From the Departament de Biologia Molecular i Cel.lular, Institut de Biologia Molecular de Barcelona, Centre d'Investigació i Desenvolupament, Consejo Superior de Investigaciones Científicas, Jordi Girona Salgado 18-26, 08034 Barcelona, Spain
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ABSTRACT |
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The Drosophila GAGA factor
self-oligomerizes both in vivo and in vitro.
GAGA oligomerization depends on the presence of the N-terminal POZ
domain and the formation of dimers, tetramers, and oligomers of high
stoichiometry is observed in vitro. GAGA oligomers bind DNA
with high affinity and specificity. As a consequence of its multimeric
character, the interaction of GAGA with DNA fragments carrying several
GAGA binding sites is multivalent and of higher affinity than its
interaction with fragments containing single short sites. A single GAGA
oligomer is capable of binding adjacent GAGA binding sites spaced by as
many as 20 base pairs. GAGA oligomers are functionally active, being
transcriptionally competent in vitro.
GAGA-dependent transcription activation depends strongly on
the number of GAGA binding sites present in the promoter. The POZ
domain is not necessary for in vitro transcription but, in
its absence, no synergism is observed on increasing the number of
binding sites contained within the promoter. These results are
discussed in view of the distribution of GAGA binding sites that, most
frequently, form clusters of relatively short sites spaced by small
variable distances.
The GAGA factor is a sequence-specific DNA-binding protein, which
participates in the regulation of the expression of a variety of
different classes of genes in Drosophila such as many
developmentally regulated genes, stress induced genes, and cell cycle
regulated genes, as well as housekeeping genes (for reviews see Refs. 1 and 2). GAGA binds repeated d(GA·TC)n sequences. As judged
from the size of the regions protected from DNase I cleavage, GAGA
binding sites are variable in length, ranging from 10 bp1 to more than 40 bp, with
an average of 15 bp (1). GAGA can also bind to the (AAGAG)n
and (AAGAGAG)n satellite DNAs, which constitute exceptionally
long GAGA binding sites (3, 4). Although GAGA binding sites frequently
show a degenerated nucleotide composition, a "consensus" binding
site containing 3.5 dinucleotide repeats was derived from the analysis
of about 50 different sites found at promoter regions (1). A remarkable feature of the GAGA binding sites is their multiplicity so that, in
general, promoters contain multiple sites spaced by relatively short
variable distances (5). The DNA binding domain of GAGA spans 82 amino
acid residues (310-391) and contains a single zinc-finger flanked by
three regions of basic amino acids (6). In addition, GAGA contains a
C-terminal glutamine-rich domain and a highly conserved N-terminal POZ
domain that has been shown to occur in a number of other
Drosophila proteins as well as in proteins from other
eukaryotes and viruses (7-13). The POZ domain was reported to be
involved in self-oligomerization in a number of other POZ domain
containing proteins (7-10). In this study, we have shown that, also in
the case of the GAGA protein, the N-terminal POZ domain mediates the
formation of oligomers both in vitro and in vivo.
GAGA oligomers bind DNA with high affinity and specificity, being
capable of binding adjacent sites spaced by as many as 20 bp. The
interaction of GAGA with DNA fragments carrying several d(GA·TC)n sequences is of higher affinity than its
interaction with fragments containing a single short binding site.
These observations are discussed in view of the clustered organization
of GAGA binding sites at promoter regions and might also have
consequences in the context of the efficient contribution of GAGA to
nucleosome disruption.
Proteins and DNAs--
Recombinant proteins used in these
experiments correspond to gaga519 form (GAGA) (5) and several peptides
derived from it spanning residues 310-391 (BDGAGA), 122-519
(
DNA fragments GA22, GA10, and GA5 were obtained as described elsewhere
(14). DNA fragments GA10/5, GA10/10, and GA10/20 were obtained by
insertion of double-stranded oligonucleotides of sequence 5'-AGC TT(G
A)10CG TAC (GA)10/AGC T(TC)10 GTA
CG(T C)10A-3', 5'-AGC TT(G A)10CG TAC CGT AC(G
A)10/AGC T(TC)10 GTA CGG TAC
G(TC)10 A-3', and 5'-AGC T(GA)10 CGT ACC GGT
AAC CAC CGT AC(G A)10/AGC T(TC)10 GTA CGG TGG
TTA CCG GTA CG(T C)10A3' into the
HindIII site of pUC18. Fragments were then excised from the
corresponding pUC18 constructs by digestion with
EcoRI+PvuI.
Yeast Two-hybrid Assays--
For the yeast two-hybrid assays the
GAGA constructs described in the text were cloned into plasmid pGBT9
(CLONTECH), to express fusion proteins containing
the GAL4 DNA binding domain, or into plasmid pGAD424
(CLONTECH), to express fusion proteins containing the GAL4 activation domain. Appropriate plasmids were then transformed into the yeast HF7c strain (MATa, ura3-52,
his3-200, ade2-101, lys2-801,
trp1-901, leu2-3, 112,
gal4-542, gal80-538,
LYS2::GAL1UAS-GAL1TATA-HIS3, URA3::GAL417MERSX3-CyC1TATA-LacZ),
and the transformants were tested for their ability to grow on
selective medium lacking histidine. For quantitative analysis,
transformants were tested for activation of the
GAL4-dependent LacZ reporter gene.
EMSA Experiments--
For EMSA experiments, the
32P-labeled DNA fragments were incubated with recombinant
GAGA, BDGAGA or DNase I Footprinting Experiments--
DNase I digestions were
performed with 0.01 units of enzyme (Roche Molecular Biochemicals) for
2 min at room temperature in a final volume of 20 µl. The enzyme was
added in 8 mM MgCl2, 4 mM
CaCl2 and the reaction stopped by the addition of 200 µl
of a solution containing 50 mM Tris-HCl, pH 8, 0.1 M NaCl, 0.5% SDS. After phenol extraction and ethanol
precipitation, samples were analyzed in 7% polyacrylamide, 7 M urea sequencing gels.
Sedimentation Experiments--
For sedimentation experiments,
purified recombinant proteins were dialyzed overnight against 20 mM Hepes, pH 7.9, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 0.1% Nonidet P-40, and the KCl concentration indicated in
each case. After dialysis, about 200 µg of the protein in 75 µl
were loaded onto 10-30% sucrose gradients at the same buffer
conditions of the dialysis. Centrifugation was carried out in a Beckman
SW41 rotor at 40,000 rpm for 23 h at 4 °C. Fractions of about
0.6 ml were collected from top to bottom and analyzed for the presence of protein by Western blot using a rabbit Transcription Experiments--
The templates used for in
vitro transcription assays were derived from a minimal promoter
containing a TATAAA box followed by a G-less cassette as described in
(15). A double-stranded oligonucleotide of sequence TTG GGA GCG
AGA GGG AA, encompassing the GAGA binding site found in region C
of the engrailed promoter (16), was multimerized by
self-ligation and inserted at the AccI site at position The POZ Domain of GAGA Mediates Oligomerization Both in Vitro and
in Vivo--
The ability of the POZ domain of GAGA to mediate protein
oligomerization in vivo was determined in a yeast two-hybrid
assay. As judged from its homology to other POZ domain containing
proteins, the POZ domain of GAGA extends for about the first 120 amino
acid residues. Fig. 1A
summarizes the constructs used in these experiments. Two different
peptides,
The sedimentation behavior through 10-30% sucrose gradients was
analyzed to determine the stoichiometry of the GAGA oligomers. As shown
in Fig. 2, the sedimentation profile of
GAGA shows three relatively well defined peaks when the gradients are
performed in the presence of 50 mM KCl (Fig.
2A). A first peak is found at the light fractions of the
gradient that must correspond to GAGA monomers, because it shows an
apparent molecular mass (M) close to that of GAGA (67 kDa). A second
peak is detected at the middle of the gradient showing an apparent M
close to what would be expected for a GAGA dimer, and a third peak is
found at the dense fractions of the gradient that, from its apparent M,
is likely to correspond to tetramers. In addition to these three peaks,
part of the molecules are found at the bottom of the gradient corresponding to complexes of stoichiometry higher than tetramers. The
sedimentation experiments described above were performed in the
presence of 0.1% Nonidet P-40. When the gradients are run in the
absence of detergent, all the molecules are found at the dense
fractions corresponding to oligomers of high stoichiometry (not shown).
The stability of the complexes described above depends on the ionic
strength so that, at 200 mM KCl, GAGA molecules are found
all throughout the gradient (Fig. 2B). Under these
conditions, the monomeric forms predominate and the complexes of high
stoichiometry are less abundant. When the sedimentation behavior of the
The Interaction of GAGA Oligomers with DNA Is of High Affinity and
Specificity--
Fig. 3 shows the
interaction of GAGA with d(GA·TC)n sequences of increasing
length as analyzed by EMSA and DNase I footprinting. The DNA fragments
used in these experiments were described in detail elsewhere (14).
Briefly, fragments GA5, GA10, and GA22 are all about 180-bp long,
differing in the size of the repeated d(GA·TC)n sequence they
contain, n = 5, 10, and 22, respectively. Several
features of the EMSA experiments presented in Fig. 3A
indicate that the protein-DNA complexes detected arise from the binding
of GAGA oligomers. First of all, the GAGA-DNA complexes formed with all
three DNA fragments show an anomalous low electrophoretic migration,
corresponding to a very large apparent M, much higher than would be
expected for a polypeptide of 67 kDa such as GAGA. These protein-DNA
complexes can only be resolved in low percentage (0.8%) agarose gels
where they show a slow electrophoretic mobility. They cannot be
resolved in native polyacrylamide gels, barely entering gels of very
low percentage (3.5%) (not shown). As a comparison, complexes formed
with the same DNA fragments by proteins of similar M are easily
resolved in 4-6% polyacrylamide gels (14). Furthermore, regardless of
the actual length of the d(GA·TC)n sequence contained within
the fragment, the formation of only one protein-DNA complex is
detected, indicating that increasing the length of the binding site
4-fold, from 10 bp in fragment GA5 to 44 bp in fragment GA22, does not
result in an increased number of GAGA molecules entering the complex. As judged by DNase I footprinting (Fig. 3B), the interaction
of GAGA oligomers with all three DNA fragments takes place at the repeated d(GA·TC)n sequence. In all three cases, the footprints are centered around the d(GA·TC)n sequence (Fig.
3B), spanning similar regions. Even at high protein
concentration, the footprints do not significantly extend beyond the
d(GA·TC)n sequence, and no footprints are detected in other
regions of the DNA fragments (Fig. 3B, lanes 2).
These results indicate that GAGA oligomers bind DNA sequence
specifically. The affinity of GAGA for fragment GA22, carrying long
d(GA·TC)n sequences, is significantly higher than for
fragments GA10 or GA5 containing shorter GAGA binding sites (Fig.
3A, right panel). This difference in affinity is
better reflected by the amount of protein required to induce 50%
retardation, which is about 3-fold higher for fragments GA10 and GA5
than for fragment GA22. The higher affinity of GAGA for fragment GA22
is consistent with the protein being an oligomer capable of
establishing a higher number of productive interactions with long
d(GA·TC)n DNA sequences than with short ones.
The EMSA experiments reported above were performed under conditions (50 mM KCl in the absence of Nonidet P-40) where, according to
its sedimentation behavior, GAGA exists as complexes of high stoichiometry. Similar results are obtained when the EMSA experiments are carried out in the presence of Nonidet P-40 at either 50 mM KCl (Fig. 4A)
or 200 mM KCl (Fig. 4B) where, according to the sedimentation experiments described in Fig. 2, a percentage of the GAGA
molecules exists as complexes of low stoichiometry, monomers, and
dimers. Also under these conditions, the formation of protein-DNA complexes of high M is detected, with both the GA22 and GA5 fragments, and the affinity of GAGA for the fragments carrying long
d(GA·TC)n DNA sequences is higher. In addition to the high M
complexes, the formation of complexes of fast electrophoretic mobility
can also be observed. Although these complexes are detected at
different experimental conditions, they appear to be more abundant when the binding is performed under conditions favoring disassembly of the
GAGA oligomers (200 mM KCl in the presence of Nonidet P-40) (Fig. 4B), suggesting that they arise from the binding of
the GAGA species of low stoichiometry. Similar results were obtained when sedimentation experiments, similar to those described in Fig. 2,
were performed in the presence of radioactively labeled GA5 fragment.
When the gradients are run at 200 mM KCl, the
32P-labeled fragment is distributed into two peaks (Fig.
4C, center panel). One peak is found at the heavy
region of the gradient, as expected for the binding of GAGA oligomers
of high stoichiometry, but in addition a second peak is found at the
light region of the gradient, sedimenting significantly faster than the
free DNA fragment (Fig. 4C, left panel), as would
be expected for the binding of GAGA forms of low stoichiometry.
Consistent with this interpretation, when the gradients are run at 50 mM KCl, most of the radioactivity is recovered at the heavy
fractions and only a low percentage is found at the light region of the
gradient (Fig. 4C, right panel). Under these low
ionic conditions, a slow sedimenting peak is also detected at the
position corresponding to the free DNA fragment.
The characteristic DNA binding behavior of GAGA described above is not
observed in the absence of the POZ domain. When the interaction of the
Altogether these results show that GAGA oligomers bind DNA with high
affinity and specificity and corroborate the important contribution of
the N-terminal POZ domain to the formation of these oligomers and,
thereby, to their DNA binding properties.
A Single GAGA Oligomer Can Interact with Two Adjacent Binding Sites
Spaced by as Many as 20 bp--
In general, GAGA binding sites at
promoters show a peculiar distribution in which relatively short sites,
spaced by small distances, form clusters (5). Most frequently, the
individual binding sites of each cluster are spaced between 10 and 30 bp. Promoters might contain several clusters of GAGA binding sites, which are then spaced by larger distances. To analyze the ability of
GAGA to interact with adjacent binding sites, the binding of GAGA to
DNA fragments containing two d(GA·TC)10 sequences spaced by either 5 bp (GA10/5), 10 bp (GA10/10), or 20 bp (GA10/20) was determined. As shown by the EMSA experiments in Fig.
6A, the affinity of GAGA
oligomers for fragments GA10/5, GA10/10 and GA10/20 is higher than for
fragment GA10, which contains a single d(GA·TC)10 site,
but very similar to the affinity for fragment GA22, which for this
purpose can be considered as containing two immediately adjacent sites
of about the same size. Furthermore, regardless of the distance between
the two binding sites, the formation of only one protein-DNA complex of
high M is detected. This behavior depends on the presence of the POZ
domain, because binding of Multiple GAGA Binding Sites Are Required for Efficient
Transcription Activation in Vitro--
GAGA is known to enhance
transcription from promoters containing d(GA·TC)n sequences,
both in vitro and in vivo (5, 18). To analyze the
contribution of the presence of multiple binding sites to the
transcription activity of GAGA, we determined the rate of
GAGA-dependent transcription activation from promoters containing an increasing number of GAGA binding sites. For these experiments, the GAGA binding site found at the C-region of the engrailed promoter (16) was multimerized and fused to a
minimal promoter, which was shown earlier to efficiently drive
transcription of a G-less cassette (15). As shown in Fig.
8A, the constructs used in
these experiments contained from 1 to 6 copies of this engrailed site. The extent of maximal activation obtained in
the presence of GAGA strongly depends on the number of binding sites present at the promoter (Fig. 8B). No significant activation
is observed from constructs containing only one or two GAGA binding sites, and only a moderated 3-fold activation is observed in the presence of three binding sites. However, a strong increase in activation, to about 8-9-fold, is seen from constructs containing five
or six binding sites. Similar results were reported earlier by others
(5). This behavior depends on the presence of the POZ domain. When the
transcription activity of the We have shown that GAGA forms oligomers both in vitro
and in vivo. Others (19) have also reported the formation of
GAGA-DNA complexes compatible with the formation of GAGA oligomers.
Here, however, we have been able to show that formation of these
oligomers, which exist in solution in the absence of DNA binding,
requires the contribution of the N-terminal POZ domain and that GAGA
can form different types of oligomers, dimers, but also tetramers and
complexes of high stoichiometry. Several results indicate that these
oligomeric forms do not arise from the unspecific in vitro
self-aggregation of the protein. First, they bind d(GA·TC)n sequences with high specificity as reflected by the fact that their
DNase I footprints do not extend beyond the d(GA·TC)n sequence, even when binding to DNA fragments carrying two independent binding sites spaced by as few as 5 bp (Fig. 7). Second, the formation of these GAGA oligomers requires the contribution of specific protein-protein interactions involving the N-terminal POZ domain, deletion of the first 6 or the last 21 amino acid residues of the
domain abolishing the interaction in vivo. Finally, these GAGA oligomers are functionally active, being transcriptionally competent in vitro. Altogether, these observations indicate
that GAGA oligomers are functionally active forms of the protein
arising from specific protein-protein interactions.
The crystal structure of the POZ domain of the human promyelocytic
leukemia zinc finger protein was recently reported (20). In the
crystal, the promyelocytic leukemia zinc finger POZ domain forms a
dimer that is held together by interactions involving the most N- and
C-terminal regions of the monomers. In particular, the five most
N-terminal residues of one monomer form a Our results indicate that in solution GAGA can form dimers and
tetramers, as well as oligomers of higher stoichiometry. This behavior
is not influenced by the source of the protein used that, in the
experiments reported here, was expressed in E. coli as a
His-tagged protein. Non-His-tagged recombinant GAGA also binds DNA as
an oligomer (21), and native GAGA protein obtained directly from
Drosophila cells (22, 23) or by cell-free in
vitro transcription-translation (24) behaves also as an oligomer
of high M. Other POZ domain-containing proteins have also been shown to
form oligomers (7-13). Although, in general, the stoichiometry of
these complexes was not determined, the formation of both dimers and
tetramers was observed in some cases (12). Interestingly, though the
promyelocytic leukemia zinc finger POZ domain appears to form only
dimers in solution, the formation of a short four-stranded antiparallel
The binding experiments reported here were performed under conditions
where GAGA exists mainly as tetramers and oligomers of higher
stoichiometry. Consistent with these results, the GAGA-DNA complexes
formed under these conditions are of high M. However, when the binding
was performed under conditions where a significant percentage of the
GAGA molecules exists as oligomers of low stoichiometry, the formation
of protein-DNA complexes of low M could also be observed (Fig. 4),
indicating that the GAGA species of low stoichiometry, monomers and
dimers, can also bind DNA. Nevertheless, the formation of GAGA-DNA
complexes of high M is also observed under these conditions, predominating at high protein concentration (Fig. 4B). These
results suggest that the GAGA oligomers of high stoichiometry bind DNA very efficiently or, alternatively, that DNA binding facilitates GAGA oligomerization.
Others (7) had reported earlier that GAGA, through its POZ domain,
forms in vitro heteromeric complexes of unknown
stoichiometry with other POZ domain-containing proteins, such as
tramtrack. Several other Drosophila proteins,
including the enhancer of variegation E(var)3-39D, contain
similar POZ domains (13, 25), but it is not known whether they can also
interact with GAGA. These observations indicate that the POZ domain is
likely to play a key role in the control of GAGA function(s) in
vivo, by regulating its oligomerization state and its interaction
with other proteins. Interestingly, GAGA associates with the
centromeric (AAGAG)n heterochromatin during mitosis (3, 4). The
formation of large oligomers might play a role in this context. The POZ
domain might also regulate DNA binding. Earlier studies indicate that
the POZ domain inhibits the interaction of GAGA with a short 17-bp-long
DNA fragment carrying a single GAGA binding site (7). We have also
observed that, compared with the BDGAGA peptide, GAGA binds
with high difficulty to short synthetic oligonucleotides (not shown).
However, as reported here and elsewhere (5, 26), GAGA binds efficiently to DNA fragments carrying longer or multiple GAGA binding sites. Our
results also show that the affinity of GAGA for DNA depends strongly on
the length and number of binding sites, being significantly lower for
fragments carrying single short d(GA·TC)n sequences than for
fragments carrying either several short sites or a long one, providing
a reasonable interpretation for the apparently contradictory results
mentioned above.
We have shown here that a single GAGA oligomer can bind two adjacent
GAGA binding sites spaced by as many as 20 bp. Others have recently
reported similar results (34). As judged from EMSA and DNase I
footprinting experiments, this interaction is of high affinity and
specificity. As mentioned before, most frequently, promoters contain
clusters of independent d(GA·TC)n sequences that, as derived
from our results, could be bound by a single GAGA complex. Previously
reported results suggest that this situation might occur both in
vitro (16, 27) and in vivo (28). Moreover, a single
short d(GA·TC)n sequence is not a good substrate for GAGA
binding in vitro. These results suggest that several adjacent d(GA·TC)n sequences are required to create a GAGA
binding site of high affinity. Given the repetitive character of the
sequences that GAGA recognizes, this mode of interaction, requiring the
presence of several independent d(GA·TC)n sites for efficient
protein binding, provides an additional factor that increases the
specificity of the interaction of GAGA with DNA.
Several observations suggest that, to some extent, GAGA functions at
the chromatin level, participating in the formation of an open
chromatin structure. GAGA is the product of the
Trithorax-like(Trl) gene (29) which, being a member of the
Trithorax group, antagonizes the chromatin-mediated
repression that Polycomb genes induce on the expression of
the homeotic genes. A more direct link to chromatin structure is
indicated by the fact that Trl is an enhancer of position
effect variegation (29). Moreover, in collaboration with nucleosome
remodeling factor, GAGA was shown to help nucleosome disruption at
specific regions of the hsp70 promoter, encompassing GAGA binding sites
(26, 30, 31). At present, little is known about the specific
contribution of GAGA to chromatin remodeling and, although other
DNA-binding proteins such as HSF (heat shock factor) or GAL4-HSF can
also help disrupting chromatin organization (29, 31, 32), GAGA appears
to be particularly efficient in this respect (31, 33). Although a
direct interaction with the chromatin remodeling machinery cannot be
excluded, the simultaneous interaction of GAGA oligomers with multiple
adjacent sites could significantly contribute to the higher efficiency
of GAGA in disrupting nucleosomes. In this context, it would be
interesting to know whether a functional POZ domain is required for
efficient nucleosome disruption. GAGA can also activate transcription
in vitro suggesting a possible interaction with the basal
transcription machinery. Our results indicate that the presence of
several independent GAGA sites is required for efficient transcription
activation in vitro, indicating that the oligomeric
character of GAGA might also be functionally relevant in this context.
Interestingly, in the case of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
POZ122), and 245-519 (
POZ245). All the
proteins were expressed as 6× His-tagged proteins in Escherichia
coli strain BL21-LysE using pET14-b or, in the case of
POZ122, pET29-b expression vectors. Recombinant GAGA
protein was purified on a DEAE-SepharoseFF column followed by a
heparin-Sepharose column and a Ni2+-HiTrap (Amersham
Pharmacia Biotech) column. BDGAGA,
POZ122, and
POZ245 proteins were purified on a Ni2+-NTA
column (Qiagen) and
POZ245 was further purified on a
Mono-S column (Amersham Pharmacia Biotech).
POZ245 proteins in 50 mM KCl, 15 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.5 mM dithiothreitol, and 5%
glycerol in the presence of 10 µg of bovine serum albumin and a
50-100-fold excess (w/w) of E. coli DNA for 20 min at room temperature in a final volume of 10 µl. The amount of protein used in
each experiment is expressed as units of activity defined as the amount
of protein inducing about 50% retardation of 1 ng of fragment GA22.
GAGA-DNA complexes were analyzed on 0.8% agarose, 0.5× TBE gels, but
POZ245-DNA and BDGAGA-DNA complexes were run on
4.5 and 6% polyacrylamide, 0.5× TBE gels, respectively. When
indicated, 0.1% Nonidet P-40 was added to the binding reaction and to
the gel and electrophoresis buffer.
GAGA polyclonal antibody (a gift of Dr. C. Wu) that was shown to recognize all GAGA peptides used in these experiments. Bovine serum albumin (M = 67 kDa) (Amersham Pharmacia Biotech), yeast alcohol dehydrogenase (M = 150 kDa) (Sigma), and bovine liver catalase (M = 232 kDa) (Amersham
Pharmacia Biotech) were used as molecular weight markers. When the
sedimentation behavior of the GAGA-DNA complexes was analyzed, about 40 ng of 32P-labeled GA5 fragment were incubated with 35 µg
of protein in the presence of a 50-fold excess (w/w) of E. coli DNA at room temperature for 30 min at the buffer conditions
indicated in each case in a final volume of 200 µl. Samples were then
loaded onto the gradients and, after centrifugation, the radioactivity
recovered in each fraction was determined by Cerenkov counting.
40
of the minimal promoter described above. Transcription reactions were
carried out as described (17) using 200 ng of each supercoiled
template, 50 µg of nuclear HeLa cell extract, and increasing amounts
of either recombinant GAGA or
POZ245 proteins.
Quantitative analysis was performed by laser densitometry of the
corresponding autoradiographs in a Molecular Dynamics densitometer and
normalized using the recovery controls for each case. For each
template, maximal activation rates with respect to controls receiving
no protein were determined and compared.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
POZ122 and POZ245, were fused to
the GAL4 activation domain (ADGAL4).
POZ122 is
missing the first 122 amino acid residues of GAGA, whereas
POZ245 contains the POZ domain plus about half of the
region located between the POZ domain and the DNA binding domain. These
two constructs were tested for interaction with fusion proteins
containing the GAL4 DNA binding domain (BDGAL4) and either the
122 amino acid long POZ domain of GAGA (POZ122), the
POZ245 construct, or full GAGA. None of these fusion
proteins were able to activate by themselves the
GAL4-dependent expression of the HIS3 or
LacZ genes used as reporters (not shown). However, as judged
from the growth obtained in the absence of histidine (Fig.
1B), the POZ245-ADGAL4 fusion interacts
with all three POZ122-BDGAL4,
POZ245-BDGAL4, and GAGA-BDGAL4 fusion
proteins. On the other hand, none of these three fusion proteins were
found to interact with the
POZ122-ADGAL4 fusion.
Similar results were obtained when the LacZ gene was used as
reporter (Fig. 1C). Also in this case, all three constructs
interact with the POZ245-ADGAL4 fusion but not with
the
POZ122-ADGAL4 fusion. These results show
that the POZ domain mediates GAGA oligomerization in vivo.
In agreement with these results, a positive interaction is also
detected between POZ122-ADGAL4 and
POZ122-BDGAL4 (Fig. 1C). This
interaction is of the same magnitude as the interaction between
POZ245-ADGAL4 and POZ245-BDGAL4
but weaker than between POZ245-ADGAL4 and
POZ122-BDGAL4, which is the strongest. Terminal
deletions of the POZ domain have a strong effect on its oligomerization
potential, and no interaction is detected upon removal of the last 21 amino acids (POZ1-101), the first 30 amino acids
(POZ30-122), or simply the first 6 amino acids
(POZ6-122) (Fig. 1C).
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Fig. 1.
The POZ domain mediates GAGA oligomerization
in vivo. A, constructs used
in the yeast two-hybrid assays. The POZ245 and
POZ122 constructs were fused to the GAL4 activation
domain (ADGAL4). Full GAGA, the POZ122, and
POZ245 constructs were fused to the GAL4 binding domain
(BDGAL4). B, growth in selective medium lacking
histidine of strains carrying the POZ245-ADGAL4 or
the
POZ122-ADGAL4 constructs and either the
GAGA-BDGAL4, the POZ122BDGAL4, or the
POZ245-BDGAL4 constructs. C,
-galactosidase activities, expressed as Miller units, corresponding
to strains carrying several combinations of constructs as indicated. In
all cases, the different constructs were fused to the BDGAL4
and were tested for interaction with the
POZ245-ADGAL4, the
POZ122-ADGAL4, or the
POZ122-ADGAL4 constructs. When the interaction
between POZ122 and the POZ1-101 and
POZ30-122 constructs was studied, the former was fused to
the BDGAL4 and the latter to the ADGAL4.
POZ122 peptide was analyzed, a single peak was detected
at the light region of the gradient (Fig. 2C), indicating
that in the absence of the POZ domain only monomers are formed. This
behavior is independent of the KCl concentration at which the gradients
are run and of the presence or absence of Nonidet P-40 (not shown).
Similar results were obtained with the
POZ245 peptide.
These results demonstrate that GAGA oligomerization also occurs
in vitro and the formation of dimers, tetramers, and
complexes of higher stoichiometry is detected at low ionic
strength.
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Fig. 2.
Sedimentation profiles through 10-30%
sucrose gradients of full GAGA (A and
B) or 5-20% sucrose gradients of
POZ122 (C).
Gradients were run in the presence of 0.1% Nonidet P-40 at either 50 mM KCl (A and C) or 200 mM KCl (B). Fractions were collected from top to
bottom, and the presence of the proteins was determined by Western
blotting (inserts), using rabbit
GAGA polyclonal
antibodies. Bands of higher electrophoretic mobility arise from
proteolytic degradation of the recombinant protein occurring during the
dialysis. The positions corresponding to markers of known M: 67, 150, and 232 kDa are indicated at the top of each graph.
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Fig. 3.
The interaction of GAGA with
d(GA·TC)n sequences of increasing
length. A, the binding of GAGA to fragments GA22, GA10, and
GA5 is analyzed by EMSA in 0.8% agarose gels as a function of
increasing protein concentration expressed as units of activity (see
"Experimental Procedures"). All lanes contain 0.5 ng of the labeled
DNA fragment. Panel GA22: 0 units (lane 0); 0.3 units (lane 1); 0.6 units (lane 2); 1.1 units
(lane 3), 1.5 units (lane 4), and 2.25 units
(lane 5). Panels GA10 and GA5: 0 units
(lane 0); 0.6 units (lane 1); 1.1 units
(lane 2), 1.5 units (lane 3), 2.25 units
(lane 4), and 3 units (lane 5). Quantitative
analysis of the results is shown on the right: , GA5;
, GA10; and
, GA22. B, DNase I footprinting analysis of the binding
of GAGA to fragments GA22, GA10, and GA5 as a function of increasing
protein concentration: 0 units (lanes 0); 1.5 units
(lanes 1); and 3 units (lanes 2). Lanes
L correspond to G + A sequencing ladders. The position of the
d(GA·TC)n sequence is indicated. The 5' to 3' direction is
also indicated.
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Fig. 4.
The interaction of GAGA with fragments GA22 and GA5
is analyzed by EMSA (A and B) in the presence of
0.1% Nonidet P-40 at either 50 mM KCl (A) or
200 mM KCl (B) as a function of increasing
protein concentration expressed as units of activity: 0 units
(lane 0); 0.6 units (lane 1); 0.9 units
(lane 2); 1.2 units (lane 3); 1.5 units
(lane 4); and 1.8 units (lane 5). All lanes
contain 1 ng of the labeled DNA fragment. The arrows in
B point to the complexes of fast electrophoretic mobility
discussed in the text. C, the sedimentation profiles of the
GAGA-GA5 complexes through 10-30% sucrose gradients performed in the
presence of 0.1% Nonidet P-40 at 200 mM KCl (center
panel) or 50 mM KCl (right panel). The
sedimentation profile of the free GA5 DNA fragment is shown in the
left panel.
POZ245 peptide with the GA5, GA10, and GA22 DNA
fragments was studied, a totally different binding behavior was
observed (Fig. 5). In this case, the
apparent M of the protein-DNA complexes corresponds to what would be
expected from the size of the polypeptide. These protein-DNA complexes
are resolved in native 4.5% polyacrylamide gels where they show an
electrophoretic mobility consistent with the binding of the 274-amino
acid residue-long
POZ245 peptide (Fig. 5A).
In the presence of low amounts of
POZ245 a single
protein-DNA complex is detected, but on increasing the protein
concentration, the formation of additional bands of slower electrophoretic mobility is observed reflecting the binding of additional
POZ245 peptides to the DNA fragments. As
judged from the number of complexes detected, fragment GA5 accommodates
basically only one
POZ245 molecule, whereas fragments
GA10 and GA22 can accommodate up to two and three independent
molecules, respectively. When the interaction of the DNA binding domain
of GAGA (BDGAGA) with the same DNA fragments described above
was studied, a binding behavior very similar to that of the
POZ245 peptide was observed (not shown). As observed
with full GAGA, binding of
POZ245 results in intense
DNase I footprints centered around the repeated d(GA·TC)n sequence (Fig. 5B). The binding of
POZ245 to
fragment GA22 is slightly more efficient than to fragment GA5, as would
be expected from the higher number of binding sites present in fragment
GA22 (Fig. 5A, right panel), but the difference
in relative affinity in this case is much lower than that observed with
full GAGA. These results indicate that, contrary to full GAGA, which
binds DNA as an oligomer,
POZ245 binds as a monomer
whose interaction with DNA fragments containing long
d(GA·TC)n sequences, capable of accommodating several
POZ245 molecules, is noncooperative.
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Fig. 5.
The interaction of
POZ245 with
d(GA·TC)n sequences of
increasing length. A, the binding of
POZ245 to fragments GA22, GA10, and GA5 is analyzed by
EMSA in 4.5% native polyacrylamide gels as a function of increasing
protein concentration expressed as units of activity. All lanes contain
1 ng of the labeled DNA fragment: 0 units (lanes 0); 0.6 units (lanes 1); 1.25 units (lanes 2); 2.5 units
(lanes 3); and 5 units (lanes 4). Quantitative
analysis of the results is shown on the right: (
) GA5, (
) GA10,
and (
) GA22. B, DNase I footprinting analysis of the
binding of
POZ245 to fragments GA22, GA10, and GA5 as a
function of increasing protein concentration: 0 units (lanes
0), 2.4 units (lanes 1), and 6 units (lanes
2). Lanes L correspond to G + A sequencing ladders. The
position of the d(GA·TC)n sequence is indicated. The 5' to 3'
direction is also indicated.
POZ245 to fragments GA10/5,
GA10/10, and GA10/20 is noncooperative showing a similar affinity as to
fragments GA10 or GA22 (Fig. 6B). BDGAGA shows a
similar binding behavior as
POZ245 (not shown). These
results indicate that a single GAGA oligomer is binding to the
fragments containing two independent d(GA·TC)10 sequences
even when spaced by 20 bp, suggesting that GAGA oligomers can bind
simultaneously to at least two, and likely more, adjacent binding
sites. Interestingly, binding of GAGA does not protect from DNase I
cleavage the region located between the two adjacent d(GA·TC)10 sequences of fragments GA10/5, GA10/10, and
GA10/20 (Fig. 7A), indicating
that, in these DNA fragments, the region between the two GAGA binding
sites is not in intimate contact with the protein. Actually, the
footprints obtained with full GAGA are very similar to those obtained
with the
POZ245 peptide (Fig. 7B). These
results show that the simultaneous interaction of GAGA with adjacent
sites is also highly specific.
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Fig. 6.
The interaction of GAGA (A)
and POZ245 (B)
with fragments GA22, GA10/5, GA10/10, GA10/20, and GA10 is analyzed by
EMSA as a function of increasing protein concentration expressed as
units of activity. Panel A: 0 units (lanes 0),
0.8 units (lanes 1), 1.2 units (lanes 2), and 1.6 units (lanes 3). All lanes contain 0.5 ng of the labeled DNA
fragment. Panel B: 0 units (lanes 0), 0.17 units
(lanes 1), 0.35 units (lanes 2), 0.7 units
(lanes 3), and 1.1 units (lanes 4). All lanes
contain 0.25 ng of the labeled DNA fragment. Quantitative analysis of
the results is shown on the right. Solid lines correspond to
GA10 (
) and GA22 (
). Broken lines correspond to GA10/5
(
), GA10/10 (
), and GA10/20 (
).
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Fig. 7.
DNase I footprinting analysis of the
interaction of GAGA (A) and
POZ245 (B) with
fragments GA22, GA10/5, GA10/10, GA10/20, and GA10 as a function of
increasing protein concentration. Panel A: 0 units
(lanes 0), 1.1 units (lanes 1), and 3 units
(lanes 2). Panel B: 0 units (lanes 0),
0.6 units (lanes 1), and 1.5 units (lanes 2).
Lanes L correspond to G + A sequencing ladders. The 5' to 3'
direction is indicated.
POZ245 peptide was
analyzed, a significant activation was observed in the presence of two
GAGA binding sites, which increases only slightly as the number of
binding sites do (Fig. 8B). In this case, a low though reproducible activation is detected even in the presence of a single
site. The synergism in transcription activation detected upon
increasing the number of binding sites is consistent with the higher
affinity of GAGA oligomers for fragments carrying multiple GAGA sites.
Consistent with this hypothesis, this synergism depends on the presence
of the POZ domain.
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Fig. 8.
The effect of GAGA and
POZ245 on the rate of transcription
in vitro. A, templates used in these
experiments containing either 1 (1x), 2 (2x), 3 (3x), 5 (5x), or 6 (6x) copies of the
GAGA binding site contained in region C of the engrailed
promoter. The arrows indicate the relative orientation of
the copies. B, effect of the addition of 1 µl of GAGA
(lanes GA) or 2 µl of
POZ245
(lanes
) on the in vitro transcription rate
obtained from the templates shown in A. WT
corresponds to the construct containing no GAGA binding site.
Lanes
correspond to the rates of transcription obtained in the
absence of any added protein. Arrows indicate the G-less
transcripts. The bands on the top correspond to the recovery
controls. For each construct, maximal activation rates obtained from
titration experiments are shown on the right for GAGA
(solid columns) and
POZ245 (hatched
columns).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-strand that interacts
with an internal
5'-strand on the second monomer. One face of this
1/
5' sheet is then packed against the
6' helix of the second
monomer, which spans the last 15 residues, establishing a number of
stabilizing hydrophobic interactions. These interactions provide a
reasonable interpretation for the strong effect that terminal deletions
have on GAGA oligomerization in vivo, because given its high
degree of conservation, it is reasonable to assume that all POZ domains
will have a similar fold.
-sheet between two symmetry related dimers is observed in the
crystal (20). This interaction involves four different peptide chains
and, therefore, can give rise to the formation of tetramers and
oligomers of higher stoichiometry.
POZ245 peptide,
significant transcription activation is detected in the presence of a
single binding site, and no synergism is observed upon increasing the
number of GAGA binding sites. These results suggest that the synergism
observed with full GAGA arises from specific features of the GAGA-DNA
complex rather than from the simple recruitment of multiple GAGA
molecules to the promoter.
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ACKNOWLEDGEMENTS |
---|
We are thankful to Dr. C. Wu for providing us
with GAGA antibodies and to Dr. M. Ortiz-Lombardía for
purified GAGA protein. We are also thankful to Dr. B. Piña for
helpful discussions and advice.
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FOOTNOTES |
---|
* This work was supported by Grants PB95-64 and PB96-812 from the Spanish Dirección General de Euseñanza Superior, the European Union (BIO2-CT94-3069), and the Comissió Interdepartamental de Recerca i Innovació Tecnològica (CIRIT) of the Generalitat de Catalunya (SGR97-55).This work was carried out in the context of the Centre de Referència en Biotecnologia of the CIRIT of the Generalitat de Catalunya.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.
Recipient of doctoral fellowship from the CIRIT.
§ Recipient of doctoral fellowship from the Dirección General de Euseñanza Superior.
¶ To whom correspondence should be addressed: Dpt. Biologia Molecular i Cel.lular, Institut de Biologia Molecular de Barcelona, Centre d'Investigació i Desenvolupament, Consejo Superior de Investigaciones Científicas, Jordi Girona Salgado 18-26, 08034 Barcelona, Spain. Tel.: 343-4006137; Fax: 343-2045904; E-mail: fambmc{at}cid.csic.es.
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair(s); Ni2-NTA, nickel nitrilotriacetic acid; EMSA, electrophoretic mobility shift assay; M, molecular mass.
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REFERENCES |
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