation for 30
minutes on ice and microfuge centrifugation for 10 minutes at 4°C.
Immunoprecipitation of TBP with monoclonal antibody 2Cl and elution with
peptide were as described (Mengus et al.,
1995
). Immunoblotting and chemiluminescence detection were
performed by standard methods.
Yeast two hybrid screen
A yeast two-hybrid screen was performed essentially as described
(Fimia et al., 1999). As bait,
full length TAF7L was cloned in frame with the DNA-binding domain of the yeast
activator GAL4 (GAL4 DBD). This plasmid was transformed in the
Saccharomyces cerevisiae strain sc1945 with a murine adult testis
cDNA library (Clontech). Approximately 2x106 transformants
were plated on selection medium lacking Leu, Trp and His. Clones expressing
HIS3 were isolated, plasmid DNA was recovered and transformed in the E.
coli HB101 strain to isolate the prey plasmid. The specificity of each
clone was tested further by co-transformation of the different clones with the
TAF7L bait or the vector encoding only the GAL4 DBD in yeast strain Y190,
containing an integrated GAL4 responsive ß-galactosidase gene.
Quantitative ß-galactosidase assays were performed as described
(Fimia et al., 1999
).
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Results |
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To selectively detect TAF7L, monoclonal and polyclonal antibodies were
generated against a synthetic peptide (underlined in
Fig. 1A). These antibodies
recognised a 70 kDa protein in extracts from Cos cells transfected with a
TAF7L expression vector and in extracts from mouse testis
(Fig. 1B and data not shown).
In agreement with the published RT-PCR analysis
(Wang et al., 2001), TAF7L was
not detected in extracts from several other organs and is therefore
testis-specific, whereas as described, TBP and other TAFs are widely expressed
although at different levels (Fig.
1C) (Perletti et al.,
1999
).
The intracellular localisation of TAF7L is regulated during male
germ-cell differentiation
The polyclonal antibodies were used to examine the expression of TAF7L
during spermatogenesis. Segments of mouse seminiferous tubules corresponding
to each developmental stage were isolated by transillumination-assisted
microdissection to make squash preparations where the exact stage of the cycle
was identified (Parvinen and Hecht,
1981). Double immunostaining was performed with the polyclonal
anti-TAF7L antibodies and the 3G3 monoclonal antibody against TBP
(Lescure et al., 1994
). We
have previously used this antibody to establish a precise map of TBP
expression in male germ cells (Martianov
et al., 2002
).
TAF7L was strongly expressed in the nucleus of haploid round spermatids,
but was excluded from the heterochromatic chromocenter
(Fig. 2A, red image and 2B, green image, see also Fig. 4A).
Expression in these cells was seen between stages I and VII and disappeared
early in the elongation phase (Fig
2A,B, Fig. 3, and
data not shown, summarised in Fig.
5A). Strong nuclear TAF7L expression at these stages coincides
with strong TBP expression [Fig.
2A green image (see also
Martianov et al., 2002);
summarised in Fig. 5B]. TAF7L
is also strongly expressed along with TBP in the nuclei of late pachytene
spermatocytes from stage VIII onwards (Fig.
2A,B). Strikingly however, at stage XII, all the cellular TAF7L is
found associated with the condensed chromatin in meiotically dividing cells,
while TBP is excluded (Fig. 2A,
Fig. 3). Thus, at this stage
TBP and TAF7L do not colocalise and hence cannot be associated. The
differential localisation of TAF7L and TBP at this stage can be more clearly
seen in confocal sections (Fig.
4B). Close examination of TAF7L localisation indicates that it is
excluded from the most dense heterochromatin. In agreement with this TAF7L is
obviously excluded from the heterochromatic foci in early round spermatids
(Fig. 4B) in which the
chromocenter is forming and is excluded from the chromocenter in later stage
spermatids (Fig. 4A).
Localisation of TAF7L with what appear to be specific chromatin domains can
also be seen in the stage XII sections of
Fig. 2A,B and
Fig. 3.
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Contrary to later stage pachytene spermatocytes, where TAF7L is nuclear, in
spermatogonia, preleptotene, leptotene and zygotene cells, TAF7L is restricted
to the cytoplasm and is excluded from the nucleus
(Fig. 2A,B and
Fig. 3, and data not shown).
This can be clearly seen in confocal sections of stage VI tubules, where TAF7L
is present in the cytoplasm of a type B spermatogonia
(Fig. 4A), but is present in
the nucleus of round spermatids (Fig.
4A). As previously reported
(Martianov et al., 2002),
perinuclear TBP expression in late elongating spermatids can also be seen in
these images.
In mid-stage pachytene spermatocytes, there is a switch in TAF7L
localisation as it begins to enter the nucleus and becomes uniquely nuclear
only in late stage pachytene cells. This change in TAF7L intracellular
localisation shows an interesting correlation with the increase in TBP
expression. In early primary spermatocytes, TBP expression is low and nuclear
[see double staining in Fig. 2A
(see also Martianov et al.,
2002)], while TAF7L is cytoplasmic. Soon after TAF7L enters the
nucleus in mid-stage pachytene cells, TBP expression strongly increases
(compare TBP expression in zygotene and pachytene cells at stage IX in
Fig. 2A) such that both
proteins are found strongly expressed in the nucleus in later stage pachytene
and haploid cells (summarised in Fig.
5A,B).
Differential expression of TAF7 and TAF7L during spermatogenesis
We next compared the testis expression of TAF7L with that of its somatic
paralogue TAF7. Staged squash preparations were double stained with a
polyclonal antibody against TAF7L (green, in
Fig. 2B) and the previously
characterised anti-TAF7 monoclonal antibody
[(Lavigne et al., 1996) red in
Fig. 3B].
While TAF7L is uniquely located in the cytoplasm in early primary spermatocytes, TAF7 is strongly expressed in both the cytoplasm and the nucleus at this stage (Fig. 2B, stage XI, see zygotene spermatocytes, summarised in Fig. 5A,C). TAF7 is expressed in both the cytoplasm and the nucleus up until the late pachytene stage, where its expression declines (Fig. 2B, stages II and VII). In marked contrast to TAF7L, which is associated with the condensed chromatin in stage XII dividing spermatocytes, TAF7, like TBP, is excluded from the chromatin (Fig. 2B, stage XII). Furthermore, while TAF7L is strongly expressed in haploid cells, TAF7 expression is virtually undetectable in these cells. Hence, TAF7 and TAF7L exhibit a reciprocal mirror-like expression pattern. At early stages TAF7, but not TAF7L, is present in the nucleus, while at the pachytene stage both proteins are nuclear and in haploid cell nuclei TAF7L is strongly expressed, whereas TAF7 disappears (summarised in Fig. 5A,C).
TBP-associated and TBP-independent forms of TAF7L
To confirm the differential expression of TAF7 and TAF7L and to determine
whether TAF7L is associated with TBP, extracts from mouse testis and from
centrifugal elutriation purified populations of spermatocytes and haploid
cells were prepared. The presence of TBP, TAF7L and TAF7 in cytoplasmic and
nuclear extracts from whole mouse testis was verified. TAF7L and TAF7 were
present in the nuclear extract and a significant fraction was also present in
the cytoplasmic extract (Fig.
6A). While some TBP was also detected in the cytoplasmic extract,
the majority was present in the nuclear extract
(Fig. 6A). In addition, TBP,
TAF7 and TAF7L could all be readily detected in extracts from purified
spermatocytes, but, in agreement with the immunofluorescence, significantly
lower amounts of TAF7 were seen in haploid cell extracts
(Fig. 6D). As a control, the
transcriptional coactivator ACT was detected only in the haploid cell extract
showing efficient separation of the two cell polulations
(Fimia et al., 1999).
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To determine whether the cytoplasmic TAF7L is associated with TBP, this
fraction was precipitated with the anti-TBP antibody that has been previously
used to purify and characterise TFIID in HeLa cell extracts
(Brou et al., 1993;
Mengus et al., 1995
).
Immunoprecipitation of the cytoplasmic extract depleted almost all the TBP
which was eluted using a peptide corresponding to the antibody epitope
(Fig. 6B). Neither the
cytoplasmic TAF7L nor TAF7 were coprecipitated with TBP (lane 3), showing that
they are not associated with TBP. In contrast, a fraction of both TAF7L and
TAF7 was coprecipitated with TBP from the nuclear extract
(Fig. 6C, lane 3), indicating
that a sub-population of these molecules is tightly associated with TBP.
However, a significant amount of these proteins remains in the supernatant
showing the existence of a TBP-independent form(s). Similar results were seen
with other known TFIID subunits, TAF4 and TAF6, which were also present in
both the supernatant and immunoprecipitated fractions from total testis
extracts (Fig. 6C).
To determine whether TAF7L is associated with TBP in spermatocytes and/or haploid cells, the corresponding extracts were precipitated with the anti-TBP antibody. In extracts from spermatocytes, TAF7L was coimmunoprecipitated along with TBP, but no TAF7 was precipitated (Fig. 6E, upper panel). Similarly, TAF7L was also coprecipitated with TBP in extracts from haploid cells, however only trace amounts of TAF7 present in this extract were precipitated with TBP (Fig. 6F, upper panel). These results show that TAF7L, but not TAF7, is associated with TBP in spermatocytes, and in haploid cells much more TAF7L than TAF7 is precipitated with TBP. However, in each immunoprecipitation, a significant amount of TAF7L remains in the supernatant fraction indicating the existence of a TBP-independent form of TAF7L in both cell types.
The above immunoblots were reprobed for the presence of TAF6. Unlike TAF7 and TAF4, comparable amounts of TAF6 were present in both the spermatocyte and haploid cell extracts (Fig. 6E,F, lane 1, lower panel). TAF6 was coprecipitated with TBP from the spermatocyte extract, but little or no TAF6 was precipitated with TBP from the haploid cell extract and in both extracts a TBP-independent population of TAF6 exists.
Interaction of TAF7L with TAF1
To identify potential molecular targets of TAF7L a two hybrid screen was
performed using a fusion of TAF7L to the DNA-binding domain of the yeast GAL4
and a testis cDNA library fused to the GAL4 activation domain
(Fimia et al., 1999). The cDNA
library was cotransformed along with the vector expressing the GAL4-TAF7L
fusion into a yeast strain harbouring an integrated HIS3 gene under the
control of GAL4 DNA-binding sites. Transformants were grown in the absence of
histidine to select for clones that had activated the HIS3 gene. From
2x106 transformants, more than 180 HIS3-expressing clones
were isolated. However, only two of these clones, which also grew most
rapidly, displayed a significant expression of an integrated
ß-galactosidase gene under the control of GAL4 binding sites
(Fig. 7A). A strong interaction
of TAF7L with the proteins encoded by each of these clones was observed, while
no significant ß-galactosidase activity was observed in control
experiments with the GAL4 activation domain alone, or when each clone was
expressed in the presence of only the GAL4 DNA binding domain
(Fig. 7A, and data not shown).
Sequencing of these clones revealed them to be identical, both encoding amino
acids 1070-1226 of TAF1, the largest TFIID subunit. This region overlaps with
the previously described RAPiD domain (Fig.
7B), which interacts with the RAP74 subunit of transcription
factor TFIIF (Ruppert and Tjian,
1995
)
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We also tested the ability of TAF7L to interact with the testis-specific
activator CREM and its coactivator ACT
(Fimia et al., 1999;
Sassone-Corsi, 1998
). However,
in two hybrid assays, neither of these proteins showed significant interaction
with TAF7L (Fig. 7A).
Differential expression of TAFs and TBP during male germ-cell
differentiation
TBP and many TAFs are overexpressed in testis
(Perletti et al., 1999).
However, as shown above, TAF7 and TBP are strongly expressed at different
stages of differentiation. This is contrary to what one may have expected for
two subunits of the same complex. We therefore decided to investigate the
expression of another TFIID component to evaluate whether its expression was
coordinated with that of TBP. TAF10 is a subunit of both TFIID and the
TFTC/STAGA complexes (Jacq et al.,
1994
; Martinez et al.,
2001
; Wieczorek et al.,
1998
). A monoclonal antibody against TAF10 (red), was used to
determine its expression in staged squash preparations where strong expression
is seen in leptotene, zygotene and early-stage pachytene spermatocytes,
whereas it is only very weakly expressed in haploid cells
(Fig. 3 and data not shown). At
stage XII, TAF10 is not associated with the condensed chromatin of meiotically
dividing cells and therefore cannot be associated with TAF7L at this time.
Hence, TAF10 expression differs significantly from that of both TAF7L and TBP
(summarised in Fig. 5D).
Lastly, we also investigated the expression of TAF4, a subunit of TFIID and
TFTC (Brand et al., 1999;
Gangloff et al., 2001b
;
Mengus et al., 1997
). TAF4 is
strongly expressed in the nucleus of early stage spermatocytes, however a
weaker expression is visible in haploid round spermatids
(Fig. 8, data not shown). The
downregulation of TAF4 is also seen by comparing its presence in extracts from
purified pachytene spermatocytes and haploid spermatids
(Fig. 6D, summarised in
Fig. 5E). Similar results were
observed for TAF3 (data not shown summarised in
Fig. 5F). Therefore, the TAF3
and TAF4 expression profiles are similar to those of TAF7 and TAF10, but are
different from that of TBP.
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Discussion |
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We report here the amino acid sequence of mouse and human TAF7L. The
sequence of Wang et al. predicted the translation of a mouse TAF7L protein
beginning at M94 or M97 that would have a molecular mass similar to that of
TAF7 or human TAF7L (Wang et al.,
2001). However, our sequence predicts the presence of an upstream
in-frame methionine encoding a mouse TAF7L with an additional N-terminal
region not found in human TAF7L. The molecular mass of the endogenous mouse
testis TAF7L is consistent with the use of this methionine, but not with that
of the downstream methionines.
Besides this domain, the amino acid sequences of both human and murine TAF7L show strong similarity to those of other TAF7 family proteins. The apparently minor differences in the sequences of TAF7 and TAF7L do however lead to a differential intracellular localisation of these proteins in primary spermatocytes and in meiotically dividing cells. One of the most striking features is the dynamic control of TAF7L intracellular localisation. In early stage spermatocytes, TAF7L is excluded from the nucleus and begins to enter only at the pachytene stage, whereas TAF7 is always present both in the cytoplasm and nucleus. Entry of TAF7L into the nucleus coincides with increased TBP expression and a wave of transcriptional activity in the pachytene cells. Both TAF7L and TBP are subsequently strongly expressed in the nucleus of haploid round spermatids, where they associate to form a TFIID complex with a unique composition specifically adapted to control the gene expression programme in these cells. Developmental regulation of intracellular localisation is unique to TAF7L as expression of TBP, TAF10, TAF4 and TAF3 are nuclear at all stages.
It is also striking that somatic TAF7 expression is strongly downregulated
in haploid cells leading to a developmental switch between the somatic and
germ-cell specific paralogues. In terms of association with TBP, this switch
takes place in pachytene cells where TAF7L, TAF6 (and TAF11 and 13; J.-C.P.,
G.M. and I.D., unpublished), but not TAF7, is associated with TBP. This
indicates that TFIID composition has been remodelled in these cells to include
TAF7L and exclude TAF7. Thus, if TAF7 plays a role in pachytene cells, this
function is TBP-independent. The function of TAF7L is likely to be predominant
in haploid cells where its expression is significantly higher than that of
TAF7, and much more TAF7L than TAF7 is associated with TBP. This suggests that
TAF7L has a unique function in these cells that cannot be fulfilled by somatic
TAF7. A unique function for TAF7L is also implied by the observation that it
remains associated with the condensed chromatin during meiotic division, while
TAF7 and TBP do not. TAF7L associates with specific chromatin domains at this
time, but not with the dense heterochromatin. While its function at this stage
is unknown, it is interesting to speculate that it may `bookmark' specific
promoters or chromatin domains for activation (or repression) in haploid cells
as has been suggested for TBP in somatic cells
(Christova and Oelgeschlager,
2002).
TAF7L was first reported to be expressed in spermatogonia
(Wang et al., 2001). The
detailed study presented here shows that it is present in spermatocytes and in
round haploid spermatids. Its expression and potential function at these later
stages are interesting as the gene encoding TAF7L is present on the X
chromosome (Wang et al.,
2001
), which is transcriptionally silenced in the XY body,
possibly by the Xist RNA (Ayoub et
al., 1997
) in pachytene cells. As a consequence, at later
developmental stages the TAF7L must be translated from a pre-existing mRNA
store. Moreover, only around 50% of the haploid spermatids carry an X
chromosome. This unique regulatory mechanism allows cells to express a
potentially critical factor, despite the loss of the corresponding gene.
TAF7L has TAF-like properties
As discussed above, a TBP-TAF7L complex exists in both pachytene and
haploid cells. The existence of these complexes is supported by biochemical
analyses of pachytene spermatocyte and haploid cell extracts indicating that a
sub-population of TAF7L is tightly associated with TBP. As TAF7 does not
directly interact with TBP (Lavigne et
al., 1996), it is probable that TAF7L interacts indirectly with
TBP in the context of a complex with other TAFs. The TBP-TAF7L association may
also be dynamic, as these proteins are physically separated during the meiotic
divisions. In contrast, however, TAF7 is not associated with TBP in pachytene
cell extracts. This shows that a specialised TFIID complex containing TAF7L,
but lacking TAF7, exists in these cells. Hence, during spermatogenesis, TFIID
composition is modulated by the inclusion of TAF7L and the exclusion of TAF7
and this process is probably regulated through changes in the intracellular
localisation of TAF7L.
Further evidence for association of TAF7L with a TBP-TAF complex comes from
a two-hybrid screen revealing a TAF7L-TAF1 interaction. TAF1 is the largest
TFIID subunit and is TFIID-specific not being present in the TFTC-type
complexes (Brand et al., 1999).
The TAF7L bait independently isolated twice the same clone encoding the
central region of TAF1 containing the RAPiD domain that has been shown to
interact with the RAP74 subunit of transcription factor TFIIF
(Ruppert and Tjian, 1995
). A
region of TAF1 overlapping with RAPiD was previously used in a screen of a
HeLa cell cDNA library which isolated somatic TAF7
(Gegonne et al., 2001
). The
study of Gegonne et al. showed that TAF7 interacts with TAF1 amino acids
1120-1279 (Gegonne et al.,
2001
). The clone isolated here encodes amino acids 1070-1226, thus
more precisely mapping the required TAF1 region to amino acids 1120-1226. The
ability to interact with TAF1 is therefore common to TAF7 and TAF7L.
Together, the conserved TAF7L-TAF1 interactions and the coprecipitation of
TAF7L with TBP indicate that TAF7L is a TFIID subunit in pachytene and haploid
cells where the two proteins colocalise. This is the first demonstration that
the testis-specific TAF paralogues are in fact bona fide TAFs. It remains to
be determined whether the Drosophila Can protein (TAF5L), which
shares strong sequence similarity with TAF5, is also associated with TBP
(Hiller et al., 2001).
Although TAF7L can be coprecipitated with TBP, a significant fraction of TAF7L is not associated with TBP and hence may also function in a TBP-independent manner (we excluded the possibility that TAF7L associates with TLF, as these two proteins do not co-purify with one another; J.-C.P., G.M. and I.D., unpublished). Gel filtration analysis of this TAF7L fraction suggests that it is present in a high molecular mass complex(s) (J.-C.P., G.M. and I.D., unpublished). Purification of this complex(s) will allow the characterisation of its subunit composition and its functional properties.
Somatic TAF7 has been reported to interact with several transcriptional
activators in vitro (Chiang and Roeder,
1995), however the functional significance of these interactions
has not been demonstrated in vivo. Additionally TAF7 interacts with the
nuclear receptors for vitamin D3 and thyroid hormone in mammalian cells
(Lavigne et al., 1999
). One
possibility therefore is that TAF7L exerts its function by interacting
specifically with haploid cell-specific transcriptional activators or
coactivators such as CREM or ACT (Fimia et
al., 1999
; Sassone-Corsi,
1998
). However, two-hybrid assays with these factors failed to
reveal such interactions. Moreover, our two-hybrid screen with TAF7L did not
isolate any known transcriptional activators, nor other obvious targets for
TAF7L (J.-C.P., G.M. and I.D., unpublished). Although, by analogy with the
somatic TAF7, we are presuming that TAF7L action is limited to the nucleus, we
cannot exclude that TAF7L also has a function in the cytoplasm of early
spermatocytes. Further genetic studies will be required to understand the role
of TAF7L during spermatogenesis.
Remodelling of TFIID composition during spermatogenesis
Previous reports have shown that both TBP and TAFs are strongly
overexpressed in testis (Perletti et al.,
1999; Schmidt and Schibler,
1995
). As these proteins are all part of the TFIID complex in
somatic cells, it may have been assumed that they were overexpressed at the
same developmental stage leading to a net increase in TFIID concentration.
Here we show that this is not the case. TAF3, TAF4, TAF7 and TAF10 are
strongly expressed in early stage spermatocytes and are downregulated in
haploid cells, just the opposite of TBP. While the diminished levels of TAF7
may be compensated by the presence of TAF7L, no testis-specific paralogues of
the other TAFs have yet been described in mammals which could substitute for
the somatic TAFs in haploid cells. The low TBP/TAF ratio suggests that the
above TAFs may in part function independently from TBP in primary
spermatocytes. These TAFs are present in the TFTC-type complexes
(Brand et al., 1999
;
Martinez et al., 2001
;
Ogryzko et al., 1998
),
suggesting that these complexes play an important role at this stage. However,
the existence of novel TAF-containing complexes with specialised functions
during germ-cell differentiation cannot be excluded.
An opposing situation is observed in haploid cells where expression of a
key TFIID and TFTC component TAF10 is strongly diminished, while TBP is
strongly upregulated. These changes in protein abundance suggest that the
compositions of TFIID and TFTC in haploid cells may radically change both by
the presence of TAF7L and the absence of TAF10. Given the key role of the
histone-fold-containing TAF10 in TFIID organisation
(Gangloff et al., 2001a;
Gangloff et al., 2001b
;
Gangloff et al., 2001c
;
Kirschner et al., 2002
;
Leurent et al., 2002
;
Mohan et al., 2003
), the loss
of this protein as well as the diminished expression of the other TAFs should
lead to a disruption of TFIID structure and a consequent reduction in the
amount of TFIID with the full complement of TAFs in haploid cells. Direct
evidence for this comes from the observation that, while TAF7L can be found
associated with TBP in haploid cell extracts, almost no TAF6 coprecipitates
with TBP, whereas TAF6 is found associated with TBP in spermatocyte extracts.
Hence, in haploid cells, partial TBP-TAF sub-complexes exist and TBP may also
act in a TAF-independent manner, either alone or complexed with other factors.
This is the first documentation of how the composition of a general
transcription factor can change during a developmental process. The changes in
TBP and TAF expression and the resulting remodelling of TFIID composition and
that of other TAF-containing complexes highlight the unique nature of the
general transcriptional machinery in haploid cells.
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Acknowledgments |
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References |
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