From the Departments of Medicine and Pathology and the Molecular Biology Program, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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The B-isoform of human progesterone receptors (PR) contains three activation functions (AF3, AF1, and AF2), two of which (AF1 and AF2) are shared with the A-isoform. AF3 is in the B-upstream segment (BUS), the far N-terminal 164 amino acids of B-receptors; AF1 is in the 392-amino acid N-terminal region common to both receptors; and AF2 is in the C-terminal hormone binding domain. B-receptors are usually stronger transactivators than A-receptors due to transcriptional synergism between AF3 and one of the two downstream AFs. We now show that the N terminus of PR common to both isoforms contains an inhibitory function (IF) located in a 292-amino acid segment lying upstream of AF1. IF represses the activity of A-receptors but is not inhibitory in the context of B-receptors due to constraints imparted by BUS. As a result, IF inhibits AF1 or AF2 but not AF3, regardless of the position of IF relative to BUS. IF is functionally independent and strongly represses transcription when it is fused upstream of estrogen receptors. These data demonstrate the existence of a novel, transferable inhibitory function, mapping to the PR N terminus, which begins to assign specific roles to this large undefined region.
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INTRODUCTION |
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Transcriptional control in response to extracellular signals involves the binding of regulatory proteins to specific enhancer elements of target genes. These proteins contain activation functions (AFs)1 through which contact is made with the basal transcription machinery either directly or indirectly by means of intermediary coregulatory proteins (1). Progesterone receptors (PR) are members of the nuclear receptor family of ligand-inducible transcription factors. These are structurally complex proteins containing multiple functional domains, including a highly conserved central DNA-binding domain (DBD), a moderately well conserved C-terminal hormone-binding domain (HBD), and a poorly conserved, N-terminal region whose function is largely unknown (1).
There are two naturally occurring isoforms of PR. The 933-amino acid B-receptors contain an N-terminal 164-amino acid upstream segment (BUS) that is missing in the truncated 769-amino acid A-receptors (2-5). The two PR isoforms have AF1 and AF2 in common (5, 6). AF1 maps to a 91-amino acid "proline-rich" segment located just upstream of the DBD and AF2 is located in the HBD (6). BUS, restricted to B-receptors, contains AF3 (5). In general, B-receptors are stronger transactivators than A-receptors (5, 7-9), and only B-receptors can activate transcription in the presence of antiprogestins (9-11). On the other hand, A-receptors can dominantly inhibit B-receptors (9, 12, 13) as well as other members of the steroid receptor family (14).
In addition to AFs, some transcription factors also contain inhibitory domains (IDs) that modulate the activity of the AFs. Such IDs have been identified by deletion mutagenesis that generate proteins with enhanced transcriptional activities. Examples include members of the AP1 family c-Jun (15), c-Fos, and the related protein, FosB (16); ATF-2, a member of the ATF/cAMP regulatory element-binding protein subfamily of basic region leucine zipper (bZIP)-containing transcription factors (17); and the lymphoid-specific transcription factor, Oct-2a (18). An ID has also been found in the proto-oncogene c-Myb, which plays a key role in hematopoesis (19). Finally, IDs have been characterized in two yeast transcription factors: PHO4, which is regulated by phosphate levels (20), and ADR1, which regulates glycerol metabolism genes (21). To date, no ID has been described in the nuclear receptor family of transcription factors.
The IDs are structurally distinct from the AFs that they regulate (15-21). In some cases, inhibition is transferable to heterologous AFs, suggesting that the IDs are functionally independent. For example, when fused to the Escherichia coli polypeptide B42, the inhibitory regions of ADR1 repress transcription (21). Similarly, the IDs of c-Myb and c-Jun can inhibit the activity of VP16, a potent transactivator (15, 19). Inhibition by other IDs, however, is restricted to either the cognate AFs or a certain subset of AFs. For example, the bZIP domain of ATF-2 inhibits the related AFs of ATF-2 and E1a but not the acidic AF of VP16 or the glutamine-rich AF of Sp1 (17). Similarly, the N-terminal ID of c-Fos specifically silences the HOB1 subset of AFs found in c-Fos and c-Jun but not other phosphorylation-dependent AFs such as that found in cAMP regulatory element-binding protein (16).
Because A-receptors are weak transactivators compared with B-receptors and are trans-dominant inhibitors of other nuclear receptors, we postulated that A-receptors contain inhibitory sequences distinct from the three defined AFs and that these sequences are inoperative in B-receptors. We sought this inhibitory function (IF) in a previously uncharacterized 292-amino acid region of the A-receptor N terminus. In this paper we have compared the activity of several PR constructs that either contain or lack IF. We show that IF expresses a novel inhibitory function, distinct from the AFs, that inhibits AF1 and AF2 but not AF3. Therefore, IF removal converts A-receptors from weak into strong transactivators. Additionally, IF is transferable and suppresses estrogen receptor (ER) activity when it is cloned upstream of ER.
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MATERIALS AND METHODS |
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Recombinant Plasmids-- Complementary DNAs, hPR2 and hPR1, encoding human A- and B-receptors, respectively, and HEGO, encoding human estrogen receptors, cloned into pSG5 (4), were gifts from P. Chambon (Strasbourg, France). Construction of BUS-DBD, N-terminal B (NTB)-DBD, N-terminal A (NTA)-DBD, DBD-HBD, and BUS-DBD-HBD expression vectors, all containing a nuclear localization signal (NLS), was described in Sartorius et al. (5).
NTA-Immunoblotting--
Whole-cell 0.5 M KCl extracts
were prepared from COS cells transiently transfected with the
expression vectors described. The expressed PR fragments were resolved
by electrophoresis on 7.5% or 10% SDS-containing denaturing
polyacrylamide gels and transferred to nitrocellulose. Protein blots
were probed with our anti-PR monoclonal antibodies, AB-52 and B-30
(24), and the anti-DBD polyclonal antibody 266 (25) provided by D. Toft (Rochester, MN). For detection of ER or IF-ER, the anti-ER
antibody SRA 1,000 was used (StressGen, Victoria, BC). Bands were
detected by enhanced chemiluminescence (Amersham Corp.) as described
previously (11).
Transfection and Transcription Assays--
HeLa cells were
plated in 100-mm tissue culture dishes in 10 ml of minimum essential
medium supplemented with 5% twice charcoal-stripped, heat-inactivated
fetal calf serum (DCC-MEM). Duplicate plates were transfected by
calcium phosphate coprecipitation with 2 µg of the reporter plasmid,
variable amounts (indicated in the figures) of the receptor expression
vectors, 3 µg of the -galactosidase expression plasmid pCH110
(Pharmacia Biotechnology Inc.) to correct for transfection efficiency,
and Bluescribe (Stratagene, La Jolla, CA) carrier plasmid for a total
of 20 µg/plate (11). 24 h later, the medium was changed to 7.5%
DCC-MEM, and cells were either left untreated or were incubated with 10 nM of the synthetic progestin R5020 (Roussel UCLAF, France)
or 17
-estradiol, for an additional 24 h. Cells were harvested,
and lysates were normalized to
-galactosidase activity and analyzed
for CAT activity by TLC as described previously (9, 10). Standard
deviations of phosphorimaging (Image Quant, Molecular Dynamics,
Sunnyvale, CA) data were determined using Microsoft Excel, version 5.0 (Microsoft Corporation, Seattle, WA) for the number of sets indicated
in the figure legends.
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RESULTS |
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A series of expression vectors was constructed in which each
region of PR that contains an AF was fused, either alone or in combination with another AF, to the PR DBD-NLS (5). Additionally, the
constructs contained or lacked IF, the 291 amino acids lying upstream
of AF1. IF was also cloned upstream of full-length ER. The detailed
structure of all the constructs is shown in Fig. 1.
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Expression of the receptor proteins was verified by SDS-polyacrylamide gel electrophoresis and immunoblotting of whole-cell extracts derived from COS cells (Fig. 2). They range in size from 191 to 933 amino acids and are all well expressed. The presence of multiple bands for some constructs, particularly ones that contain BUS (Fig. 2A, lane 8, for example), is due to phosphorylation (5, 26). Interestingly, this multiple banding pattern is amplified by removal of the HBD (Fig. 2A, lanes 4 and 8) and is reduced by juxtaposition of IF upstream of BUS (Fig. 2, compare lane 6 in A and lane 1 in B). Each of the receptors shown in Fig. 2 binds to a perfect palindromic PRE in an electrophoretic mobility shift assay (data not shown).
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An Inhibitory Function--
To search for an inhibitory function,
an A-receptor variant was constructed (A-IF) that lacks the
N-terminal 292 amino acids located upstream of AF1 (Fig. 1). This
previously uncharacterized domain, designated IF (amino acids
165-455), although common to both receptor isoforms, has the potential
to function differently when free at the N terminus of A-receptors but
constrained by BUS in B-receptors. Strikingly, on the
PRE2-TATAtk promoter (Fig. 3A) or on the MMTV promoter
(Fig. 3B), deletion of IF converts A-receptors from weak
into strong transactivators equivalent to B-receptors. However, unlike
B-receptors, but like A-receptors, A-
IF displays strong
"self-squelching" behavior. Therefore, as the concentration of
A-
IF is increased, the high levels of CAT activity fall.
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IF Inhibits AF1 and AF2 but Not AF3--
Fig. 3 shows that IF
contains a potent inhibitory function that suppresses the activity of
A-receptors. However, because B-receptors, which also contain IF
sequences, are strong transactivators, we postulated that IF does not
influence AF3 but that its inhibitory effects are restricted to AF1
and/or AF2 (Fig. 4). To test this hypothesis, constructs were made that contained each AF alone, with or
without IF (Fig. 1). Dose-response data using
PRE2-TATAtk-CAT are shown in Fig. 4 for AF1
(NTA-DBD) with (+) and without () IF (Fig.
4A); AF2 (DBD-HBD) with (+) and without (
) IF (Fig. 4B); and AF3 (BUS-DBD) with (+) and without (
) IF (Fig.
4C) compared with full-length B-receptors. Analogous to its
role in full-length A-receptors, we find that IF has its strongest
effect on AF1 and AF2 at low receptor concentrations.
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Mechanisms of PR Auto-inhibition by IF--
Several possible
mechanisms can be invoked for auto-inhibition of A-receptors by IF. One
is binding of a repressor at IF. However, we find that overexpression
of an IF-NLS construct does not squelch the putative repressor (not
shown). Another possible mechanism is steric hindrance of AF1 and AF2
by IF due to the latter's upstream position. To test this hypothesis,
IF was cloned upstream of AF3. We reasoned that if IF acts by steric
hindrance in A-receptors, then perhaps juxtaposition of IF upstream of
BUS would inhibit AF3 activity. To that end, IF-BUS-DBD was constructed and compared with NTB-AF1 (BUS-IF-DBD) on
PRE2-TATAtk-CAT (Fig. 4D). The only
difference between these two constructs is the position of IF relative
to AF3. BUS-DBD was used as a control. At all concentrations tested,
the two IF-containing constructs had equivalent transcriptional activity. Therefore, BUS appears to be insensitive to the inhibitory effects of IF, regardless of the position of IF. In addition, we find
that IF has no effect in other B-receptor derivatives. Specifically,
constructs containing AF2 plus AF3 (B-
AF1 and BUS-DBD-HBD) had
identical transcriptional profiles with and without IF (not shown).
Taken together, these data suggest that IF does not act simply by
steric hindrance of any AF to which it is linked; rather IF inhibition
is specific for AF1 and AF2. We therefore asked whether IF could
suppress AF1 and AF2 of another member of the steroid receptor family.
IF Is Transferable to the Heterologous AFs of ER--
Inhibitory
domains, like activation domains, can be discrete and modular. To
determine whether IF effects were transferable, we tested the ability
of IF to inhibit the heterologous AFs of ER. ERs contain AF1 and AF2
and, in this respect, structurally resemble A-receptors (14, 27, 28).
However, ER have no sequences homologous to IF. To test the effects of
IF on ER, an IF-ER chimera was constructed (Fig. 1) in which IF was
cloned upstream of ER. Fig. 5A
shows transcription by wild-type ER or IF-ER of the
ERE2-TATAtk-CAT reporter in the absence
(open symbols) or presence of 10 nM
17-estradiol (solid symbols). CAT activity induced by ER
is maximal at 0.1 µg of the expression vector and then decreases at
higher concentrations due to self-squelching. This has previously been
described (7, 14). At the same cDNA concentrations, IF cloned
upstream of ER markedly reduces transcription. Fig. 5 (B and
C) compares the transcriptional efficacy of ER and IF-ER
when the two are expressed at similar protein levels. We find that
IF-ER is expressed at lower efficiency than ER. Thus, 1 µg of the
IF-ER expression vector and 0.1 µg of HEGO produce equivalent amounts
of immunoreactive protein (Fig. 5B). Note that the expected
molecular mass of ER is 65 kDa and that of IF-ER is 97 kDA. Fig.
5C shows that at these equivalent protein concentrations,
wild-type ER strongly activate transcription, whereas little or no
transcription is produced by IF-ER. We conclude that when IF is
transferred upstream of ER, it silences ER-dependent
transcription.
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DISCUSSION |
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This paper describes a novel, transferable inhibitory function, designated IF, which lies in the 292-amino acid N-terminal region upstream of the PR AF1 but operates only in the context of A-receptors.
A- versus B-receptors-- Why progesterone target tissues contain two receptor isoforms remains an intriguing physiological puzzle. They were first described in chick oviducts (29) and then in human cells (2). In humans, the two proteins are the products of a single gene that has two promoters, from which at least nine messages, two of which are A-receptor specific, are transcribed (30). An internal AUG present in some messages may also encode A-receptors (30). Thus, there is complex regulatory control over protein levels of the two isoforms, the details of which are still unclear. In initial studies using breast cancer cell lines, the two isoforms were found in approximately equimolar amounts (3). However, it is now clear that their relative levels are under tight developmental and hormonal control in chicken oviducts (31-33) and the female rat brain (34), and preliminary data in the human uterus also show a discordance, with A:B ratios ranging between 50:1 and 2:1 during the menstrual cycle due to large excursions in the levels of B-receptors (35). In breast cancers 25% of tumors have a significant excess of A-receptors (36). Given the functional transcriptional differences between the two isoforms, their unequal distribution in tissues and tumors could be biologically important. For example, an excess of B-receptors in the uterus may mark those patients at greatest risk of developing tamoxifen-induced endometrial cancers (37).
Transferable Inhibition of AF1 and AF2-- Much of the work devoted to understanding regulation of transcription by steroid receptors has focused on AFs and their stimulatory actions. However, transcriptional inhibition may be equally important as a way of preventing or terminating activation. Studies that deal with inhibition have focused on composite DNA elements and invoke mechanisms in which receptor occupancy at one DNA site interferes with transcription by an activator at an adjoining site (38, 39). Heterodimerization of an activator by a repressor and recruitment of corepressors are other silencing mechanisms (40). We now demonstrate that negative signaling elements can exist in the receptor molecule itself.
We show that IF markedly suppresses the transcriptional activity of AF1 and AF2 of A-receptors (Fig. 4). The ability of IF to also strongly suppress AF1 plus AF2 of ER (Fig. 5) suggests that its inhibitory mechanisms involve general steroid receptor-related processes. It is tempting to speculate that IF prevents the binding of key AF1 or AF2 transcriptional coregulators that are shared by all steroid receptors (40). However the inability of soluble IF (i.e. IF-NLS) to squelch such activity suggests that IF acts structurally, perhaps through intramolecular contacts. Our data show that the inhibitory activity can be transferred to the cognate AFs of ER. In that respect, IF resembles the bZIP domain of ATF-2 and the N-terminal ID of c-Fos (16, 17). Whether IF can also suppress heterologous AFs remains to be determined.IF Cannot Inhibit AF3-- B-receptors also contain the IF element, but its repressor activity appears to be constrained by BUS, which is located further upstream. Therefore, IF specifically inhibits AF1 and AF2 of PR but not AF3 (Fig. 4). Furthermore, IF cannot inhibit AF3 regardless of its position relative to BUS (Fig. 4, C and D). We have previously demonstrated that AF3 transcriptional activity is unusual in that it is critically dependent on the presence of the PR DBD. In gel mobility shift studies, BUS-DBD binds to a PRE only if a bivalent monoclonal antibody is added, which appears to supply a dimerization function. The possibility exists that BUS and the DBD of PR are linked through intramolecular contacts so that the mechanisms of AF3 action may be quite different from those of AF1 and AF2.
There is now compelling evidence that alterations in the three-dimensional structure of steroid receptors modifies their transcriptional behavior. Most of that work comes from analyses of the HBD. For example, using protease accessibility as a probe for receptor structure, it has been shown that PR (41) and ER (42, 43) assume altered conformational states when the HBD is occupied by agonists or antagonists. More recently, crystallographic analyses of the HBDs of unliganded RXR ![]() |
ACKNOWLEDGEMENTS |
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We are grateful to Pierre Chambon for the
gift of pSG5-hPR1, hPR2, and MMTV-CAT, to David Toft for the gift of
266 antibody, to Roussel UCLAF for R5020, and to our colleague David
Bain for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by Grants CA-26869 and DK-48238 from the National Institutes of Health, by a grant from the U. S. Army, and by the National Foundation for Cancer Research. Cell culture support was provided by the Tissue Culture Core Laboratory of the University of Colorado Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a graduate student stipend from the Lucille P. Markey Charitable Trust.
§ To whom correspondence should be addressed: Depts. of Medicine and Pathology and the Molecular Biology Program, University of Colorado Health Sciences Center, 4200 East 9th Ave., Box B-151, Denver, CO 80262. Tel.: 303-315-8443; Fax: 303-315-4525; E-mail: kate.horwitz{at}uchsc.edu.
1 The abbreviations used are: AF, activation function; PR, progesterone receptor; DBD, DNA-binding domain; HBD, hormone-binding domain; ID, inhibitory domain; bZIP, basic region leucine zipper; IF, inhibitory function; ER, estrogen receptor; NTA, N-terminal A; NTB, N-terminal B; NLS, nuclear localization signal; PCR, polymerase chain reaction; nt, nucleotide(s); MMTV, mouse mammary tumor virus; CAT, chloramphenicol acetyltransferase; PRE, progesterone response element; h, human; BUS, B-upstream segment.
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REFERENCES |
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