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| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cell Growth & Differentiation |
Section of Microbiology, Division of Biological Sciences, University of California at Davis, Davis, California 95616
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and MethodsReferences |
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with portions of the
promyelocytic leukemia protein (PML) or promyelocytic leukemia
zinc-finger protein (PLZF). The wild-type RAR readily forms
heterodimers with the retinoid X receptors (RXRs), and these RAR/RXR
heterodimers appear to be the principal mediators of retinoid signaling
in normal cells. In contrast, PML-RAR and PLZF-RAR display an
enhanced ability to form homodimers, and this enhanced homodimer
formation is believed to contribute to the neoplastic properties of
these chimeric oncoproteins. We report here that the DNA recognition
specificity of the RXR/RAR heterodimer, which is presumed to be
the dominant receptor species in normal cells, differs from that of the
PML-RAR and PLZF-RAR homodimers, which are thought to
prevail in the oncogenic cell. We suggest that differences in
target gene recognition by the normal and oncogenic RAR proteins may
contribute to the leukemogenic phenotype. |
Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Mutant nuclear hormone receptors are involved in several forms of
neoplastic diseases. For example, aberrant forms of RAR are found in
over 95% of patients with APL (17, 18, 19, 20, 21, 22, 23, 24, 25)
. These aberrant
proteins are the result of chromosomal translocations wherein a portion
of the NH2-terminal region of RAR is replaced
with novel NH2-terminal sequences (Refs.
19, 20, 21, 22, 23, 24, 25
; Fig. 1B
). Although the location of the breakpoint in the RAR
sequence is highly conserved in these leukemias, the nature of the
novel NH2 terminus can differ. The clinically
most common form of APL is associated with a t(15;17) chromosomal
translocation, resulting in expression of a PML-RAR chimeric
receptor (17, 18, 19, 20, 21)
. Less frequently observed are t(11;17),
t(5;17), or t(11;17) chromosomal translocations, which result in
PLZF-RAR, NPM-RAR, or NuMA-RAR chimeric receptors,
respectively (18, 19, 20, 21)
. Intriguingly, the PML, PLZF, NPM,
and NuMA open reading frames do not share significant sequence homology
with one another and have distinct functions in the normal organism
(18, 19, 20, 21)
. The PML-RAR, PLZF-RAR, NPM-RAR, and
NuMA-RAR chimeras themselves appear to play a central role in the
etiology of APL, although other factors may also contribute. When
introduced into transgenic mice, for example, PML-RAR and
PLZF-RAR constructs induce myeloproliferative disorders that can
advance to neoplasias similar in phenotype to those observed in human
patients (26, 27, 28, 29)
.
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and PLZF-RAR chimeras can function as dominant
negative inhibitors of normal RAR function, apparently due to
defects in the ability of these chimeric receptors to release
corepressor under physiological hormone concentrations (28
, 30, 31, 32, 33)
. These defects in corepressor release are closely
associated with the leukemogenic phenotype, suggesting that repression
of normal RAR function plays an important role in oncogenesis. However,
several observations suggested to us that the DNA recognition
properties of PML-RAR and PLZF-RAR might be different from those
of wild-type RAR and that, in addition to defects in corepressor
release, the chimeric oncoproteins might also display a distinct target
gene specificity from that of the normal RAR progenitor. First, we
and others have noted that the NH2 terminus of
the nuclear hormone receptors, which is lost in the leukemogenic RAR
variants, plays an important role in defining the DNA recognition
properties of the normal nuclear hormone receptors
(34, 35, 36, 37, 38, 39)
. Second, it has been noted that the chimeric
RAR oncoproteins exhibit an enhanced ability to bind to DNA as
homodimers, rather than heterodimers with RXR, and that this enhanced
homodimerization appears to correlate with oncogenesis
(40, 41, 42, 43)
. This enhanced homodimerization by RAR chimeras
has been implicated in the aberrant corepressor interaction properties
of these oncogenic receptor derivatives (43)
, but any
alteration in the dimerization properties of the chimeric RAR
oncoproteins is also likely to influence their DNA recognition
properties.
In the present study, we show that the in vitro DNA binding
specificities of PML-RAR and PLZF-RAR were indeed modestly
altered from that of RAR when these receptors were tested as
homodimers. More significantly, perhaps, we found that the
heterodimeric interaction of RAR with RXR conferred an enhanced
binding to a broader range of DNA sequences relative to that seen for
the corresponding homodimers. The wild-type RAR is believed to
function in cells almost exclusively as a heterodimer with RXR
(44, 45, 46, 47)
and would therefore be expected to display this
broadened range of DNA recognition characteristic of the RXR/RAR
heterodimer. In contrast, PML-RAR and PLZF-RAR have been proposed
to function in leukemogenesis as homodimers or perhaps as higher order
homo-oligomers (40, 41, 42, 43
, 48)
, indicating that PML-RAR
and PLZF-RAR in cells would exhibit the more restrictive DNA
recognition specificity that we observe for homodimers in
vitro. Results from our in vivo transactivation studies
are consistent with this proposal: transcriptional regulation by RAR
is enhanced by cointroduction of RXR; whereas transcriptional
regulation by PML-RAR is impaired by cointroduction of RXR. Our
results therefore suggest that not all genes regulated by RXR/RAR
in normal cells may be recognized or subject to repression by the
chimeric receptor homodimers found in APL. PML-RAR and PLZF-RAR
homodimers may therefore participate in oncogenesis by aberrantly
regulating only a subset of the total gene repertoire normally
controlled by RXR/RAR heterodimers.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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and PLZF-RAR
Homodimers Are Similar but not Identical to Those of RAR Homodimers.s involved in APL might confer an altered DNA half-site
recognition on these chimeric receptors. In prior experiments on T3Rs,
RARs, and retinoid orphan receptors, alterations in the
structure of the receptor NH2 terminus were
manifested as changes in the ability of the receptor to recognize DNA
elements bearing nonconsensus half-sites (34, 35, 36, 37, 38, 39)
. We
therefore tested the ability of RAR, PML-RAR, and PLZF-RAR to
bind to an assortment of different oligonucleotide probes, each bearing
a distinct half-site sequence. Many nuclear hormone receptors recognize
extended half-sites that are 8 or more bases in length (reviewed in
Ref. 1
). A P-box recognition helix in the receptor’s
zinc-finger domain contacts the major groove of the DNA over bp 1–6 in
the half-site, whereas a more COOH-terminal A-box, which is also
present in many nuclear receptors, can make additional contacts in the
minor groove with bases located at positions -2 and -1 of the
half-site [Fig. 1A
; we use this numbering system to permit
comparison with earlier work based on the premise of a hexanucleotide
half-site (1)
]. We therefore began our experiments with
an optimized consensus half-site sequence for RAR derived by a PCR
selection procedure and consisting of an AGAGGTCA sequence
(8)
, and we systematically altered each position to each
of the alternative three bases. Each of the 25 possible permutated
half-sites was synthesized as a direct repeat element with a 5-bp
spacer, an arrangement found both in the experimentally defined
consensus sequence and in many naturally occurring response elements
for RAR (Fig. 1A)
. Corresponding bp changes were made
concurrently in both half-sites in the direct repeat element.
In our first series of experiments, we tested the ability of human
RAR to bind to our panel of consensus and variant half-site DNA
elements by using a gel electrophoretic mobility shift assay. Using
RAR and the consensus AGAGGTCA repeat element as a probe, a single
specific protein-DNA complex was detected by this methodology (Fig. 2
A, Lane 2). This complex was specifically supershifted using
anti-RAR antibodies and not by anti-RXR antibodies (Fig. 2
A, Lanes 3 and 4), whereas an equivalent complex
was not detected with otherwise comparable protein preparations lacking
RAR (Fig. 2
A, Lane 1). We therefore identify this
protein-DNA complex as representing a RAR/RAR homodimer bound to
the DNA. This characterization is also consistent with the apparent
mobility of the complex in the gel, the alteration in mobility of the
complex on formation of a RXR/RAR heterodimer (Fig. 2B)
, and the inability of RAR to bind detectably to DNA
elements bearing only a single half-site (i.e., as a protein
monomer).4
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to bind to the corresponding response element (Fig. 3A)
. This ability of RAR to accept nonconsensus sequences
was particularly evident for substitutions at the -2 and -1 positions
of the half-site, all of which were bound with at least 50% of the
ability of the consensus half-site element (Fig. 3A
;quantified in Fig. 4A
). Indeed, under our conditions, a -1A half-site
substitution element was bound somewhat better than was the consensus
element itself. In contrast, base substitutions at the +2 position were
uniformly destabilizing, with any departure from consensus at this
location significantly reducing RAR binding (Figs. 3A
and 4A)
. Substitutions at the remaining +1, +3, +4, +5, and +6
positions displayed an intermediate phenotype: certain substitutions at
these positions exerted relatively minimal effects on DNA binding by
RAR (e.g., the +3T element); whereas other substitutions
at the same sites exhibited strongly destabilizing effects on RAR
binding (e.g., the +3A or +3C elements). The specific
activities of the different probes were comparable to one another, and
free probe was in excess in all reactions.
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in our single point assay. In all cases, these
titrations paralleled the results obtained by single point assays:
RAR bound very weakly to the 2C element, bound with intermediate
strength to the 5G and 5T elements, bound stronger still to the 3T
element, and bound best to the consensus element (Fig. 5, A and B)
. We conclude that RAR, in addition
to binding with high efficiency to the consensus AGAGGTCA element, is
able to also recognize a select series of half-site sequences that
differ from the consensus by single-bp substitutions.
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and PLZF-RAR proteins. Using the same electrophoretic mobility shift
assay conditions as described for RAR, both PML-RAR and
PLZF-RAR generated specific protein-DNA complexes on the consensus
AGAGGTCA direct repeat element that were not observed using control
nuclear extracts lacking the corresponding receptors (Fig. 2
A,
Lanes 5, 8, and 1, respectively). In contrast to
RAR, however, PML-RAR and PLZF-RAR each generated two distinct
protein-DNA complexes (Fig. 2
A, Lanes 5 and 8),
one of which entered the gel and migrated at the position expected for
the corresponding receptor homodimer (Fig. 3, B
and C,
arrowheads), and the other of which remained near the very top of
the electrophoretogram (Fig. 3, B and C
,
arrows). The presence of a PML-RAR or PLZF-RAR complex
migrating at or near the top of the electrophoretic resolving space has
been reported by other researchers and is due to the formation of high
order oligomeric receptor complexes greater than dimer in size
(e.g., Refs. 41
and 49, 50, 51
). We
therefore report our results for the more readily resolved homodimeric
complexes, the identity of which was confirmed by supershift with
RAR-directed antisera (Fig. 2
A, Lanes 6 and
9). Nonetheless, when quantified, the amount of high
molecular weight complex closely paralleled the amount of the
homodimeric species for the different response elements (data not
shown); therefore, interpretation of our results would not differ were
the high molecular weight complexes included in our analysis.
Overall, the ability of the PML-RAR and PLZF-RAR proteins to
recognize the different half-site elements generally paralleled the
pattern observed with the wild-type RAR (Figs. 3
and 4
, compare
A, B, and C). Thus, substitutions at
the -2 and -1 positions had relatively modest effects on the binding
of either PML-RAR or PLZF-RAR to the DNA, whereas any base change
from the consensus sequence at the +2 position severely destabilized
the ability of both PML-RAR and PLZF-RAR to bind to the
corresponding DNA probes (Fig. 3, B and C
;
quantified in Fig. 4, B and C
). Also in common
with RAR, the ability of PML-RAR and of PLZF-RAR to bind to
half-sites bearing substitutions at the +1, +3, +4, +5, and +6
positions depended on the precise nature of the substituted base. The
pattern observed in these single point analyses was recapitulated in
titration experiments with the representative consensus, 2C, 3T, and 5G
DNA elements (Fig. 6, A and C)
.
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and PLZF-RAR have DNA
recognition properties similar to those of RAR. However,
close inspection of the PhosphorImager scan (Fig. 3)
and quantification
of the data (Fig. 4)
revealed several consistent differences in the DNA
binding properties of PML-RAR and PLZF-RAR relative to those of
the wild-type RAR. For example, wild-type RAR exhibited an
enhanced ability to bind to elements bearing substitutions at the +2
and +3 positions, as well as to the +SA and +6T elements, compared with
PML-RAR and PLZF-RAR (Fig. 4)
. Interestingly, differences between
the abilities of PML-RAR and PLZF-RAR to recognize individual DNA
sequences were also discernible and were manifested primarily as a
reduced ability of PLZF-RAR to bind to several of the base
substitutions tested.
RXR/RAR Heterodimers Recognize a Distinct Panel of Half-Site
Sequences Compared to RXR or RAR Homodimers.
RARs can form heterodimers with other members of the nuclear hormone
receptor family. RXRs are particularly important heterodimer partners
for RARs, and RXR/RAR heterodimers exhibit both an enhanced overall DNA
binding affinity and enhanced transcriptional regulatory properties
when compared with homodimers of the same receptors (1, 2, 3, 4, 5, 6
, 44, 45, 46, 47)
. PML-RAR and PLZF-RAR have been shown to retain at
least some of this ability to heterodimerize with RXRs
(40, 41, 42)
, leading us to examine the effect of this
heterodimerization on DNA recognition by these different receptor
variants.
We first tested the ability of RXRs to bind to our different DNA probes
in the absence of RARs. Although RXR homodimers bind with highest
avidity to DNA elements composed of a DR-1 spacing, we were able
to detect a modest binding of RXR to our DR-5 consensus element at
high receptor concentrations (Fig. 2A
, compare Lane
11 with the nonrecombinant extract, Lane 1). We
interpret this complex as being a RXR/RXR homodimer, and it was
shifted in mobility by anti-RXR antibodies but not by the control
anti-RAR antibodies (Fig. 2
A, Lanes 12 and
13). Significantly, the DNA recognition properties of RXR
homodimers were very different from the pattern seen for RAR,
PML-RAR, and PLZF-RAR homodimers. For example, whereas RAR and
its oncoprotein derivatives could accommodate a variety of base
substitutions at positions +3, +4, +5, and +6 of the DNA half-site,
these same substitutions virtually eliminated binding by RXR
(compare Figs. 4A
and 7A
). Thus, different
nuclear hormone receptors can display very different DNA recognition
specificities under the conditions used here.
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with RAR resulted in the appearance of a strong
electrophoretic shift complex migrating at a position distinct from
that of either the RAR homodimer or the RXR homodimer (Fig. 2B
, compare Lane 4 with Lanes 2 and
3). This novel complex migrated at a position consistent
with that of a receptor heterodimer and contained both RAR and
RXR, as demonstrated by its ability to be supershifted by the
appropriate antisera (Fig. 2
B, Lanes 5 and 6).
This RXR/RAR heterodimer exhibited two features distinct from
those of the corresponding homodimers: (a) consistent with
prior work, the overall affinity of the RXR/RAR heterodimer for
the consensus AGAGGTCA repeat element was greater than that of the
corresponding homodimers (Fig. 2B
, the RAR and RXR
protein extracts were used at a 4-fold lower concentration in the
heterodimer lane than in the homodimer lanes but generated comparable
levels of complex); and (b) distinct from this increase in
absolute affinity for the consensus element, the RXR/RAR
heterodimer also displayed reproducible changes in its relative ability
to bind to the different half-site variants compared with either the
RAR/RAR or RXR/RXR homodimers (compare Figs. 4A
, 7A
, and 8A
; the same results are expressed as a
difference plot in Fig. 7B
). It is important to note that binding to the consensus
RARE was defined as 100% for each experiment; therefore, differences
in the ability of homodimers and heterodimers to bind to the individual
DNA variants represent differences in relative avidity for the
different DNA sequences and are distinct from the overall increased
affinity displayed by the heterodimer for the consensus DNA sequence.
For example, although the RXR/RAR heterodimer bound to the
consensus DNA sequence better than did the RAR/RAR homodimer, the
heterodimer actually bound comparatively less well to the -1A element
than did the homodimer (compare Figs. 7A
, 7B
, and 8A
).
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/RAR heterodimer
typically displayed an enhanced ability to bind to nonconsensus
half-site sequences relative to the RAR/RAR homodimer; this
broadened recognition specificity was most clearly observed for the
+4C, +4G, and +5T probes, with more subtle effects on recognition at
other positions also detected (Figs. 7A
, 7B
, and 8A)
. These differences in sequence specificity between homo-
and heterodimers were reproducible from experiment to experiment. We
also confirmed the results of these single point assays by performing
titration experiments using several representative DNA elements (Fig. 5, B and C
; note the enhanced relative binding of
the +5T and +5G DNA elements by the heterodimer compared with that of
the RAR/RAR homodimer). It is noteworthy that the DNA binding
pattern of the RXR/RAR heterodimer was not simply a summation of
the individual specificities of the RXR and RAR homodimers; for
example, the enhanced recognition of the +4C, +4G, and +5T elements by
the RXR/RAR heterodimer was not predictable from the
specificities of either of the corresponding homodimers (compare Figs. 4A
, 7A
, and 8A
).
RXR/PML-RAR and RXR/PLZF-RAR Heterodimers
Display a Broader DNA Recognition Specificity than Do the
Corresponding Homodimers but Form Inefficiently Compared with
RXR/RAR Heterodimers.
We next extended our analyses of heterodimer formation to the chimeric
RAR derivatives associated with APL. Addition of RXR to
PML-RAR resulted in the formation of a novel complex that was not
observed with the parental receptors when tested individually (Fig. 2
B, Lane 14, arrowhead) and was supershifted with antisera
to either RXR or PML-RAR (Fig. 2B
, Lanes 15
and 16). In keeping with the assignments of others
(40)
, we interpret this complex as representing a
RXR/PML-RAR heterodimer. Notably, PML-RAR formed heterodimers
less efficiently than did RAR, and a significant amount of residual
PML-RAR homodimer complex was observed even when PML-RAR was
mixed with large amounts of RXR (Fig. 2B
; data not shown).
In the case of PLZF-RAR, addition of RXR resulted in the
formation of three different presumptive RXR/PLZF-RAR heteromeric
species (Fig. 2
B, Lane 8) that could be quantitatively
supershifted with antibodies to either RXR or RAR determinants
(Fig. 2
B, Lanes 9 and 10). Based on the relative
migration, the antibody supershift, and the analysis of analogous
complexes by other researchers (41
, 42)
, the fastest
migrating heteromeric complex (Fig. 2
B, Lane 8, arrowhead)
was identified as a RXR/PLZF-RAR heterodimer. This
heterodimerization of PLZF-RAR with RXR on the consensus element
was also inefficient, resulting in a mixture of homo- and heterodimers
under the conditions used here (Figs. 2B
and 9B)
.
This enhanced ability of PML-RAR and PLZF-RAR to form homodimers,
as compared with RAR, has been reported by other researchers
(40, 41, 42)
.
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/PML-RAR and RXR/PLZF-RAR heterodimers
that did form under these conditions exhibited the broadened
ability to bind to nonconsensus half-sites that was observed for
RXR/RAR heterodimers (compare Fig. 8, B and C
, with Fig. 4, B and C
). This enhancement in the recognition of nonconsensus
half-sites, such as the +3T and +5G elements, was also observed in
titration experiments (Fig. 6)
. We conclude that the DNA binding
specificities of RXR/RAR, RXR/PML-RAR, and
RXR/PLZF-RAR heterodimers are very similar to one another but are
distinct from and typically more accepting of nonconsensus half-site
sequences than the DNA recognition specificities of the corresponding
receptor homodimers.
The difference in the half-site recognition specificity of receptor
homodimers and heterodimers was particularly evident for PLZF-RAR,
which formed a mixed population of homo- and heterodimers on the
consensus element even in the presence of high levels of RXR (Fig. 2B
, compare Lane 8 with Lane 7 and
note the absence of a supershift of the homodimer when using
RXR-directed antibodies; Lane 10). This feature served as an
internal control, permitting an accurate comparison of the formation of
PLZF-RAR homodimers versus RXR/PLZF-RAR
heterodimers on each DNA sequence variant under identical conditions in
the same binding reaction (Fig. 9A
; compare the strong homodimer formation on the -1A
element, for example, to the strong heterodimer formation on the 5T
element). This type of experiment was also quantified and is presented
as a difference plot (relative binding of the heterodimer minus the
relative binding of homodimers) for the different response elements
tested (Fig. 9B)
. We conclude that heterodimerization with
RXR alters the half-site recognition properties of RAR,
PML-RAR, and PLZF-RAR from that of the corresponding receptor
homodimers. Given that both PML-RAR and PLZF-RAR may function in
oncogenesis as homodimers (40
, 41
, 43)
, our results also
imply that these aberrant receptor homodimers have a DNA recognition
specificity distinct from that of the RXR/RAR heterodimers, which
are the prevailing species in the normal cell. A comparison of
the DNA binding specificities of RXR/RAR heterodimers with that of
PLZF-RAR homodimers is presented as a difference plot to illustrate
this phenomenon (Fig. 9C)
.
Transactivation in Vivo by RAR and PML-RAR
Largely Parallels but Is not Identical to Their Ability to Bind DNA
Elements in Vitro.
We next created a series of reporter constructs containing
representative DNA response elements (i.e., those that
displayed strong, medium, or weak receptor binding in vitro)
and tested the ability of RAR, PML-RAR, and PLZF-RAR to
modulate transcription in transfected cells. At physiological hormone
conditions (10-9 to
10-8
M all-trans retinoic acid), PML-RAR
and PLZF-RAR are unable to release from corepressor and function as
dominant negative inhibitors, interfering with gene activation by the
wild-type RAR (Refs. 40
and 50, 51, 52, 53
).
However, in the presence of higher concentrations of
all-trans retinoic acid
(10-7 to
10-6
M), PML-RAR releases from corepressor and
activates reporter gene expression, whereas PLZF-RAR remains a
dominant negative even at high hormone concentrations (30
, 31
, 52
, 53)
. A characterization of the DNA recognition properties of
PML-RAR or PLZF-RAR as dominant negative inhibitors
(i.e., assaying the ability to interfere with RAR at
nanomolar hormone concentrations) would require interpreting a complex
interaction between the DNA recognition properties of the
positive-acting RAR and those of the interfering, chimeric receptor.
To allow us to characterize the autonomous DNA recognition properties
of PML-RAR, we therefore used
10-7
M hormone and measured the ability of PML-RAR
to activate transcription from the different response element
reporters. PLZF-RAR, as anticipated, failed to activate
transcription of any of the reporter constructs tested even at
micromolar hormone concentrations and was therefore excluded from the
analysis (data not shown).
CV-1 cells have low but detectable levels of endogenous RAR activity
that can be observed as a hormone-dependent activation of the consensus
reporter element in the absence of exogenous receptor (Fig. 10
, NO RECEPTOR). As anticipated, introduction of exogenous
RAR into these cells further enhanced the ability of retinoid
hormone to activate the consensus reporter (Fig. 10
, RAR).
Consistent with the receptor binding properties we observed in our
in vitro DNA binding studies (Fig. 4A)
, the -1A
DNA element functioned as a RAR response element at levels
comparable with that of the consensus element, whereas the 2T
element, which was poorly bound by RAR in vitro, was
poorly activated by RAR in our transfection experiments (Fig. 10)
.
Also consistent with our in vitro DNA binding studies,
cointroduction of a RXR expression vector in our transfection
experiments enhanced the ability of RAR to activate the reporter
construct, and this was evident on all four DNA elements tested (Fig. 10
, RAR + RXR). These results suggest that RXR/RAR
heterodimers are indeed stronger transcriptional activators than the
corresponding homodimers and that heterodimer formation with RXR can
broaden the half-site recognition properties of RAR. Despite this
general correlation between DNA binding in vitro and
transactivation in vivo, some modest differences were noted.
For example, the 4G element, which was less efficient than the
consensus element at binding RAR in vitro, was equal or
better than the consensus element in mediating transcriptional
activation in the transfected cells. Other researchers have previously
noted similar discrepancies between DNA binding and transcriptional
activation by nuclear hormone receptors when tested on different
response elements (e.g., Refs. 54, 55, 56, 57
).
|
mediated strong transcriptional activation from both the consensus and
the -1A elements (Fig. 10
, PML-RAR). In fact, activation of
the reporter construct when PML-RAR was introduced alone was
consistently higher than that observed for RAR; this was
particularly notable for the 2T and 4G elements, which mediated
reporter gene activation at levels significantly greater than those
anticipated from our in vitro DNA binding studies (compare
Fig. 4B
with Fig. 10
). Immunoblot analysis confirmed that
the levels of expression of RAR and PML-RAR in the transfected
cells were very similar (Fig. 10
, inset). These results are
in agreement with prior work indicating that at high hormone
concentrations PML-RAR can function as a stronger transcriptional
activator than RAR (51)
. In notable contrast with the
results observed for RAR, cointroduction of RXR resulted in a
decrease, not an increase, in the ability of PML-RAR to activate
transcription, and this too extended to all four DNA elements tested
(Fig. 10)
. These results are consistent with our own studies and with
prior suggestions that PML-RAR may function as a homodimer and not
as a heterodimer in cells (40
, 41
, 43)
. |
Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Can Recognize a Spectrum of Distinct Half-site
Sequences. homodimer is able to recognize a wide range of response elements
representing single-base substitutions of the AGAGGTCA consensus
sequence. In fact, only the G at the +2 position of the consensus
half-site proved to be absolutely required for RAR binding,
with any other base substitution at the +2 position incompatible with
efficient RAR recognition. In contrast, other positions in the
half-site were able to accept at least one base substitution with
relatively minimal effect on RAR binding. The effect of
substitutions at the +1, +3, +4, +5, and +6 positions was dependent on
the precise base introduced, with certain substitutions at these sites
compatible with, and other substitutions highly disruptive of, DNA
recognition by RAR. Conversely, base substitutions at the -2
and -1 position were quite permissive, with virtually every
substitution at these positions compatible with RAR binding.
The ability to bind to a spectrum of half-site sequences might appear
inconsistent with structural studies indicating that very specific
amino acid-bp contacts can occur between nuclear hormone receptors and
their consensus DNA response elements. However, binding to nonconsensus
sequences appears, at least in part, to be accommodated by relatively
modest rearrangements of receptor conformation (58, 59, 60, 61)
.
This ability of RAR to recognize a spectrum of nonconsensus
artificial half-sites in our experiments is also consistent with the
diversity of half-site sequences found in naturally occurring retinoic
acid response elements (47)
. Our artificially permuted
half-site sequences emphasize, however, that not all single base
variations from the consensus sequence are compatible with response
element function, and they help define the set of half-site sequences
that are acceptable RAR binding sites. As such, our results
complement prior work defining optimal response element sequences and
may assist in the identification of authentic response elements in the
promoters of retinoid-regulated genes.
The DNA Recognition Specificities of PML-RAR and PLZF-RAR
Homodimers Are Similar but not Identical to That of RAR Homodimers.
The nature of the NH2 terminus of the T3Rs and
retinoid orphan receptor receptors plays an important role
in the ability of these receptors to recognize nonconsensus DNA
half-sites (34, 35, 36, 37, 38, 39)
. In fact, as observed here, the
replacement of the normal RAR NH2 terminus
with the novel PML or PLZF sequences does result in reproducible,
modest alterations in the DNA recognition properties of these chimeric
receptors, relative to those of RAR. These changes were manifested
principally as alterations in the recognition of base substitutions at
the +2, +3, +4, +5, and +6 positions of the half-site, and notably, the
PML-RAR and PLZF-RAR recognition patterns differed somewhat from
one another, as well as differing from that of the wild-type RAR.
The precise molecular mechanisms by which the receptor
NH2 terminus participates in DNA recognition by
nuclear hormone receptors have not yet been fully determined. Available
evidence suggests that the receptor NH2 terminus
may act by influencing the three-dimensional structure of the receptor,
thereby affecting the conformation of and precise DNA contacts made by
the regions of the receptor that interact directly with the response
element (34
, 37, 38, 39)
. The modest differences in DNA
recognition by PLZF-RAR relative to RAR and PML-RAR may
therefore reflect subtle effects on receptor conformation that are
conferred by the different NH2 termini of
these different receptor derivatives.
Heterodimerization with RXR Expands the Half-Site Repertoire.
Many nuclear hormone receptors efficiently heterodimerize with RXRs,
and the resulting receptor heterodimers exhibit enhanced DNA binding
and transcriptional regulatory properties compared with the parental
homodimers. RXR/RAR heterodimers have been proposed to be the
physiologically dominant receptor species in the biology of retinoid
signaling in many cell types (44, 45, 46, 47)
. We report here that
heterodimer formation with RXR not only enhances the absolute
affinity of RAR for DNA but also alters the relative ability of
RAR to bind to a variety of nonconsensus half-site elements. These
changes in relative specificity for the different half-site sequences
are distinct from the overall increase in affinity of the RXR/RAR or
RXR/T3R heterodimers for DNA. For example, it is striking that
recognition of certain half-site substitutions is relatively unaffected
by heterodimerization with RXR, whereas recognition of other response
elements is significantly enhanced, and that the DNA binding
specificity of the heterodimer is not a simple additive combination of
the specificities of the parental homodimers. These results suggest
that heterodimerization with RXR may actually alter the bp contacts
made by the RAR or T3R partner. This hypothesis is supported by
demonstrations that RXR/T3R heterodimers generate DNA footprint
patterns that differ from those generated by T3R/T3R homodimers
(62)
.
PML-RAR and PLZF-RAR share the ability of RAR to bind DNA both
as homodimers and as heterodimers with RXR (40, 41, 42)
.
Also in common with RAR, the formation of RXR heterodimers by
PML-RAR and PLZF-RAR expands the half-site repertoire of DNA
binding compared with the corresponding homodimers. Interestingly,
however, PLZF-RAR and PML-RAR retain the ability to form
homodimers even at high RXR concentrations. The enhanced
homodimerization capability of these oncoproteins has been mapped to
protein-protein interaction domains located within the PML- and
PLZF-derived sequences (41
, 49) . The capacity for enhanced
protein dimerization is a hallmark of the chimeric RARs found in APL
and has been implicated in mediating the oncogenic phenotype
(17, 18, 19, 20, 21
, 43)
. Thus, in contrast to RAR, which is
believed to function in cells exclusively as a RXR/RAR heterodimer
(44, 45, 46, 47)
, PML-RAR and PLZF-RAR have the potential to
exert at least some of their functions in the neoplastic cell as
receptor homodimers or as still higher order oligomers (43
, 48)
. Our own experiments indicate that this enhanced ability of
PML-RAR and PLZF-RAR to form homodimers would further shift the
DNA recognition specificities of these chimeric oncoproteins away from
those of the RXR/RAR heterodimers that are prevalent in normal cells;
the implications of this observation for leukemogenesis are discussed
below.
Transactivation from DNA Elements Generally Correlates with but Is
Not Identical to Binding by Receptors in Vitro.
We found that the ability of RAR and PML-RAR to activate
transcription from a given DNA response element in transfection
experiments generally correlated with the ability of these receptors to
bind to the response element in vitro. Typically,
transcriptional activation was weak with DNA elements that were bound
poorly by receptor in vitro, whereas transcriptional
activation was strong from DNA elements that were bound with high
avidity by receptor in vitro. However, there are some
exceptions to this generic rule. Both RAR and PML-RAR displayed
better activation on the 4G element than would have been expected from
our in vitro binding studies, and, in addition, PML-RAR
exhibited an unusually strong ability to activate transcription from
the 2T element, which was bound very poorly by any of the RAR
derivatives in vitro. Similar discrepancies between in
vitro binding and in vivo transcriptional activation
have been reported in previous studies, and response element sequences
that bind nuclear hormone receptors poorly in vitro may
nonetheless serve as efficient response elements in vivo
(54, 55, 56, 57)
. This "uncoupling" between DNA avidity and
transcriptional activation may reflect differences in the ability of
nuclear hormone receptors to activate transcription once bound to
different response elements. Alternatively, posttranslational
modifications of the receptor in vivo may confer novel
sequence binding properties not seen with unmodified receptor in
vitro, or response elements may bind additional transcription
factors in vivo that assist in the binding or function of
the nuclear hormone receptor.
Implications for the Role of PML-RAR and PLZF-RAR in APL.
It has been proposed that the chimeric RAR proteins involved in APL
function by perturbing normal retinoid signaling, leading to a block in
differentiation and in an accumulation of leukemic cells at a
promyelocytic stage. This hypothesis is consistent with observations
that retinoids can regulate differentiation of normal myeloid cells and
that supraphysiological levels of retinoic acid can induce
differentiation of leukemic cells bearing the PML-RAR translocation,
driving the corresponding leukemias into clinical remission
(21)
. Also consistent with these ideas, one consequence of
the translocations that produce the PML-RAR and PLZF-RAR proteins
is an impairment in the ability of these chimeric receptors to release
corepressor on addition of retinoic acid (28
, 30, 31, 32, 33)
.
Thus, PML-RAR and PLZF-RAR can function as dominant negative
inhibitors of normal retinoid signaling, and this dominant negative
activity correlates with a number of important aspects of the
leukemogenic phenotype.
The results described here, however, raise the possibility that the
panel of target genes that are subject to this aberrant regulation by
PML-RAR and by PLZF-RAR may be only a subset of the full panel of
genes that represent the targets of regulation by RAR in normal
cells. Three hypotheses can be advanced in this regard, depending on
the nature of the RARs actually operative in the leukemic cell.
(a) RAR, PML-RAR, and PLZF-RAR may all exert their
functions in the cell as homodimeric species. If this is true, then
given their overlapping but not identical DNA recognition specificities
as homodimers, PML-RAR and PLZF-RAR would be expected to function
as dominant negatives on many of the response elements that are
regulated by normal RAR. This hypothesis, however, appears unlikely:
RAR does not appear to homodimerize in solution, and RAR
homodimers are relatively inefficient at binding to DNA in
vitro. Instead, analysis both in vitro and by genetic
dissection in vivo suggests that RAR functions principally
as a RXR/RAR heterodimer (44, 45, 46, 47)
.
(b) Alternatively, RAR, PML-RAR, and PLZF-RAR may
all carry out their roles as heterodimers with RXR. Arguing against
this hypothesis, however, are the observations that both PML-RAR and
PLZF-RAR bind DNA efficiently as homodimers, that PML-RAR also
forms stable homodimers in solution, that the addition of RXR can
actually impair transcriptional regulation by PML-RAR, and that the
ability of PML-RAR to form homodimers appears to be both necessary
and sufficient for its ability to block myeloid differentiation
(40, 41, 42, 43)
.
(c) A third hypothesis, which we favor, is that PML-RAR
and PLZF-RAR homodimers play an important role in the leukemic cell
and act by interfering with the actions of RXR/RAR heterodimers.
Significantly, the DNA recognition properties of PML-RAR and
PLZF-RAR homodimers are similar to one another but different from
that of RXR/RAR heterodimers. Therefore, our data would suggest
that PML-RAR and PLZF-RAR homodimers, given their more narrow DNA
binding specificity, may be able to target only a subset of the
response elements that are recognized by RXR/RAR heterodimers.
This prediction appears to account for observations that the dominant
negative effects of PML-RAR and PLZF-RAR are promoter specific
(49, 50, 51)
. Importantly, NuMA, NPM, PML, and PLZF, although
otherwise structurally unrelated, all have dimerization domains that
are retained in the fusion protein (18
, 19)
, which may
suggest that homodimerization is an important common factor in the
function of all of the RAR-fusion oncoproteins. Of course, reality
may represent an amalgamation of these reductionalist views, and a
mixture of receptor homodimers, heterodimers, and heterotetramers may
participate in conferring the leukemic cell phenotype.
|
Materials and Methods |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
|---|
, human PML-RAR, human PLZF-RAR, and human RXR
proteins were obtained as nuclear extracts from Sf9 insect cells that
were infected with the appropriate recombinant baculoviruses. Briefly,
full-length clones of the human RAR, PML-RAR, PLZF-RAR,
and RXR genes were independently cloned into the baculovirus
transfer vector pVL1393 as NotI/NotI,
Blunt/EcoRI, EcoRI/EcoRI, or
EcoRI/EcoRI fragments, respectively
(63)
.4
Recombinant plasmids or a
nonrecombinant pVL1393 plasmid was individually transfected into Sf9
cells by lipofection together with a suitable linearized baculovirus
genome; stocks of virus originating by recombination with the transfer
vector in vivo were identified and isolated by plaque
purification by use of the BaculoGold system (PharMingen). The
plaque-purified, recombinant viruses were subsequently amplified in Sf9
cells, and nuclear extracts were prepared from the infected cells
(36)
. Expression of the correct proteins was confirmed by
SDS-PAGE analysis. The functionality of the baculovirus-encoded RAR,
PML-RAR, PLZF-RAR, and RXR proteins was confirmed by
electrophoretic mobility shift assays, and their identity was confirmed
by using appropriate antibodies to supershift the corresponding
protein-DNA complexes (see "Gel Electrophoretic Mobility Shift
Assays" below).
Oligonucleotide Probes.
The oligonucleotide probes used in the electrophoretic mobility shift
assays were chemically synthesized (Operon Technologies, Inc.). Each
oligonucleotide probe was made as two complementary strands with
4-base overhangs on each end to permit radiolabeling and/or
subsequent molecular cloning. The consensus sequence (8)
consisted of the following two oligonucleotides (the half-site
sequences are underlined):
5'-TCGACAGAGGTCAACGAGAGGTCAGAG-3' and
5'-TCGACTCTGACCTCTCGTTGACCTCTG-3'.
Systematic variations of these consensus oligonucleotides were designed
such that one base at a time, concurrently in both half-sites, was
altered to all other possible bases. As an example, base changes at
position +2 (Fig. 1A)
were synthesized as the following
oligonucleotides (only the upper strand oligonucleotide is depicted):
+2A,
5'-TCGACAGAAGTCAACGAGAAGTCAGAG-3';
+2C,
5'-TCGACAGACGTCAACGAGACGTCAGAG-3';
and +2T,
5'-TCGACAGATGTCAACGAGATGTCAGAG-3'
(half-sites are underlined, substitutions from
consensus are in bold).
The two strands were annealed, and the probes were radiolabeled by a
fill-in reaction with the Klenow fragment of Escherichia
coli DNA polymerase in the presence of
[-32P]dGTP.
Gel Electrophoretic Mobility Shift Assays.
Standard gel electrophoretic mobility shift assays were performed as
described previously (34)
. Nuclear protein extracts
containing RAR, PML-RAR, or PLZF-RAR, with or without RXR,
were incubated in 13 µl of binding buffer [3% glycerol, 11
mM Tris-HCl (pH 7.5), 15 mg/ml BSA, 77 mM KCl,
2 mM MgCl2, 154 µg/ml
poly(deoxyinosine-deoxycytosine) containing 40,000–60,000 cpm
(approximately 10–20 ng) of -32P-labeled
oligonucleotide) for 25 min at room temperature. Receptors were
titrated so as to bind approximately 25% of the consensus probe at
maximum; this represented approximately 12.5 ng of RAR, 6–8 ng of
PML-RAR, 1–2 ng of PLZF-RAR, and 2.5–5 ng of RXR when the
receptors were assayed as homodimers and approximately 3 ng of RAR,
6–12.5 ng of PML-RAR (with the exception of the experiment in Fig. 2
, which used 2 ng), 0.4–0.8 ng of PLZF-RAR, and 0.6–1.2 ng of
RXR when the receptors were used as heterodimers. The protein-DNA
complexes that formed were subsequently resolved by electrophoresis on
a 5% nondenaturing polyacrylamide gel at 200 V for 75 min in 0.25x
Tris-borate EDTA buffer. The electrophoretogram was then dried,
visualized, and quantified by PhosphorImager analysis (Molecular
Dynamics STORM system). Supershifts were performed in a similar manner,
with the addition of either 0.5 µl/lane of RXR-specific antibody
4RX-1D12 (1.0 µg/µl IgG; provided by P. Chambon, Pasteur Institute,
Strassbourg, France) or 1.5 µl/lane of RAR-specific
antibody sc551 (1.5 µg/µl IgG; Santa Cruz Biotechnology, Inc.). No
detectable cross-reaction of the RXR-specific antibody with RAR,
PML-RAR, or PLZF-RAR or of the RAR-specific antibody with
RXR was observed (Fig. 2B)
.
Titration Experiments.
Titration experiments were performed in two different ways: either a
constant amount of radiolabeled DNA probe was titrated with differing
amounts of protein nuclear extract (0.02, 0.05, 0.1, 0.4, 1.3, or 4
µl; approximately 50 ng/µl protein); or a constant amount of
protein was titrated with differing amounts of a radiolabeled DNA probe
(0, 0.35, 1.4, 5.6, 23, or 90 ng). The protein-DNA complexes that
formed were subsequently resolved by gel electrophoretic mobility shift
assays, as described above.
Transient Transfections.
Representative DNA elements were cloned into a thymidine kinase
promoter/reporter vector for use in transient transfection assay.
Oligonucleotides consisted of two retinoic acid response elements, each
comprised of the consensus, -1A, 2T, or 4G DR-5 sequence.
Oligonucleotides were made as two complementary strands bearing 4-base
overhangs compatible with SalI and XhoI sites.
For example, the plus oligonucleotide for the consensus sequence was
composed of the following sequence (half-sites are
underlined): 5'-TCGACAGAGGTCAACGAGA-
GGTCAGAGCTCAGAGGTCAACGAGAGGTCAGAG-3'
.
These oligonucleotides were then individually cloned into the XhoI and SalI sites of a thymidine kinase/luciferase reporter construct (64) , and appropriate recombinant vectors were screened by restriction mapping and confirmed by DNA sequence analysis.
Transient transfections of CV-1 cells were performed in 12-well tissue
culture plates with 9 x 104
cells/well.
Transfections were performed using a liposome/LipofectAMINE plus
technology (Life Technologies, Inc.); 20 ng of a pSG5-plasmid (either
an empty vector or a vector encoding RAR, PML-RAR, PLZF-RAR,
or RXR) were transfected per well, together with 100 ng of the
luciferase reporter plasmid, 100 ng of a pCH110-promoter-lacZ reporter
plasmid (used as an internal control), and 280 ng of pUC18 (to
standardize the total amount of transfected DNA to 500 ng/well). Three
h after transfection, all-trans retinoic acid was added (or
not added) to 400 nM, and the cells were
subsequently incubated for an additional 24 h. Thereafter, the
cells were harvested, and the luciferase and ß-galactosidase
activities were determined as described previously (30)
.
Transfected cell extracts were also analyzed by immunoblotting to
determine the levels of expression of the different nuclear receptor
derivatives.
|
Acknowledgments |
|---|
-specific antibodies. We thank S. Lee-Bond for
initiating the construction of the baculovirus vector clones of human
RAR, PML-RAR, and PLZF-RAR used in these experiments.
We are also grateful to Behnom Farboud for assistance in transient
transfection assays. |
Footnotes |
|---|
1 Supported by USPHS/NIH Grants CA53394 and
DK54064. 
2 To whom requests for reprints should be
addressed, at Section of Microbiology, Division of Biological Sciences,
University of California at Davis, One Shields Avenue, Davis, CA 95616.
Phone: (530) 752-3013; Fax: (530) 752-9014; E-mail: mlprivalsky{at}ucdavis.edu
3 The abbreviations used are: T3R, thyroid hormone
receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; ROR,
retinoid orphan receptor; DR-1, direct repeat-1; PML, promyelocytic
leukemia protein; PLZF, promyelocytic leukemia zinc-finger protein;
NuMA, nuclear mitotic apparatus protein; APL, acute promyelocytic
leukemia; NPM, nucleophosmin.
Received for publication 10/ 4/99. Revision received 10/12/00. Accepted for publication 11/30/00.
|
References |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
|---|
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transgenic mice.. Proc. Natl. Acad. Sci. USA, 94: 5302-5307, 1997. under control of cathepsin G regulatory sequences.. Blood, 89: 376-387, 1997. and PLZF-RAR with co-repressors determine differential responses to RA in APL.. Nat. Genet., 18: 126-135, 1998.[Medline]
transgene initiates murine acute promyelocytic leukemia.. Proc. Natl. Acad. Sci. USA, 94: 2551-2556, 1997. (RAR) and PLZF-RAR oncoproteins associated with acute promyelocytic leukemia.. Proc. Natl. Acad. Sci. USA, 94: 9028-9033, 1997., and BCL-6.. J. Biol. Chem., 273: 27695-27702, 1998. recruit histone deacetylase in promyelocytic leukaemia.. Nature (Lond.), 391: 815-818, 1998.[Medline]
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developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis.. Cell, 78: 987-1003, 1994.[Medline]
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(RAR) and PLZF-RAR fusion proteins on retinoic acid signalling.. Mol. Cell. Biol., 17: 4859-4869, 1997.This article has been cited by other articles:
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, without all-trans
retinoic acid; 
, with all-trans retinoic acid.
Inset, extracts of the transfected cells were also
analyzed by immunoblotting to confirm that the different receptors were
expressed at comparable levels