1 Department of Developmental Genetics, National Institute of Genetics and
Department of Genetics, Graduate University for Advanced Studies, Mishima,
Shizuoka-ken 411-8540, Japan
2 Institute of Entomology, Czech Academy of Sciences, Ceske Budejovice, 37005
Czech Republic
Author for correspondence (e-mail:
shirose{at}lab.nig.ac.jp)
Accepted 19 November 2002
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SUMMARY |
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Key words: TDF, MBF1, Co-activator, Trachea, CNS, Drosophila
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INTRODUCTION |
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Two classes of transcriptional co-activators have been described to date.
The first class comprises proteins that possess or recruit enzymatic
activities to modify chromatin proteins, e.g. by acetylation of histones.
Resulting alteration in the chromatin structure causes a switch in the `state'
of chromatin between transcriptionally inactive and active. Co-activators of
the second class act more directly to recruit the general transcription
machinery to a promoter where a transcription factor is bound. Among the
latter class are TATA element-binding protein (TBP)-associated factors (TAFs)
that are subunits of TFIID, and others that serve as adaptors to mediate the
contact between transcription factors and the basal transcriptional complex.
Although the importance of the first type co-activators for gene expression
has been demonstrated (Akimaru et al.,
1997; Brownell et al.,
1996
; Chakravarti et al.,
1996
; Grant et al.,
1997
; Kamei et al.,
1996
; Ogryzko et al.,
1996
; Waltzer and Bienz,
1999
), little is known about how the second class co-activators
function in vivo, or how the two types interact to achieve elaborate
regulation of gene expression.
Multiprotein bridging factor 1 (MBF1) was first identified from the
silkmoth as a co-factor necessary for transcriptional activation in vitro by a
nuclear receptor FTZ-F1 (Li et al.,
1994; Takemaru et al.,
1997
). The ability of MBF1 to bind both FTZ-F1 and TBP suggested a
mechanistic model in which MBF1 recruits TBP to a promoter carrying the
FTZ-F1-binding site by interconnecting FTZ-F1 and TBP. The MBF1 sequence is
highly conserved across species from yeast to human
(Takemaru et al., 1997
). In
the yeast Saccharomyces cerevisiae, MBF1 functions as a co-activator
of a bZIP transcription factor GCN4, by bridging between GCN4 and TBP in
response to amino acid starvation
(Takemaru et al., 1998
).
To analyze the biological role of MBF1 in a multicellular organism, we
characterized its ortholog in Drosophila. The Drosophila
mbf1 gene partially rescued the phenotype of a S. cerevisiae
mbf1- mutant, establishing that both the structure and the
function of MBF1 remained well conserved during evolution. In nuclear extracts
of Drosophila embryos, we identified a bZIP protein Tracheae
Defective (TDF; APT FlyBase)
(Gellon et al., 1997;
Su et al., 1999
;
Eulenberg and Schuh, 1997
) as a
new partner of MBF1. We show that MBF1 mediates transcriptional activation by
TDF through directly binding both TDF and TBP. Although mbf1 null
mutants can survive to adulthood, tdf becomes haploinsufficient in
mbf1- background, resulting in severe tracheal and central
nervous system (CNS) defects. These results show that MBF1 acts as a crucial
bridging co-activator during tracheal and CNS development.
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MATERIALS AND METHODS |
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A 4574-bp genomic EcoRI fragment encompassing the entire mbf1 gene was subcloned from the P1 clone DS05624 into pBluescript II (Stratagene) and sequenced on both strands in its entirety (DDBJ/EMBL/GenBank Accession Number, AB031273). Comparison of this genomic sequence with the mbf1 cDNA reveals that the gene consists of four exons, the first one being non-coding. The clone contains 2.3 kb of DNA upstream of the mbf1 transcription unit and 54 bp downstream of the mbf1 mRNA polyadenylation site.
Fly stocks
Using P-element-mediated germline transformation
(Rubin and Spradling, 1982),
we made a transgenic Drosophila line expressing a FLAG epitope-tagged
MBF1 (FLAG-MBF1) protein under the control of the constitutive hsp83
promoter. Expression levels of the tagged MBF1 protein were comparable with
those of endogenous MBF1 during embryogenesis and also in the salivary glands.
An mbf1-null mutant was generated by isolating a P-element insertion
in the mbf1 locus and its subsequent imprecise excision (M. J., M.
Okabe, Y. H. and S. H., unpublished). A rescue construct
P[MBF1+], mbf1 was made by P-element-mediated insertion of
the 4574 bp mbf1 genomic sequence to the mbf1 mutant
chromosome. tdfP2 allele corresponds to the P-element
insertion line l(2)k15608 (Torok
et al., 1993
). tdfP
3 has a deletion
that includes the first exon and 1 kb of the first intron of the tdf
gene (Eulenberg and Schuh,
1997
). The sgP[Gal4] line has been described by Brand and
Perrimon (Brand and Perrimon,
1993
). Transgenic lines harboring fPE-lacZ or its mutant
derivatives have been described elsewhere
(Han et al., 1998
). Other
stocks have been described previously
(Lindsley and Zimm, 1992
).
Identification of FLAG-MBF1 associated proteins
Embryos 0-20 hours after egg laying (AEL) were collected from a population
of the hsp83-FLAG-MBF1 transgenic line reared at 25°C. Nuclear
extracts were prepared from the embryos as described previously
(Ueda et al., 1990). For
isolation of FLAG-MBF1-associated proteins, nuclear extracts (15 ml) were
loaded onto a 2 ml column of anti-FLAG M2-Agarose affinity gel (Sigma). The
column was washed extensively with TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4),
until no proteins were detectable in the wash fraction. Bound proteins were
then eluted with the FLAG peptide (50 µg/ml) (Sigma). Chromatography was
carried out at 4°C.
To identify FLAG-MBF1 associated proteins, the eluted proteins were concentrated by acetone precipitation and then resolved on a preparative 10% SDS-PAGE. The proteins were transferred to a PVDF membrane (Boehringer Mannheim) on a Trans-Blot Semi-Dry apparatus (BioRad). The membranes were subsequently stained with Coommassie Brilliant Blue and selected bands were subjected to N-terminal sequencing. Peptide sequence of 63 kDa band VNKQYSATDLEAFMKIAANWQNSN showed an unambiguous match with that of TDF.
Antibodies
For the production of anti-TDF serum, a 1455 bp cDNA fragment encoding the
entire TDF was inserted into a pET-28a vector (Novagen) and the
polyhistidine-tagged protein was expressed in Escherichia coli BL21
(DE3). The His-TDF protein was purified by Ni2+ affinity
chromatography and used to generate polyclonal antibodies in rabbit. The serum
was used directly for immunostaining at a 1000-fold dilution. For western
analyses, it was used at a 10,000-fold dilution. The production of the MBF1
antibody will be described elsewhere (M. J., M. O., Y. H. and S. H.,
unpublished). This antibody detects a single band of 16 kDa in normal embryos,
but not in mbf1- embryos
(Fig. 3).
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Other antibodies include a rabbit anti-Drosophila TBP antibody (a gift from Yoshihiro Nakatani); mouse anti-ß-galactosidase mAb 40-1a; rabbit anti-ß-galactosidase antibody (Cappel); mouse anti-FLAG M2 (Kodak); mouse mAb BP102; and mouse mAb 2A12 that recognizes an unknown luminal component of the trachea (a gift from Shigeo Hayashi).
The secondary antibodies used were as follows: biotinylated anti-mouse IgG; Cy3-conjugated anti-rabbit IgG; Cy3-conjugated anti-mouse IgG (Jackson Laboratory); and Alexa 488 goat anti-rabbit IgG conjugate (Molecular Probes). When biotinylated anti-mouse IgG was used for the 2A12 staining, signals were amplified by adding biotinylated tyramide (NEN Life Science Products) as a substrate, followed by application of the ABC kit (Vector Lab), and finally visualized by Cy3-conjugated streptavidin (Amersham Biosciences).
Immunostaining
For staining of embryos, 0-12 hours or 12-15 hours AEL embryos were
collected and fixed as described (Tautz
and Pfeifle, 1989). After blocking with 5% normal goat
serum/PBS-0.1% Triton X-100, the embryos were incubated with a primary
antibody, followed by a secondary antibody.
Staining of larval tissues was performed as described previously
(Liu et al., 2000). Larvae
were dissected in PBS, fixed in 25 mM PIPES-KOH (pH 7.0), 0.5 mM EDTA, 0.25 mM
MgSO4 and 4% formaldehyde for 40 minutes on ice and then
permeabilized for 15 minutes at room temperature in PBS containing 0.5% NP-40.
In the case of salivary glands, the dissected tissues were first treated with
PBS-0.5% NP-40 for 8 minutes before the fixation and the permeabilization step
after the fixation was omitted.
Staining of polytene chromosomes using indirect immunofluorescence was
performed as described (Shopland and Lis,
1996) except that salivary glands were treated with PBS, 0.5%
NP-40 for 5 minutes before fixation. FLAG-MBF1, TBP and TDF were detected with
anti-FLAG M2, anti-TBP and anti-TDF antibodies, respectively, and visualized
with the mouse Cy3- or rabbit Alexa 488-conjugated secondary antibodies. After
staining, samples were washed with PBS, 0.2% NP-40, 0.2% Tween 20 and
containing subsequently 300 mM NaCl and 400 mM NaCl.
GST pull-down assay
GST fusion proteins were purified on Glutathione Sepharose 4B beads
(Amersham Biosciences). GST pull-down assays were performed as described
(Takemaru et al., 1998). Bound
proteins were detected on western blots using either the anti-MBF1 or the
anti-TBP antibody.
Immunoprecipitation
For co-immunoprecipitation assays, 20 µl of IgG-conjugated Dynabeads
(Dynal, Oslo, Norway) containing 2 µg of IgG were incubated at 4°C for
2 hours with 40 µl of a primary antibody or the corresponding preimmune
serum and then washed with TBS [50 mM Tris-HCl (pH 7.4), 60 mM NaCl]. Nuclear
extracts were prepared from yw or mbf1 embryos (0-20 hours
AEL) as described (Ueda et al.,
1990) and 30 µl of the nuclear extract was added to the
antibody-loaded beads. After incubation at 4°C for 2 hours, the beads were
washed with TBS containing 0.01% NP-40. Bound proteins were eluted from the
beads with SDS loading buffer, resolved by SDS-PAGE (8% for TBP and TDF, 12.5%
for MBF1) and analyzed on a western blot.
In vitro DNA-binding experiments
The TDF-binding consensus was obtained from a pool of oligonucleotides
containing 16-nucleotide random sequences in the middle as described
(Gogos et al., 1992). Binding
reactions were performed as described elsewhere
(Pollock and Treisman, 1990
).
The binding mixture (25 µl) contained buffer E [20 mM Hepes (pH 7.9), 100
mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 0.1% NP-40, 5
µg/ml leupeptin, 5 µg/ml pepstatin and 0.75 µg/ml aprotinin], 1 µl
anti-TDF, 2 µl nuclear extract (0-20 hours AEL), 0.1 pmole of the
32P-labeled oligonucleotide probe, 200 ng poly(dIdC)-poly(dIdC) and
200 ng bovine serum albumin (Sigma). The mixture was incubated on ice for 30
minutes and oligomers bound by TDF were collected on protein A-Sepharose
beads. After washing the beads with buffer E, the oligomers were recovered by
phenol treatment, amplified using PCR and then used for the next selection
cycle. After the fifth round of selection, DNA fragments were subcloned into a
T-vector (Novagen) and sequenced. The consensus sequence was deduced from
sequences of 54 clones.
Gel mobility retardation assays were performed as described previously
(Ueda and Hirose, 1991),
except that electrophoresis was carried out on a 4% polyacrylamide gel in 50
mM Tris-HCl (pH 7.5), 2 mM EDTA, 380 mM glycine and 5% glycerol. The
double-stranded oligomers used for the mobility retardation assay are
described below. The probe or functional competitor was
5'-GAGCTCGGATCCGAATTCCAATTGGAATCTCGAGAAGCTTGATCA
G-3'.
Mutant competitor was 5'-GAGCTCGGATCCGAATTCCCCTTGTGATCTCGAGAAGCTTGATCAG-3'. Underlined regions indicate the recognition sequence of TDF and its mutant derivative respectively.
TDF binding site-dependent reporter assays
Reporter constructs were fusions of the lacZ gene with three
tandemly repeated copies of either the functional TDF-binding site
(TDS-lacZ) or the mutated site (TDMS-lacZ)
(Fig. 4A), inserted into the
hsCaSpeR-AUG-ß-gal vector. The functional and mutant binding sites were
those used in the DNA binding assays (above). These constructs were introduced
into the yw host by the P-element transformation method of Rubin and
Spradling (Rubin and Spradling,
1982). A line carrying the TDS-lacZ reporter at the
cytological position 34F on the second chromosome was used for most
experiments. The position of the reporter gene was determined by
circularization of Sau3AI-digested genomic DNA with DNA ligase,
followed by sequencing of PCR-amplified fragment with P-element primers.
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To examine the effect of the loss of tdf function on the reporter expression, the TDS-lacZ transgene was introduced into the tdfP2 chromosome. The TDS-lacZ expression in mbf1- and heterozygous tdfP2 background were examined in TDS-lacZ/CyO; mbf1 and TDS-lacZ, tdfP2/CyO; mbf1, respectively. ß-Galactosidase activities in embryonic extracts were measured using a Galacto-Light Plus System (Applied Biosystems) and a luminometer LUMAT LB9507 (Berthold Technologies). For scoring of X-gal-stained embryos, we excluded embryos that died earlier and only counted embryos that developed to stage 16.
To misexpress TDF in the salivary glands, a UAS-tdf transgene was
controlled by a Gal4 driver sgP[Gal4] on the X chromosome.
sgP[Gal4] is one of the enhancer trap lines isolated by Brand and
Perrimon (Brand and Perrimon,
1993) and is active in the salivary gland cells from embryonic
through larval stages. To localize TDF on its binding site in vivo, the
TDS-lacZ reporter was introduced into the UAS-tdf chromosome
and the y+ sgP[Gal4]/Binscinsy females were
crossed with the yw:UAS-tdf, TDS-lacZ males. After culturing at
18°C, y+ third-instar larvae were dissected and stained with
anti-TDF serum and the anti-ß-gal mAb 40-1a.
Genetic analyses
To analyze genetic interactions between mbf1 and tdf, we
made two double mutant stocks, homozygous for mbf1 deletion:
tdfP3/CyOP[ftz-lacC]; mbf1 and
tdfP2/CyOP[ftz-lacC]; mbf1. To compensate for the
mbf1 deletion, the rescue construct P[MBF1+] was
introduced into the mbf1 mutant chromosome. A double mutant
tdfP
3/CyO; P[MBF1+], mbf1 was also
prepared and used for rescue experiments. To rescue the CNS phenotype of the
tdf mutant, UAS-tdf was introduced into the
tdfP
3 chromosome. elav-Gal4;
tdfP
3/CyO females were crossed with
UAS-tdf, tdfP
3/CyO males, then embryos
were collected at 12-15 hours AEL and stained with the mAb 2A12 and mAb BP102
antibodies. The ability of Drosophila MBF1 to rescue the yeast
mbf1 disruptant was tested using aminotriazole sensitivity assay.
Yeast MBF1 mediates transcriptional activation by GCN4 that governs induction
of many genes in the amino acid synthetic pathways; hence, a yeast
mbf1 disruptant is sensitive to aminotriazole, an inhibitor of the
His3 gene product (Takemaru et
al., 1998
).
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RESULTS |
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In situ hybridization revealed that a large amount of maternal mbf1 mRNA was deposited to the egg (data not shown). Likewise, MBF1 protein was present in preblastoderm embryos and was later expressed in many tissues, including the CNS and the trachea (Fig. 1C). Widespread expression of MBF1 was also seen in post-embryonic stages, with particularly high levels in the larval salivary glands, gonads (Fig. 1C) and adult gonads (data not shown).
To address the role of MBF1 in vivo, we generated a null mutant of mbf1 (see Materials and Methods). This allele lacks a 2082 bp DNA segment encompassing the entire coding region and most of the 3'-noncoding region of mbf1 (from 211 bp upstream of the initiation codon to 857 bp downstream of the stop codon). mbf1- animals derived from mbf1-/+ parents were viable, with no detactable loss in viability. mbf1 mutant females, however, had a maternal effect phenotype with incomplete penetrance; a variable fraction of progeny from mbf1- homozygous females died before stage 5, regardless of whether they were mated to mbf1+ or mbf1- males. As escapers developed to adults with no obvious morphological defects, a stable mbf1 homozygous line could be maintained under laboratory conditions. No MBF1 protein was detectable in the mbf1 mutant by western analyses (Fig. 3A, lane 2; Fig. 3B, lane 2). Based on the requirement of MBF1 as an co-activator for FTZ-F1-dependent transcription in vitro, we had expected to see a ftz-f1-like segmentation defect among the dead mbf1 homozygous embryos. However, no such pair-rule phenotype was observed. Furthermore, we were unable to detect any decrease in the expression of a FTZ-F1-dependent reporter gene in mbf1- mutant embryos (Fig. 1D). These results showed that MBF1 is not a crucial co-activator for FTZ-F1-dependent transcription in vivo. This led us to search for new partners of MBF1.
Identification of TDF as an MBF1-associated protein
To isolate new partners of MBF1 in vivo, we used the transgenic line
expressing FLAG-MBF1 and pulled out MBF1-associated factors from embryonic
nuclear extracts. Complexes including MBF1 were captured on anti-FLAG antibody
beads, eluted with the FLAG peptide and resolved by SDS-PAGE. While the
nuclear extract of a strain lacking FLAG-MBF1 yielded no specific protein
bands (Fig. 2A, lane 1), many
proteins were recovered from the FLAG-MBF1 transgenic embryos
(Fig. 2A, lane 2). As
anticipated, TBP was identified among the MBF1-associated proteins
(Fig. 2B, lane 4). The above
procedure is also expected to pull down TAFs; indeed some of the bands had
sizes corresponding to Drosophila TAFII32, TAFII60, TAFII110 and
TAFII150, but we did not examine them further.
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We focused on the most prominent protein at 63 kDa that could not be
accounted for by any TAF. Its N-terminal amino acid sequence identified it as
the bZIP protein TDF (Gellon et al.,
1997; Eulenberg and Schuh,
1997
). This was supported by western analysis with an anti-TDF
antibody (Fig. 2B, lane 6). As
yeast MBF1 has been known to interact with the bZIP protein GCN4
(Takemaru et al., 1998
), TDF
appeared as a good candidate for a new functional partner of MBF1. Indeed, GST
pull-down assays using bacterially expressed purified proteins showed that
MBF1 bound directly to TDF (Fig.
2C) and to TBP (Fig.
2D). Yeast GCN4 binds MBF1 with its bZIP domain
(Takemaru et al., 1998
).
Similarly, the bZIP domain of TDF was sufficient for the binding of
Drosophila MBF1 (Fig.
2C).
If MBF1 bridges TDF and TBP, association between these two proteins should depend on the presence of MBF1. To examine the requirement of MBF1 for tethering TBP to TDF in vivo, we carried out co-immunoprecipitation using nuclear extracts prepared from mbf1+ or mbf1-null mutant embryos. From the mbf1+ nuclear extract, MBF1 and TDF were co-immunoprecipitated with the anti-TBP antibody, but not with the pre-immune serum (Fig. 3A, lanes 3 and 5). By contrast, when we started from the extract from mbf1- embryos, TDF was not co-immunoprecipitated with the anti-TBP antibody (Fig. 3A, lanes 2 and 6). In a reciprocal test, MBF1 and TBP were co-immunoprecipitated with the anti-TDF antibody from the mbf1+, but not from the mbf1- nuclear extracts, or from the mbf1+ extract using the pre-immune serum (Fig. 3B, lanes 3, 5 and 6). These results demonstrate that MBF1 forms a bridge between TDF and TBP and that in its absence, a stable association of TDF and TBP does not take place.
TDF binds DNA in a sequence-specific manner
The physical interaction between MBF1 and TDF, as well as the presence of
bZIP domain in TDF suggests that TDF is a transcription factor that uses MBF1
for transcriptional regulation of its target genes. To test this possibility,
we first determined the binding sequence of TDF. The full-length TDF protein
or its C-terminal 56 amino acids harboring the bZIP domain were expressed in
bacteria and purified. In a preliminary gel mobility retardation experiment,
both the full-length and the C-terminal proteins showed DNA-binding activities
(data not shown). The optimal binding sequences were selected from a pool of
random oligonucleotides (see Materials and Methods). The consensus sequence
deduced from the selected oligonucleotides was (A/G) TTC (C/T)(A/T) AT (T/A)
(G/A) GA (A/T)(T/C) (Fig.
4A).
The specificity of TDF binding to the consensus DNA sequence was verified by a competition assay. Incubation of 32P-labeled double-stranded DNA carrying the sequence 5'-ATTCCAATTGGAAT-3' with the full-length TDF protein yielded a slowly migrating complex. The complex formation was efficiently competed with the unlabeled oligonucleotide of the same sequence (Fig. 4B, lanes 2-5) but not with a mutant oligonucleotide carrying four base substitutions (Fig. 4B, lanes 6-8). Essentially the same results were obtained with the C-terminal 56 amino acid fragment of TDF (data not shown). These data indicate that TDF is a sequence-specific DNA binding protein that recognizes a 14 bp palindrome (A/G) TTC (C/T) (A/T) AT (T/A) (G/A) GA (A/T) (T/C).
TDF is a sequence-specific activator of transcription
We then tested whether TDF regulates transcription from a promoter carrying
its binding site. A reporter gene was constructed in which three tandemly
repeated TDF-binding sites (TDS) were placed upstream of a hsp70
basal promoter (-40 +90)-lacZ fusion gene (TDS-lacZ;
Fig. 5A). We also made another
reporter gene containing three tandemly repeated mutant binding sites
(TDMS-lacZ). Transgenic fly lines carrying these reporter genes were
analyzed for lacZ expression by staining with
anti-ß-galactosidase antibody. TDS-lacZ was expressed in the
tracheae, heart precursor cells, head and CNS of stage 16 embryos
(Fig. 5B,C), i.e. in the
pattern of the endogenous TDF protein
(Eulenberg and Schuh, 1997
;
Gellon et al., 1997
;
Su et al., 1999
) (Q.-X. L., M.
J., H. U., Y. H. and S. H., unpublished). By contrast, the TDMS-lacZ
reporter with mutated binding sites showed no activity
(Fig. 5D). Expression of
TDS-lacZ was not detectable in the tdf loss-of-function
mutants tdfP2 (Fig.
5E) and tdfP
3 (data not shown).
Conversely, when TDF was ectopically expressed in the posterior salivary gland
cells, it induced expression of the TDS-lacZ reporter in these cells
(Fig. 5I). Such induction of
lacZ was not observed when we used the line carrying
TDMS-lacZ (data not shown). These results clearly show that TDF is
required and sufficient to activate transcription by binding to its specific
recognition sequence TDS.
|
If MBF1 and TDF form a complex to activate the reporter gene, both proteins should localize to TDS. When TDF was ectopically expressed in the salivary glands of a transgenic line harboring a TDS-lacZ insertion at the polytene chromosome position 34F, TDF was detected at the TDS-lacZ insertion site. Visualization of MBF1 using FLAG-MBF1 strain revealed that MBF1 also accumulates at 34F, colocalizing with TDF (Fig. 6A). The localization of MBF1 to the TDS site at the position 34F occurred only when TDF was expressed in the salivary gland. These data support the idea that MBF1 is recruited to TDS owing to the sequence-specific DNA-binding activity of TDF.
|
MBF1 plays a role in the TDF-dependent activation
As animals that lack MBF1 can survive to adulthood while null mutants of
TDF are embryonic lethal, TDF must be able to carry out its function even when
no stable association with TBP occurs. This suggests that, in the absence of
MBF1, transcriptional activation by TDF may be reduced, but not abolished. To
measure the transcriptional activity of TDF, we quantitated the expression of
the TDS-lacZ reporter in normal and MBF1-deficient embryos
(Fig. 6B). We found that
ß-galactosidase expression from TDS-lacZ was reduced by 80% when
MBF1 was absent. This effect was reverted by expressing a transgenic MBF1
(data not shown). The reduction was even more pronounced when only one copy of
the tdf+ gene was present, while removing one copy of the
tdf+ gene in mbf1+ background had no
effect on TDS-lacZ reporter activity. This indicates that the loss of
MBF1 has a quantitative effect on TDF-mediated transcriptional activation.
Interestingly, when a population of stage 16 mbf1- embryos
carrying TDS-lacZ gene was stained for ß-galactosidase
expression, there was a considerable individual variation in the expression
levels; about 15% of embryos had no detectable levels of ß-galactosidase
expression, while the rest exhibited varying levels. Such variation was not
observed in mbf1+ embryos. It is possible that MBF1
function becomes essential for TDF-mediated transcription under conditions
that are not completely controlled in our experiments.
Role of TDF and MBF1 in the development of the tracheae and CNS
The involvement of MBF1 in TDF-mediated transcription suggests that loss of
mbf1 may also affect a tdf mutant phenotype. In addition to
the tracheal system (Eulenberg and Schuh,
1997; Gellon et al.,
1997
), TDF is strongly expressed in the CNS
(Eulenberg and Schuh, 1997
).
Although a defect in neural function has been reported in a tdf
mutant (Takasu-Ishikawa et al.,
2001
), the role of TDF in the CNS development remained to be
investigated. We found that tdfP
3 embryos had
breakages in the CNS axon tracts and tracheal networks
(Fig. 7C,D). CNS and tracheal
phenotypes were individually rescued when TDF was expressed in all neurons or
in all tracheal cells, respectively, indicating that these phenotypes
represent independent requirement for TDF in two tissue types, rather than one
being a secondary consequence of another
(Fig. 7E,F)
(Eulenberg and Schuh, 1997
)
(Q.-X. L., M. J., H. U., Y. H. and S. H., unpublished). The breakages in the
CNS and tracheal networks were also observed in the mbf1 mutant,
although the penetrance of these phenotypes was only 2-3%
(Fig. 7G,H).
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Although the developmental defect caused by the lack of MBF1 was rather
subtle, mbf1 mutation exhibited a strong genetic interaction with
tdf mutants. Animals that were heterozygous for the
tdfP3 allele had no detectable defects in either
the CNS or tracheae (Fig.
7A,B). However, when MBF1 was removed from the
tdfP
3 heterozygotes, we observed a significant
increase in the number of defective embryos (17-18% in
Fig. 7I,J). Moreover, the
degree of defects was also enhanced: increase in the number of breakages and
in their size (Fig. 7I,J versus
Fig. 7G,H). This effect was
completely reverted by expressing MBF1 from a transgene carrying the genomic
mbf1 locus (Fig.
7K-N). These genetic interactions between mbf1 and
tdf strongly suggest that MBF1 participates in TDF-mediated
transcription during normal development of the tracheae and CNS.
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DISCUSSION |
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Two pathways for transcriptional activation by TDF
The relationship between Drosophila MBF1 and TDF is similar to
that between yeast MBF1 and its partner transcription factor GCN4
(Takemaru et al., 1998). Just
as yeast MBF1 contacts GCN4 through its bZIP domain, Drosophila MBF1
binds the bZIP domain of TDF. Moreover, the lack of GCN4-dependent activation
in yeast mbf1 mutant can be partially restored by expressing
Drosophila MBF1. The sequence and functional conservation between
yeast and Drosophila MBF1 indicates that the interaction with bZIP
proteins is a conserved feature of the bridging factor MBF1.
Genetic studies of mbf1 in yeast and Drosophila suggest
that MBF1-associated transcription factors have two pathways for activation.
In addition to the MBF1-mediated recruitment of TBP via its bZIP domain
(Takemaru et al., 1998), GCN4
also recruits the SAGA complex with its N-terminal activation domain and
effects transcription through chromatin modification
(Grant et al., 1997
).
Likewise, Drosophila TDF has a region similar to the glutamine-rich
transactivation domain (Eulenberg and
Schuh, 1997
) and may employ an activation pathway independent of
recruiting TBP through MBF1. Such pathway may account for the residual
expression of the TDS-lacZ reporter gene in the absence of MBF1.
Although MBF1 is essential for GCN4-dependent transcription of its target gene
HIS3 (Takemaru et al.,
1998
), low level of TDF-dependent transcription of the
TDS-lacZ gene can still occur in the absence of MBF1. This suggests
that the relative importance of the two pathways is different between GCN4 and
TDF. The DNA-binding domain of FTZ-F1 carries a basic region homologous to
those in bZIP proteins and binds MBF1 through this region
(Takemaru et al., 1997
).
However, loss of mbf1 showed no effect on FTZ-F1-dependent
transcription in vivo, suggesting that the activation by FTZ-F1 relied solely
on the pathway through its transactivation domain. In our in vitro
transcription system, the transactivation domain does not seem to be
functional because FTZ622 polypeptide bearing only the DNA-binding domain of
FTZ-F1 showed the same transcriptional activity as the intact FTZ-F1
(Takemaru et al., 1997
). This
may explain the difference in the MBF1 requirement between FTZ-F1-dependent
transcription in vivo and in vitro.
It is possible that the role of MBF1 becomes more critical under certain
circumstances, when rapid induction of gene expression is demanded by
environmental conditions. The expression of the TDS-lacZ reporter in
mbf1- background varied considerably from embryo to
embryo, suggesting that certain conditions that are uncontrolled in our
experiments may render transcription particularly dependent on the
MBF1-mediated pathway. In the natural environment, there are many stimuli that
alter gene expression profile: UV radiation, poison agents, nutrient
starvation and so on. Therefore, direct recruitment of TBP by MBF1 may become
essential for rapid activation of transcription under such conditions. In
agreement with this idea, the yeast mbf1 disruptant is viable under
normal culture conditions, but sensitive to amino acid starvation
(Takemaru et al., 1998).
The biological role of MBF1
Studies on MBF1 homologs also support the idea that MBF1 may function when
gene expression is required in response to developmental or environmental
signals. Rat MBF1 has been isolated as a calmodulin-associated peptide 19
(CAP-19) (Smith et al., 1998)
and human MBF1 (Kabe et al.,
1999
) has been identified as endothelial differentiation-related
factor 1 (EDF1) (Dragoni et al.,
1998
). EDF1/MBF1 is downregulated when endothelial cells are
induced to differentiate. Interestingly, EDF1/MBF1 binds to calmodulin in the
cytoplasm under low Ca2+ conditions but the two proteins dissociate
when intracellular Ca2+ is high. The released EDF1/MBF1 is then
phosphorylated and shuttled into the nucleus, where it binds TBP
(Mariotti et al., 2000
).
Nuclear translocation of MBF1 has also been observed at a specific stage of
molting in the silkworm B. mori
(Liu et al., 2000
).
Considering the Ca2+ elevation upon exposure to the molting hormone
ecdysteroid (Biyasheva et al.,
2001
), these data raise an intriguing possibility that MBF1 is
involved in Ca2+-induced gene activation. Although in this study we
have analyzed only the developmental roles of MBF1 associated with TDF
function, Drosophila MBF1 may also be involved in other biological
processes, such as stress response, homeostasis and longevity.
Several lines of evidence suggest that Drosophila MBF1 has partners other than TDF. MBF1 is expressed in a wide spatiotemporal pattern, including tissues and stages where TDF is absent. Although TDF is not expressed in the salivary gland, immunolocalization of MBF1 on salivary gland chromosome revealed a large number of loci associated with MBF1 (Q.-X. L., M. J. and S. H., unpublished). Furthermore, FLAG-tagged MBF1 pulled down many proteins besides TDF. Although mbf1-null mutants were viable under laboratory conditions, tdf became haploinsufficient in mbf1- genetic background, clearly indicating the importance of MBF1 in the expression of the genomic information. This finding opens a way to identify new partners of MBF1 through genetic screening for loci that exhibit dominant phenotypes in the absence of MBF1. Characterization of MBF1 partners will contribute to our knowledge on how co-activators mediate specific biological events.
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ACKNOWLEDGMENTS |
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Footnotes |
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