(Received for publication, February 22, 1996)
From the
The 9-cis-retinoic acid (9cRA)-inducible enhancer of
the rat cellular retinol-binding protein type II gene (CRBP II) was
shown to be differentially regulated by the murine retinoid X receptor
(RXR
) as compared with RXR
. Transient transfection
assays performed in NIH 3T3 fibroblast cells demonstrated that RXR
yielded a high level of 9cRA-dependent transcription of a reporter gene
linked to the CRBP II enhancer, when compared with RXR
. This
effect was cell type-dependent, since both receptors elicited
comparable transcriptional activation of the same reporter in P19
embryonal carcinoma cells. To further explore the structural
determinants responsible for the differences between these two
receptors, a series of chimeric receptor constructs were made.
Co-transfection assays utilizing these chimeras demonstrated that both
the N terminus and the hinge region connecting the DNA binding domain
with the ligand binding domain of RXR
were responsible for the
high level of 9cRA-dependent transcription observed in NIH 3T3 cells.
Furthermore, the hinge region of RXR
was shown to be necessary to
repress, in the absence of hormone, the transcriptional activation
function located in the N-terminal domain of RXR
. These results
stress the importance of functional links between different RXR domains
and suggest an RXR subtype and cell type-dependent specificity in the
control of the 9cRA response.
The molecular mechanisms by which members of the steroid and
thyroid hormone receptors superfamily regulate the expression of target
genes are still unclear. RXRs, ()in particular, exert
pleiotropic functions due to their ability to heterodimerize with a
variety of other receptors within this family, including retinoic acid
receptors, thyroid hormone, and vitamin D receptors. Thus, RXRs are
assumed to play a central role in modulating the cellular response to
multiple signaling
pathways(1, 2, 3, 4, 5, 6, 7) .
In addition, RXR homodimers can transactivate RXR-responsive elements
(RXREs) such as that found within the rat CRBP II
gene(8, 9, 10) .
A further element of
complexity is added by the presence of multiple subtypes of the
receptors (,
, and
) that show dissimilar
transcriptional activities on the same response element(11) .
In addition, murine RXR receptor subtypes have been shown to be
differentially expressed during development (see (12, 13, 14) for reviews). However, the
structural feature(s) involved in subtype-specific gene regulation are
unknown.
Nuclear hormone receptors have a modular structure that consists of at least five regions (denoted A-E in Fig. 2; for reviews see (13, 14, 15) ). The most evolutionary conserved regions are the DNA binding domain (DBD, region C) and the ligand binding domain (region E). The C region directly contacts their cognate response elements, while the E domain is responsible for ligand binding, transcriptional activation function (AF2), and homo- and heterodimerization(1, 2, 3, 4, 5, 6, 7, 13, 14, 15, 19) . The D domain (hinge region) exhibits much less sequence similarity among receptor subtypes and has recently been shown to play a role in the specificity and polarity of binding of receptor heterodimers to DNA response elements(14, 15, 16, 17, 18, 24, 25) . The N-terminal or A/B domain appears to be partially responsible for specific differences among retinoic acid receptor subtypes(11, 19) . This region contains a transactivation domain function (AF-1), which can synergize with AF-2, located in the ligand binding domain (reviewed in (14) ).
Figure 2:
Chimeric constructions between RXR
and RXR
subtypes. A, homology between domains of RXR
and -
. Shown is a schematic depicting the percent of amino acid
homology (numbers inside of boxes) between the five
domains, shown by capital letters and numbers, of RXR
subtypes and the exact positions (marked by amino acid number and
endonuclease restriction sites) utilized to make the chimeras shown in
part B. B, diagrammatic representation of chimeric
constructs. C, amino acid sequence of the C end of chimeric (Chim) part 2 (open region), chimeric part 3 (shaded region), chimeric part 4 (open region), and a
portion of chimeric part 5 (shaded region).
In
this report we demonstrate that RXR and RXR
differentially
transactivate the RXRE from the rat CRBP II promoter in NIH 3T3
fibroblast cells but similarly in P19 embryonal carcinoma cells. We
have tested a series of chimeric receptors to elucidate the domains
responsible for the differences in transcriptional activation. Our
results showed that both the A/B and D domains are involved in
dictating the output of the cell-dependent transcriptional response.
Hence, these two regions cooperate with each other and potentially with
other cell-specific factors to maintain the specificity of the
response, thus providing the functional elements necessary for
eliciting subtype-specific and cell type-specific transcriptional
regulation.
Several reports have suggested that there may be RXR
subtype-specific differences in 9cRA-dependent transcriptional
activation on particular response
elements(11, 12, 19) . We compared the
transcriptional activation mediated by RXR and RXR
in
transient transfection assays in NIH 3T3 cells cotransfected with
either receptor and a reporter construct containing the luciferase
reporter gene linked to the CRBP II RXRE and the thymidine kinase basal
promoter (Fig. 1). In the presence of 9cRA, RXR
was 20-fold
more efficient in transactivating the CRBP II response element than
RXR
. Western immunoblot analysis performed after transfection of
RXR
and RXR
constructs containing C termini tagged with flag
epitope demonstrated that the difference in transcriptional activation
could not be attributed simply to differences in the levels of
expression of these receptors (data not shown). We also examined the
transcriptional activation in P19 cells. Interestingly, both RXR
and RXR
showed very similar levels of transcriptional activation
in P19 cells (Fig. 1).
Figure 1:
Cell type and subtype-specific
transcriptional activation of RXR and RXR
on the CRBP II
response element. NIH 3T3 and P19 cells were cotransfected by
electroporation or by using LipofectAMINE, respectively, with RXR
or RXR
expression plasmid and CRBPII(2)tk-luc in the presence (open bars) and absence (solid bars) of 9cRA. A
cytomegalovirus
-galactosidase plasmid was also included as an
internal control. The luciferase activity was determined on cell
extracts after 60 h (NIH 3T3) or 24 h (P19). Results
are shown as the activity relative to the reporter construct alone in
the presence of the empty expression vector
(RSVneo).
In order to better understand the structural requirements underlying the differences in transcriptional activation observed in NIH 3T3 cells, we constructed a series of chimeras between these two receptors (Fig. 2). Each receptor was subdivided into five parts. Parts 1, 2, 3, 4, and 5 correspond to the A/B, C, N and C termini of D, and the E domain, respectively. The expected molecular masses for the chimeric receptors were demonstrated following separation of in vitro transcription-translation products by SDS-polyacrylamide gel electrophoresis (data not shown). Finally, the functional integrity of each receptor was confirmed by transfection of P19 embryonal carcinoma cells. Each chimera in P19 cells showed comparable levels of transcriptional activation, differing less than 2-fold, in the presence of 9cRA on the CRBP II response element (Fig. 3).
Figure 3:
Transcriptional activation of the CRBP II
response element by RXR/RXR
chimeric receptors in P19 cells.
RXR chimeric expression plasmids were transfected into P19 cells with
the CRBPII(2)tk-luc reporter plasmid in the absence (solid
bars) or presence (open bars) of 9cRA. Transfection was
performed as described under ``Experimental
Procedures.''
Next, we tested each chimera in NIH 3T3
cells to identify the functional determinants responsible for the
differences between the two receptors (Fig. 4). As shown in Fig. 1, RXR was approximately 20-fold more active than
RXR
under identical conditions of transfection (compare lane 1 with 2 in Fig. 4A). This difference was
independent of part 2 (the C domain) since the exchange of the RXR
part 2 with the corresponding part 2 of RXR
(RXR
[
]) was as efficient as the
RXR
(compare lane 1 with 15 in Fig. 4A). Similarly, the RXR
part 2 substituted
for the RXR
part 2 in the context of the RXR
receptor
(RXR
[
]) (compare lane 2 with 9). This result was expected, since the two
receptors are more than 95% homologous in the C domain corresponding to
DBD (Fig. 2A). However, it was not possible to
completely rule out any involvement of part 2, since
RXR
[
] gave lower transactivation
than RXR
[
] (compare lane 13 with 14), suggesting that the C domain may contribute to
optimal folding.
Figure 4:
Analysis of transcriptional activation of
RXR and RXR
domains in NIH 3T3 cells. A, each RXR
chimeric expression plasmid was transfected into NIH 3T3 cells with the
CRBP II reporter plasmid in the absence (solid bars) or
presence (open bars) of 9cRA. B, enlargement of
transcriptional effect in the absence of hormone. Transfection was
performed as described for Fig. 1and under ``Experimental
Procedures.''
In agreement with a previous report(23) ,
our results point out that the A/B domain of RXR is absolutely
required for achieving the high level of transcriptional activation
observed with RXR
[
], since, when
partially deleted (RXR
[
]), a
4-fold decrease in transcriptional activation was observed (Fig. 4A, compare lane 1 with 3). In
addition, the A/B domain (part 1) of RXR
could not functionally
substitute for that of RXR
(RXR
[
]) (compare lane 1 with 13). These results confirm an earlier report (19) suggesting that the A/B domain of RXR
, but not
RXR
, exhibits a transcriptional activating function (AF-1).
The
A/B domain of RXR, however, was not sufficient to induce the high
level of 9cRA-dependent reporter activation observed with
RXR
[
], when linked to the C terminus
(D and E domains, parts 3, 4, 5) of RXR
(RXR
[
]) (Fig. 4A,
compare lane 6 with 1). In addition, the C terminus
of RXR
mediated slightly elevated transactivation above the levels
found for RXR
, when linked to the RXR
A/B domain
(RXR
[
]) (compare lane 2 with 14). Taken together, these results imply that both
the A/B domain and the C terminus of RXR
are necessary for high
transcriptional activation of the CRBP II RXRE in NIH 3T3 cells.
Next, we defined the functional part of the RXR C terminus
involved in the observed effects by testing chimeras that exchanged
both the D (parts 3 and 4) and E (part 5) domains. When the E domain of
RXR
was switched with that of RXR
, the resultant chimera,
RXR
[
] gave nearly the equivalent
amount of transcriptional activity as
RXR
[
] (compare lane 7 with 1). Therefore, the E domain does not appear to contribute to
the observed receptor subtype specificity.
Several lines of evidence
derived from our experiments suggest that the D domain (containing
chimeric parts 3 and 4) and the A/B domain are critical for the proper
functioning of RXR in NIH 3T3 cells. When the D domain of
RXR
[
] is replaced by the D domain of
, RXR
[
], the reporter activity
decreases about 75% (compare lane 7 with 6).
Furthermore, the D domain of RXR
functioned less efficiently when
linked to the RXR
A/B domain (compare lane 8 with 13). Thus, the maximum effect was only observed when the A/B
and the D domains were both from RXR
(compare lane 6 with 7 and lane 7 with 12). Taking into account
that the E domains are fully exchangeable, these results strongly imply
that the D and A/B domains function in concert to achieve optimal
transcriptional activation in NIH 3T3 cells.
Based on these results, we further dissected the D domain to identify the portion responsible for this effect. Region D includes the A box and ends at the N terminus of the E domain (Fig. 2, B and C). The A box is a seven-residue region that was originally shown to be critical for binding of the orphan receptor NGFI-B to its response element(14, 15, 16, 17, 18, 24) . The resolution of the three-dimensional structure of the RXR DBD has shown that the A box is included in a helical region beginning after the second zinc finger of the DBD(25) . This helix includes also the so-called T box that forms a dimerization interface critical in the binding of homo- and heterodimeric DBDs to some response elements(14, 15, 16, 17, 18, 24, 25) .
New chimeras, which exchanged the N terminus, including the A box
(part 3), or the C-terminal region (part 4) of the D domain (Fig. 2C), were tested for their transcriptional
activation. When part 4 of RXR was used to replace the
corresponding portion in RXR
(RXR
[
]), less than 2-fold reduction
in overall reporter activity was observed (compare lanes 1 and 4 in Fig. 4A). However, a more dramatic effect
was seen when part 3 of RXR
was substituted for the analogous
region of RXR
(RXR
[
]). Although
the transactivation in the presence of hormone was reduced less than
2-fold (Fig. 4A, compare lanes 1 and 5), a 10-fold increase in transactivation in the absence of
hormone was detected (Fig. 4B, compare lanes 1 and 5). Interestingly, this increase in basal activity
occurred only when part 3 of the
receptor was substituted for the
same part in a receptor containing RXR
A/B domain
(RXR
[
]) and not when linked to the
RXR
A/B domain (RXR
[
]) (compare lanes 6 and 9 in Fig. 4B). In
addition, when the E domain (part 5) of the
receptor was
substituted by the E domain of the
receptor
(RXR
[
]), an increase of basal
activity, although weaker than the chimeras described above, was
observed (compare lane 7 with 1, Fig. 4).
Nevertheless, the hormone-independent enhancement of basal activity
observed in these chimeras demonstrates the functional interaction of
the D domain and to some degree the E domain with the A/B domain.
Therefore, regions 3 in the D domain and the E domain must be from
RXR
to achieve repression of the N-terminal AF-1 in the absence of
hormone (Fig. 4A, lane 1).
The importance
of the RXR part 3 for 9cRA-dependent maximal activity in NIH 3T3
cells, in the absence of an increase in basal activity, was also
demonstrated using chimeras containing the RXR
A/B domain. When
construct 11 (RXR
[
]) was tested in
NIH 3T3 cells, an 8-fold 9cRA-dependent induction was observed (Fig. 4A, lane 11). However, when part 3 of
this construct was replaced with the corresponding part from RXR
(RXR
[
]), a further increase of the
reporter activity (up to 70-fold 9cRA-dependent induction) was observed (lane 12 compared with lane 11). Although the levels
of reporter activity observed in the presence of
RXR
[
] is much lower than the levels
observed with RXR
[
], the latter
containing the A/B domain of RXR
, it demonstrates again the
importance of part 3.
The fact that the D domain of RXR is
involved in both the inhibition of AF1 in the absence of hormone and in
hormone-dependent inducibility adds to the growing list of regulatory
functions attributed to this region. Other than the T and A boxes,
immediately 3` of the A box is a 70-amino acid region, which has been
shown to be responsible for the divergent transcriptional activities
between the RAR
, -
, and -
subtypes in response to
various retinoids (25) .
The data presented here also
support the conclusion that there is an AF1 present in RXR but not
in RXR
(11, 19) . Interestingly, Chen and
Privalsky (23) have provided evidence that RXR
binds to
the CRBP II RXRE as a tetramer, while the A/B domain of RXR
is
involved in inhibiting the tetrameric binding of RXR
to the CRBP
II response element. Differences in DNA binding properties between the
receptors have been invoked as the cause for the different
transactivation levels observed in SL-2 cells(23) .
Our data
indicate that, in addition to the intrinsic DNA binding properties of
the receptors, interactions with a cell-specific factor(s) or cell
type-specific post-translational modifications may be involved, given
the differences in response mediated by RXR and RXR
observed
in P19 and NIH 3T3 cells. Finally, it should be emphasized that,
although this study addresses the activities of RXR homodimers, a
differential role of RXR subtypes and cell type-specific differences in
transcriptional activation might also be found in the regulation of
gene expression mediated by RXR heterodimerization with other nuclear
receptors.