(Received for publication, November 6, 1995; and in revised form, January 11, 1996)
From the
A previously characterized retinoic acid response element (RARE1) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter confers approximately 50% of the response of this gene to retinoic acid (RA). Transient transfection experiments were performed using constructs containing progressive 5` deletions of the PEPCK promoter to locate other elements that contribute to the RA response. A second RARE (RARE2) was located between -402 and -306. Methylation interference and mobility gel shift assays indicated that RAR/RXR bound specifically to a segment of DNA located between -337 and -321. This region contains consensus and degenerate half-sites for receptor binding separated by 5 bp. Mutations in either half-site selectively decreased the RA response and diminished RAR/RXR binding in mobility gel shift assays. When both RARE1 and RARE2 were mutated, 80% of the RA response was lost. Finally, RARE2 conferred a RA response in a heterologous promoter context. We conclude that RAR/RXR binds to RARE2, and that this DR5-type element is a major contributor to the response of the PEPCK gene to RA.
The induction of transcription in response to retinoic acid is
mediated by retinoic acid receptors (RARs), ()members of the
steroid/thyroid/retinoid superfamily of nuclear hormone receptors.
RARs, as well as thyroid hormone receptors (TRs) and
1,25-dihydroxyvitamin D
receptors (VDRs) are members of a
subfamily of the nuclear hormone receptors that share structural and
functional similarities. These receptors bind specifically to their
cognate response elements and alter the transcription rates of target
genes in a ligand-dependent manner (for reviews, see (1, 2, 3) ). RAR, TR, and VDR bind DNA with
much greater affinity when complexed as a heterodimer with the
9-cis-retinoic acid receptor (RXR). The response elements
through which these receptors act are closely related and are usually
arranged as two half-sites of the sequence RG(G/T)TCA ( (4) and (5) and references therein). The spacing between the
half-sites is an important determinant of nuclear receptor binding
specificity. Typically, VDR/RXR and TR/RXR heterodimers bind to direct
repeat (DR) half-sites separated by 3 or 4 base pairs (designated DR3
and DR4), respectively. RAR/RXR heterodimers are less discriminating
since they bind DR1, DR2, and DR5 retinoic acid response elements
(RAREs)(6, 7, 8, 9) . RAR/RXR
heterodimers also bind to (and trans-activate from) inverted (10, 11) or everted repeats (12, 13) . It is also important to note that RAREs
often contain at least one degenerate half-site(5) .
Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) catalyzes the conversion of oxalacetate to phosphoenolpyruvate, the rate-limiting step of gluconeogenesis, and is, therefore, a focal point for hormonal regulation of blood glucose homeostasis (for reviews, see Refs. 14 and 15). The PEPCK gene product is apparently not allosterically or post-translationally modified to alter its activity. Rather, the activity of PEPCK is directly related to the amount of protein, which is predominantly affected by the rate of transcription of the gene (16) . PEPCK gene transcription is positively regulated by glucagon (via cAMP), glucocorticoids, and retinoic acid(17, 18, 19) . Insulin rapidly inhibits PEPCK gene transcription and is dominant over the positive effectors(20) . Thus, PEPCK is an excellent model system for the studies concerning the integration of multihormonal signals. The cis-acting elements that confer these responses, as well as developmental and liver-specific expression, all lie within the 460 bp upstream of the transcription start site of the PEPCK gene(21, 22) .
We previously demonstrated that retinoic acid (RA) increases the transcription rate of the endogenous PEPCK gene in H4IIE rat hepatoma cells. Utilizing PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion constructs in transient transfection assays, an imperfect DR1 RARE (RARE1) was localized to a segment between -451 and -433 bp relative to the transcription start site(23) . This core sequence confers approximately 50% of the PEPCK RA response in the intact PEPCK promoter and mediates a RA response when inserted upstream of a minimal thymidine kinase promoter(23, 24) . RAR/RXR binds to RARE1 with high affinity and comprises the functional trans-activating complex that mediates the RA response from this element(24) . The core sequence of the PEPCK RARE is coincident with the minimal functional boundaries of the accessory factor 1 (AF1) element, which is required for a complete glucocorticoid response and is a component of the complex PEPCK glucocorticoid response unit (GRU(19, 24, 25, 26) ). Any mutation in RARE1/AF1 that abolishes the RA response from this element also diminishes the glucocorticoid response and vice versa. Thus, the RARE1/AF1 element is required for at least two distinct hormone response pathways.
In the present paper, we report the identification of a second RARE (RARE2) within the PEPCK promoter. Mutations that decrease the binding of RAR/RXR to RARE2 also reduce the RA response of PEPCK promoter-CAT fusion constructs. When RARE2 is ligated to a thymidine kinase promoter, it confers a RA response to an associated reporter gene.
Figure 1:
Localization of a second RARE within
the PEPCK gene promoter. The complex glucocorticoid regulatory unit (GRU) is shown with its constituent accessory factor elements (AF1 and AF2) positioned upstream of two
glucocorticoid response elements (GRE). Included are elements
required for minimal basal transcription (NF1, CRE,
and TATA(49) ). A series of constructs containing
progressively less of the 5` end of the PEPCK promoter fused to CAT
were co-transfected (10 µg each) with pRShRAR (5 µg) into
H4IIE cells. CAT activity was measured in cell lysates 18 h after
treatment with or without 2 µM RA.
Figure 2:
Mutations in RARE1 and RARE2 reduce the
retinoic acid response of a PEPCK promoter-CAT fusion gene. A,
the sequence of RARE2 contains two overlapping degenerate RAR/RXR
half-sites ( and
) and a consensus half-site (
). The
half-site is underlined by an arrow to indicate
the conventional orientation of nuclear hormone receptor
half-sites(4, 5) . The RARE2m construct contains a
block mutation of the
half-site as outlined with a box.
Also shown is the sequence of RARE1 and the block mutation in the B
half-site, RARE1m, which is also highlighted with a box. B,
the plasmid pPL32 contains the wild type PEPCK promoter from -467
to +69, relative to the initiation site, ligated to CAT. The
construct RARE1m is identical with pPL32 except for a block mutation in
the RARE1 element (referred to as SDM B in (26) ). The plasmid
RARE2m is identical with pPL32 except that it contains a block mutation
in RARE2, as shown in A. The construct RARE1m/RARE2m contains
both block mutations. Results are expressed as the average fold
induction of CAT activity (with RA/without RA) ± S.E. of
4
separate determinations.
Figure 3:
RAR/RXR heterodimers bind specifically to
RARE2. Mobility gel shift assays were performed using a
double-stranded, end-labeled RARE2 oligonucleotide as probe. I and II denote complexes formed when rat liver nuclear
extract was incubated with RARE2. A, the binding reaction
mixture included 5 µg of rat liver nuclear extract (NE)
and the indicated additional components: antiRXR, 1 µl of
RXR antiserum; NS, 1 µl of nonspecific antiserum; RAR, 100 fmol of partially purified, bacterially expressed
hRAR; Mock, an equivalent volume of mock bacterial
extract. B, the indicated double-stranded competitor
oligonucleotides were added in a 100 molar excess prior to the addition
of 100 fmol of bacterially expressed hRAR
and 5 µg of rat
liver nuclear extract. RARE1 (PEPCK -460 to -425); AF2, accessory factor 2 (PEPCK -433 to -396); RARE2 (PEPCK -346 to -304); RARE2m, same
as RARE2 except for a block mutation in the
half-site (see Fig. 2A).
Figure 4: Methylation interference defines a second half-site for RAR/RXR binding within RARE2. A, mobility gel shift assays were performed using approximately 300 fmol each of bacterially expressed RAR and RXR with an end-labeled and partially methylated RARE2 element as probe. DNA was isolated from the free and RAR/RXR-bound bands and subjected to piperidine cleavage and separation on a 6% denaturing polyacrylamide gel. The methylated G residues that interfered with RAR/RXR binding are indicated with an asterisk. TS, top strand; BS, bottom strand; F, free DNA; B, RAR/RXR-bound DNA. B, a diagram of RARE2 showing the methylated G residues that interfere with RAR/RXR binding. The arrows underscore potential half sites for RAR/RXR binding.
Figure 5:
Detailed mutational analysis of RARE2. A, a series of constructs were made with mutations in each of
the potential binding motifs within RARE2, as described under
``Materials and Methods.'' Apart from the nucleotides underlined, all of the mutations are identical with pPL32 (the
full-length PEPCK promoter fused to CAT). Ten µg of the reporter
constructs and 5 µg of pRShRAR were cotransfected into H4IIE
cells. After 18 h of treatment with or without 2 µM RA,
CAT activity was measured from cell lysates. The results are expressed
as the mean fold induction of CAT activity (with RA/without RA)
± the S.E. of
4 separate experiments. B, mobility
gel shift assays were performed with a double-stranded, end-labeled
oligonucleotide containing RARE2 as probe. Double-stranded competitor
oligonucleotides were added in 100 molar excess prior to the addition
of 100 fmol each of bacterially expressed RAR and
RXR.
Mobility
gel shift assays were performed with RAR, RXR, and an RARE2 probe (Fig. 5B). Double-stranded oligonucleotides containing
wild type sequences or mutations within the half-site
(RARE2
m) effectively competed for the retinoid receptor complex.
However, oligonucleotides containing the RARE2
m or RARE2
m
mutations failed to compete completely for the binding of RAR/RXR
heterodimers. Indeed, RARE2
m competed for RAR/RXR binding more
efficiently than RARE2
m, which is in close agreement with the
observed methylation interference data (Fig. 4). Taken together,
these data demonstrate that this element is a DR5-type RARE.
Figure 6:
RARE2
confers a RA response in the context of a heterologous promoter.
Plasmids were made wherein RARE2 was ligated either in the correct (RARE2TK) or inverse (RARE2RTK) orientation, or in
tandem ((RARE2)2TK) upstream of the minimal thymidine kinase
promoter-CAT reporter construct (TK). In addition, the
half-site was mutated in the context of the RARE2TK construct (RARE2
mTK). These constructs (10 µg each) were
cotransfected with 5 µg of pRShRAR
and CAT activity was
measured 18 h after treatment with or without 2 µM RA. The
data are expressed as an average fold induction of CAT activity (with
RA/without RA) ± S.E. of
3 separate
experiments.
We have previously shown that RA increases the rate of transcription of the endogenous PEPCK gene and of PEPCK promoter-CAT fusion constructs(23) . This response is mediated, in part, by a pleiotropic element, termed RARE1, that is required for complete RA and glucocorticoid responses(19, 23, 24, 25, 26) . A second RARE in the PEPCK promoter region has now been located between -337 and -321 in relation to the transcription start site ( Fig. 1and Fig. 2). RAR/RXR heterodimers bind specifically to this sequence (Fig. 3B and 5B) which is a degenerate DR5-type RARE ( Fig. 4and Fig. 5). Furthermore, RARE2 is active in the context of a heterologous promoter, and in either orientation (Fig. 6), which is consistent with previous observations of other RAREs ( (4) and (5) and references therein).
It is worth noting that most RAREs in their natural context, including both RAREs in the PEPCK promoter ( (23) and (24) and this study), are not perfect direct repeats(5) . Additionally, RAR/RXR binds to (and transactivates through) palindromes (10, 11) and everted repeats(12, 13) . Furthermore, the mouse laminin B1(34) , rat growth hormone(34) , and human oxytocin (35) gene promoters contain complex RAREs composed of 3 or 4 repeats in various orientations that can span up to 80 bp. The work of many investigators has increased our understanding of the role of protein/DNA interactions in RA signaling(6, 7, 8, 36, 37, 38) , yet it is still vitally important to continue to identify and characterize natural RAREs so that we might understand RA signal transduction in physiologically relevant contexts.
The physiological
role of RA in the regulation of PEPCK gene transcription is unclear.
However, it is known that RA-deficient rats have impaired
gluconeogenesis (39) and, since PEPCK is the rate-limiting
enzyme of gluconeogenesis(14) , it is possible that RA is
required for maintenance of basal PEPCK expression in hepatocytes. In
fact, recent data from transgenic mice support this idea. Transgenic
mice expressing a PEPCK-promoter driven transgene and put on a
RA-deficient diet, expressed 50% less of the transgene mRNA than
control mice or RA-deficient mice supplemented with RA(40) .
Since RA levels apparently do not change appreciably in healthy adult
animals (41) , RA probably does not acutely affect PEPCK
transcription. As we have proposed before, RA may be permissive for
both generating relatively high basal levels of transcription and
elevating hormonal responsiveness of the PEPCK gene(42) .
Indeed, when RA is added together with glucocorticoids or cAMP, there
is an additive or even synergistic effect on PEPCK gene expression (43) . ()
Giralt et al.(44) identified a thyroid hormone response element (TRE)
in the PEPCK promoter. The TRE is localized between -334 and
-318 and mediates a modest 3-fold response to thyroid hormone
(T) in human HepG2 hepatoma cells, which incidentally do
not express the endogenous PEPCK gene. The element is also required for
the synergism observed when these cells are treated with both cAMP and
T
(44) . Recently, Park et al.(45) characterized this TRE in much more detail. The sequences
to which TR/RXR binds are contained within the RARE described here.
Using our nomenclature, TR/RXR binds to the
and
half-sites
of RARE2, whereas RAR/RXR binds to the
and
half-sites. The
functional significance of the observed overlap of RAR/RXR and TR/RXR
binding is not clear and we have been able to demonstrate only a very
weak thyroid hormone response in H4IIE cells, in which the endogenous
PEPCK gene is expressed(42) . However, when TR and RAR are
co-expressed with PEPCK promoter-CAT constructs, the RA response is
decreased(42) . TR may affect the RA response by sequestering
RAR in nonproductive RAR/TR heterodimers which bind to
RARE1(42) . Alternatively, TR may bind to RXR in solution, thus
preventing the formation of RAR/RXR heterodimers (46) . The
competition of TR/RXR and RAR/RXR for RARE2 could contribute to the
blunting of the RA response.
It is perhaps not surprising that the
second PEPCK RARE is required for more than one hormone response. Genes
requiring the integration of multiple environmental cues often contain
complex regulatory domains with overlapping and multiple functions (for
review, see (17) ). Within the PEPCK gene promoter, the complex
GRU serves as a prototypical example of a metabolic control domain. The
GRU is composed of two glucocorticoid receptor binding sites (GRE1 and
GRE2) and two accessory factor elements, AF1 and AF2, located just
upstream of the GREs (see Fig. 1). AF1 and AF2 are both required
for a complete glucocorticoid response; the GREs are essentially silent
without the accessory factor activity(19) . AF1 activity is
mediated by the binding of either COUP-TF or HNF-4. Both of these
proteins are members of the RAR/TR/VDR subfamily of the steroid hormone
superfamily and are orphan receptors lacking known ligands. AF2
activity is mediated by the binding of hepatic nuclear factor 3
(HNF-3), a member of the fork-head family of transcription factors. ()Remarkably, AF1 and AF2 are also required for other
hormone signaling pathways. Thus, AF1 is a RARE (via RAR/RXR
binding(23, 24) ) and AF2 is an insulin-responsive
sequence (via an unidentified insulin responsive factor, distinct from
HNF-3(47, 48) ). A similar situation may exist for
RARE2. We have evidence that the second PEPCK RARE is also an accessory
factor element required for a complete glucocorticoid response.