(Received for publication, April 7, 1995; and in revised form, July 12, 1995)
From the
The mechanisms underlying transcriptional activation are not
very well understood, and knowledge is based on experiments with a
small number of mostly viral activators. We have investigated the
mechanism underlying transactivation by the activation domain present
in the N-terminal part of retinoic acid receptor (RAR) 2 (AF-1).
We show that RAR
2 phosphorylation is not crucial for its activity
although it may modulate AF-1 activity. Sequential mutation of the
negatively charged residues (Asp) resulted in a stepwise decrease in
activity, while mutation of all aspartic acid residues resulted in
complete loss of activity. Comparison of the critical region for
activation with other activators revealed moderate homology with the
viral activator VP16. The hydrophobic amino acids surrounding the
negatively charged residues reported to be critical for activation by
VP16 are all conserved in AF-1. The hydrophobic residues are required
for AF-1, since mutation of these residues resulted in a decrease in
activity. Furthermore, the activity of this activator, VP16 and
TA
of RelA, is squelched by overexpression of an
AF-1-containing expression construct, indicating that AF-1 is an acidic
activator. Squelching experiments further indicate that AF-1 and AF-2
function by different mechanisms. Comparison of activation functions
present in the AB region of other members of the steroid/thyroid
hormone receptor family: RAR
2, RAR
2, and GR suggested that
also these receptors contain an acidic activation domain. The mechanism
underlying activation by AF-1 is discussed.
Transcription of RNA polymerase II promoters requires an
assembly of the preinitiation complex consisting of basal transcription
factors. This process begins with the binding of TFIID to the TATA box,
followed by ordered binding of the other transcription factors (TFIIA,
-B, -E, -F, -H) and RNA polymerase forming the initiation
complex(1, 2) . Transcription factors bound to
promoter or enhancer sequences modulate the activity of polymerase II
promoters. Transcription factors contain a DNA-binding domain (DBD) ()and an activation function (AF), each of which are
interchangeable units, and generally are functioning independently when
coupled to a heterologous AF or DBD(3) . Activation
functions/activators are regions of 30-100 amino acids in length
and can be classified by their sequence similarity or the presence of
predominant amino acids: acidic, glutamine-, or
proline-rich(4) . Presently, little is known about the exact
role of the predominant amino acids (Asp/Glu, Gln, or Pro) in
activators, and it is unclear whether secondary structure is required
for activation. Mutational analysis of acidic activators has shown that
negative charge per se is not sufficient for activation as
mutation of negative to neutral or even positive amino acids does not
or only marginally interferes with activation capacity(5) . An
amphipathic
-helix, with negatively charged residues on one
surface and hydrophobic residues on the other, could be a requirement
for activation(6) . However, some mutations destroying the
putative
-helix also remain active(5) . Based on
mutational analysis, the GAL4 activator was proposed to form an
antiparallel
-sheet structure (7) . Using circular
dichroism, the presence of this structure (under slightly acid
conditions) was confirmed(8) . Structural analysis using NMR
has not provided any evidence for the presence of stable secondary
structure elements in any activator analyzed so far.
The mechanism by which these activators exert their effect is currently a point of discussion. The removal of repressors interacting with a component of TFIID by activators was proposed(9) . Furthermore, it has been suggested that activators can facilitate steps in the formation of the preinitiation complex by interacting with a component of this complex (10 and references therein). Thereby, the assembly of the preinitiation complex could be enhanced, and/or the number of active transcription complexes could be increased(11, 12) . Also, the formation of an open complex following the formation of the initiation complex may be a target for an activator. Based on these models, activators may modulate transcription in several ways, whereby generally an interaction with one or more components of the basal transcription machinery seems to be necessary. Several activators have been shown to interact with TATA-binding protein(13, 14, 15) , TFIIB (16, 17, 18) , or TATA-binding protein-associated factors(19, 20) . In some cases, point mutants with reduced activity show also reduced in vitro binding(14, 21, 22) . Occasionally, however, a bridging factor/cofactor is needed for activation, possibly indirectly connecting the activator with a component of the preinitiation complex (9) . The observation that the AFs of the estrogen receptor (ER) function in a cell-specific way, and the observed promoter specificity of the AFs of ER (23) has led to the hypothesis that cofactors are required for the activity of the activators. The requirement for cofactors both in vitro and in vivo has recently been confirmed(24, 25) . A different requirement for transcriptional activation may be phosphorylation. The activity of several transcription factors, e.g. CREB and c-jun have been shown to be up-regulated by phosphorylation (for review, see (26) ). Also, steroid hormone receptors are phosphorylated in vivo(27) .
RARs belong to the steroid/thyroid hormone
receptor superfamily which share a common domain structure, denoted
A-F(28, 29) . The C region contains the DNA-binding
domain which is most conserved among the different members of this
family and consists of two zinc fingers. The hormone-binding domain is
located in the E region and contains, besides the binding domain, a
dimerization domain and a hormone-dependent transactivation function
(AF-2). The N-terminal part of the receptor (AB) also contains an
autonomous region involved in transactivation (AF-1) which functions
independently of ligand, when coupled to a heterologous DNA-binding
domain(28, 29) . We and others have previously
reported the presence of two autonomous transcriptional activation
functions in RAR which activate transcription both by different,
cell-type and promoter-dependent mechanisms(30, 31) .
The activation function present in the N-terminal part of the protein
(AF-1, formerly called TAF-1), is located in the first 32 amino acids
of the receptor, and functions both in the presence and absence of RA.
This region is negatively charged and contains putative phosphorylation
sites, but no obvious homology with known activators was
observed(30) .
Since no activation function present in the
AB region of a member of this superfamily has been analyzed in detail
so far, we decided to characterize AF-1 of RAR2 in more detail.
Here we show that AF-1 is an acidic activator, three aspartic acids
present in this region are required for its activity, and the
hydrophobic residues contribute to activity. Sequence comparison
revealed that this activation function has homology with the acidic
transactivation domain of VP16.
Figure 1:
Transcriptional activation of the
CRBPII promoter by various mutant RAR receptors. A,
schematic representation of the different RAR
2 deletion
constructs. B, activation of transcription from CRBPII-CAT by
the indicated receptors in the presence or absence of 1.0 µM retinoic acid (RA) in COS cells, depicted as the mean CAT
activity (±S.E.) of four independent experiments. All receptors
are expressed at similar levels in COS cells as judged by Western blot
using an antibody against the F region of RAR
2 (lower
panel).
To test whether
phosphorylation is involved in the activity of AF-1, we changed the
tyrosine, threonine, and all serine residues present in this region to
alanine and tested the ability of these mutants to activate
transcription, when coupled to GAL-DBD. Fig. 2shows the
quantification of CAT assays of COS cells transfected with these
mutants. It is clear from these results that all mutants are still
active; only the mutation of serines 22, 24, and 25 to alanine showed a
35% reduction in activity. These transfection data indicated that the
putative phosphorylation sites are not absolutely required for AF-1
activity, but that they can, however, influence the activity. A
decrease in the in vivo phosphorylation levels might be
expected upon mutation of the putative phosphorylation sites.
Therefore, in vivo phosphorylation experiments using the
indicated mutants in the HA-RAR
E constructs (containing a
hemagglutinin tag in front of the AB region in the RAR
expression
construct lacking the hormone-binding domain) were performed. No
obvious differences in phosphorylation levels for the various mutants
were observed (data not shown). Since we were not able to map the
phosphorylation sites within this region, it is possible that the
absence of phosphorylation is not the cause of this decrease but rather
the introduction of Ala instead of Ser residues. An alternative
explanation could be that the kinase responsible for this
phosphorylation event is induced upon RA treatment, and a 4-h RA
treatment in the in vivo phosphorylation experiment is too
short to see the differences in phosphorylation levels between wild
type and mutants. From these data we conclude that phosphorylation is
not crucial for AF-1 activity, although it may modulate the activity of
this activator.
Figure 2:
The activity of AF-1 is modulated by
putative phosphorylation sites. COS cells were transfected with various
RAR2 (1-76) point mutants (schematically depicted in Fig. 4) coupled to GAL-DBD, together with the reporter 5
GAL-CAT (32) in the presence of 1.0 µM RA. CAT
activity was determined and is presented as the mean CAT activity
(±S.E.) of five independent
experiments
Figure 4:
Transcriptional activation by the various
mutant activators/receptors. On the left, a diagram of the
various point mutants is depicted. Transfections using these mutants
were performed with either GAL-RAR(1-76) fusion constructs with 5
GAL-CAT as reporter in COS cells or with RAR
expression
constructs containing the indicated mutation, with CRBPII-CAT as
reporter in COS cells or with hRAR
-CAT (-63/+156) in
RAC65 cells, in the presence of 1.0 µM RA. On the right, results are presented as the mean of four to six
independent or three duplicate experiments with a S.E. between various
experiments smaller than 20%. Activity was calculated relative to wild
type (WT) activator/receptor; - represents the residual
activity in the absence of co-transfected activator or receptor; nd, not determined.
Figure 3:
Negatively charged amino acids are
important for AF-1 activity. Representative CAT assay of a transfection
in COS cells of the indicated point mutants (schematically depicted in Fig. 4) in GAL-RAR (1-76) together with 5
GAL-CAT as reporter in the presence of 1.0 µM
RA.
Next we asked whether negative charge per se is needed or whether the presence of these specific negatively charged amino acids is required. Mutation of Asp-3, -6 to glutamic acid, which has been shown previously to be a poor substitute for aspartic acid in case of VP16(5) , resulted in a decrease almost as strong as the corresponding Ala mutant. We then attempted to create a stronger activator by introducing extra negative charge. Changing LDF (16, 17, 18) to aspartic acid residues (DDD) did not result in a receptor with higher activity, but instead a small decrease was observed. Above we have shown that replacement of S22A,S24A,S25A resulted in a decrease in activity (Fig. 2). Upon changing the serine residues of this putative phosphorylation site to aspartic acid, a stronger activator was created (Fig. 3), showing the importance of negative charge for activation and suggesting that phosphorylation can, by introducing extra negative charge, modulate the activity of this activator.
Transfection of these mutants in P19 EC cells gave
similar results, confirming that the negatively charged residues are
most important for activation (data not shown). To confirm that the
previous results are not caused by differential stability or
accumulation of the various proteins, we performed Western blots using
both the GAL-RAR AF-1 fusion constructs and mutant receptors,
containing the same mutations in the RAR
expression construct. All
proteins migrated according to their expected molecular weight, and the
variations in expression levels were not more than 2-fold (data not
shown).
Next we examined whether the critical amino acids of AF-1,
when present in the fusion constructs, are also the most important
residues for AF-1 activity in the full-length receptor. We therefore
compared the activity of each mutant AF-1 by transfecting them as
GAL-fusion constructs as well as within the normal receptor context on
the CRBPII promoter and on the hRAR promoter. By comparing the
transactivation capacity of all mutants (Fig. 4) with the wild
type RAR
on these promoters, we observed that the amino acids
found to be critical in the GAL AF-1 fusion protein were also important
for AF-1 activity when present in RAR
. As expected, the activity
of the CRBPII promoter was most dramatically decreased by mutations
that change the negatively charged amino acids to neutral residues.
Mutations that did not give a phenotype as GAL-fusion construct also
caused no significant or only a weak decrease in activity as compared
to the wild type receptor. The only exception was the S22D,S24D,S25D
mutant which was impaired in activity on the CRBPII promoter while it
was a stronger activator as GAL-fusion protein. This is possibly caused
by disruption of the structure of the receptor which could be permitted
in the GAL-fusion protein but not in the complete receptor. The
aspartic acid residues of AF-1 were also important for activation of
the RAR
promoter. These results are unexpected as Nagpal et
al.(35) have shown that the A region of RAR
does not
contribute to RAR
promoter activation whereas we observed that
every mutant in which parts of AF-1 are deleted or mutated caused a
decrease in RAR
-dependent RAR
promoter activation, comparable
with the mutant RAR
lacking the complete AB region ( Fig. 1and Fig. 4; data not shown). We, however, used
RAC65 cells in these experiments, whereas Nagpal et al.(35) used COS cells, probably explaining the differences
in the role of AF-1 in RAR
2 promoter activation. This was
confirmed by transfection of these mutant receptors in COS cells with
both the RAR
promoter and with
RARE-tk-CAT as a reporter
showing that all mutants activate these promoters to a similar extent
(data not shown). Furthermore, we cannot exclude that regions within
the B region contribute to the activity of AF-1 although no indications
for the presence of an autonomous AF within this region were
found(30) . From these data we conclude that for activation by
AF-1 the negatively charged amino acids are required, both as an
autonomous AF and also when present in the full-length receptor.
Figure 6:
Homology comparison of RAR with other
activators showing both amino acid and structural homology with VP16
and other (acidic) activators. Sequence alignment of various activators
showing that homology is observed with AF-1 of RAR
when RAR
AF-1 is aligned around the hydrophobic residues of VP16 (5) and
other activators. The hydrophobic residues (
) A, F, I, L, M, V,
and Y are boxed, the negatively charged residues(-) D
and E are shown in bold.
Figure 5:
Overexpression of AF-1 can repress the
activity of acidic activators. A, transfection of 200 ng of
GAL-DBD fused activator AF-1 (1-76), VP16 (414-491), or
RelA (43) together with the indicated amounts (µg) of
cytomegalovirus-driven HA-RAR
E or empty CMV4 (total of 5.0
µg) and a GAL-responsive reporter (5 µg) in COS cells, in the
presence of 1 µM RA and 0.1 µM dexamethasone
in case of GAL-DBD GR AF-2. Results are presented as relative activity
compared to the activity in the absence of HA-RAR
E. B, 50 ng of the indicated GAL-DBD fused activators were
transfected together with 1.0 µg of the indicated SV40-driven
RAR
expression constructs with a GAL-responsive reporter (5
µg) in COS cells in the presence of 1 µM RA and 0.1
µM dexamethasone in the case of GAL-DBD GR
AF-2.
Comparison of
several domains which have been shown to belong to the class of acidic
activators (VP16(5) , RelA,(42, 43) ,
Rta2(44) ) revealed a striking similarity as depicted in Fig. 6. The positions of hydrophobic () and negatively
charged(-) residues is mostly conserved, with hydrophobic
residues at positions 2, 5, 7, or 8 while generally at least two of the
residues between these hydrophobic amino acids are negatively charged.
This pattern was also found in RAR
2 and RAR
2, suggesting that
these receptors also contain an autonomous activator at the N terminus
that belongs to the class of acidic activators. Also in the activation
domains of C/EBP, c-Fos, E1A, and GR, a sequence of hydrophobic
residues around the aspartic acid and/or glutamic acid residues was
found.
In this paper, we show that the autonomous activation
function (AF-1) present at the N terminus of RAR2, located between
amino acids 1 and 32, is an acidic activator. This is supported by a
number of observations. First, the activity of this activator is
dependent on the presence of three aspartic acid residues, and
hydrophobic residues are also required for activity. The behavior of
other mutant activators is in agreement with the working mechanism of
acidic activators as mutation of all nonhydrophobic/negatively charged
amino acids were permissive. Furthermore, squelching experiments
indicate that overexpression of an AF-1-containing construct interferes
with the activity of VP16 and the recently characterized acidic
activator TA
of RelA. These activators all share the
ability to activate transcription synergistically upon multimerization
of binding sites(39, 42, 43) .
Finally, we observed sequence similarity between this activator
and several other acidic activators, in which the position of critical
hydrophobic and negatively charged residues is conserved.
The
relatively high activity of VP16 and TA of RelA compared
with AF-1 of RAR
(at least 10 times less active) can be explained
by the presence of two or more regions involved in activation (40, 42, 43) and also by the presence of more
negatively charged amino acids in VP16 or RelA(43) . In the
case of RAR
, there are also two regions which contribute to the
activity of AF-1 including the region around Asp-3 and Asp-6 and the
region around Asp-17. The first region is homologous with acidic
activators, whereas in the latter region the presence of only negative
and hydrophobic residues was observed. Although we do not know how the
latter region is contributing to activity, point mutants (D17A;
L16,F18D) as well as deletion constructs (
11-22;
11-76(30) ) indicate that it does contribute to the
activity of this activator.
Figure 7:
Projection of AF-1 and VP16 in a helical
wheel model. The regions of RAR and VP16 shown to be involved in
transactivation and predicted to form an
-helix are projected
within a helical wheel model(59) . Numbers represent
the amino acid numbers of RAR
AF-1. The smaller letters represent the activation domain of VP16 aligned as shown in Fig. 6; - and
indicate that these positions, based
on the alignments of Fig. 6, are generally negatively charged or
hydrophobic, respectively.
Recently,
the activation function present in the C-terminal part of the
hormone-binding domain (AF-2) of members of the steroid/thyroid hormone
receptor superfamily has been
characterized(60, 61, 62, 63, 64) ,
shown to depend also on the presence of hydrophobic and negatively
charged residues, and was proposed to form an -helix as
well(62) . The position of these residues is, however,
different in AF-1 (
Dx
D
) as compared to AF-2
(
xE
). Furthermore, the presence of glutamic
acid cannot be altered to aspartic acid (AF-2: 61, 63) and vice versa
(AF-1: D3E,D6E) without a decrease in activity. Finally, squelching
experiments showed that the activity of AF-2 cannot be repressed by
overexpression of AF-1 and vice versa (Fig. 5B).
Together, these findings strongly suggest that these activators
function by different mechanisms, and each fulfill a different role in
the retinoid response. This is confirmed by the observation that AF-1
and AF-2 contribute differently to the activation of various
RA-dependent promoters(35) . The characterization of the two
activation functions present in these receptors will be helpful in
achieving a better understanding of the mechanism of action of these
receptors in vivo.