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Molecular Endocrinology, doi:10.1210/me.2006-0105
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Molecular Endocrinology 20 (11): 2656-2670
Copyright © 2006 by The Endocrine Society

Progesterone Receptors (PR)-B and -A Regulate Transcription by Different Mechanisms: AF-3 Exerts Regulatory Control over Coactivator Binding to PR-B

Lin Tung, Hany Abdel-Hafiz, Tianjie Shen, Djuana M. E. Harvell, Lisa K. Nitao, Jennifer K. Richer, Carol A. Sartorius, Glenn S. Takimoto and Kathryn B. Horwitz

Division of Endocrinology, Department of Medicine, University of Colorado Health Sciences Center at Fitzsimons, Aurora, Colorado 80045

Address all correspondence and requests for reprints to: Hany Abdel-Hafiz, Department of Medicine, RC1 South, 12801 East 17th Avenue, P.O. Box 6511, Aurora, Colorado 80045. E-mail: hany.abdel-hafiz{at}uchsc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The two, nearly identical, isoforms of human progesterone receptors (PR), PR-B and -A, share activation functions (AF) 1 and 2, yet they possess markedly different transcriptional profiles, with PR-B being much stronger transactivators. Their differences map to a unique AF3 in the B-upstream segment (BUS), at the far N terminus of PR-B, which is missing in PR-A. Combined mutation of two LXXLL motifs plus tryptophan 140 in BUS, to yield PR-BdL140, completely destroys PR-B activity, because strong AF3 synergism with downstream AF1 and AF2 is eliminated. This synergism involves cooperative interactions among receptor multimers bound at tandem hormone response elements and is transferable to AFs of other nuclear receptors. Other PR-B functions—N-/C-terminal interactions, steroid receptor coactivator-1 coactivation, ligand-dependent down-regulation—also require an intact BUS. All three are autonomous in PR-A, and map to N-terminal regions common to both PR. This suggests that the N-terminal structure adopted by the two PR is different, and that for PR-B, this is controlled by BUS. Indeed, gene expression profiling of breast cancer cells stably expressing PR-B, PR-BdL140, or PR-A shows that mutation of AF3 destroys PR-B-dependent gene transcription without converting PR-B into PR-A. In sum, AF3 in BUS plays a critical modulatory role in PR-B, and in doing so, defines a mechanism for PR-B function that is fundamentally distinct from that of PR-A.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AS MEMBERS OF THE steroid receptor family, human progesterone receptors (PR) are structurally unique. Two isoforms, PR-B and -A, are coexpressed in target tissues but regulate different sets of endogenous genes when expressed independently (1). Yet they have 780 identical amino acids except that PR-B contain 164 additional N-terminal residues—the B-upstream segment, (BUS) (2). Because only BUS distinguishes the large N-terminal regions of PR-B from PR-A, it can be used to study mechanistic differences between the two receptors. Liganded PR-B are not only stronger transactivators than PR-A in vitro (3) but are also stronger regulators of endogenous gene transcription (1). The in vivo differences are striking. Expression profiling (1, 4) and gene knockout studies (5, 6) show that PR-B and -A regulate distinct and nonoverlapping arrays of genes and functions; estrogen-dependent human breast tumor xenografts expressing only PR-B or PR-A exhibit isoform-specific growth phenotypes (7); and human breast cancers expressing different ratios of the two PR have different, ratio-specific clinical outcomes (8).

Both PR contain two activation functions (AF) whose location has been mapped by the classical approach of fusing receptor fragments to the DNA binding domain (DBD) of the yeast transcription factor Gal4, and assaying transcriptional activity from a Gal4 response element-driven promoter (9). By this analysis, AF1 localizes to an N-terminal region proximal to the DBD, and AF2 maps to a C-terminal region of the ligand binding domain (LBD). A third AF3 within BUS exhibits unique properties (2). Unlike AF1 and AF2, the autonomous transcriptional effects of AF3, as assessed by classical approaches, are DBD context dependent (10). That is, full transcriptional activity is achieved only if AF3 is fused to its own DBD and is reduced 70–90% if it is fused to the heterologous DBD of Gal4, of estrogen receptors (ERs), or even of the highly homologous DBD of glucocorticoid receptors (GRs). Similar properties have been reported for the AFs of USF2, SRF and ATF2, which are inactive or even repressive when tethered to heterologous DBDs (11, 12, 13).

Communication among domains within proteins is critical to their function. For example, communication between AFs and their DBDs is dependent on the sequence and architecture of the DNA response element, which acts as an allosteric ligand to modify not only DBD structure, but also structural and cooperative binding properties of distant regulatory regions (14). In the case of PR, binding of N-terminal B (NT-B) —a construct containing the entire PR-B N terminus including BUS, the DBD and downstream hinge region—to a progesterone response element (PRE) stabilizes the receptor’s structure at multiple distant sites, including the BUS/PR-A junction, residues bordering AF1, and the hinge (15). Similar influences of DNA on GR structure have been described (16). Additionally for PR, BUS has the ability to communicate with and influence the structure of downstream regions. This was demonstrated by sedimentation velocity studies, which show that NT-B paradoxically exhibits more structural homogeneity than the corresponding regions of PR-A (NT-A) lacking BUS (15). Based on this, we proposed that BUS limits the conformational ensemble of PR-B to fewer, albeit more active conformers, accounting for its strong transcriptional activity.

The ability of transcriptional regulatory domains to communicate with one another underlies the phenomenon of AF synergism exhibited by many transcription factors (17, 18). For steroid receptors, AF synergism has focused on the relationship between AF1 and AF2 (19) and N-/C-terminal interactions (20). An FxxLF sequence in androgen receptor (AR) N termini is required for direct N-/C-terminal interactions. The two LxxLL motifs in BUS, akin to nuclear receptor (NR) boxes, subserve a similar function (21). Neither of these motifs competes for binding of p160 coregulators, however, which also contain NR boxes needed to bind receptors (21). Indirect N-/C-terminal interactions are also facilitated by bridging factors like steroid receptor coactivator (SRC)-1, SRC-2, JDP-2, and cAMP response element binding protein (CREB)-binding protein (CBP)/p300 for PR (22, 23, 24, 25), Ada2, TBP, and CBP for GR (26, 27), and SRC-1, SRC-2, CBP, and the RAP74 subunit of transcription factor IIF for AR (28, 29). However, because the N termini of steroid receptors and their associated AFs are disordered with minimal secondary structure, high resolution, three-dimensional structures are not available, making it difficult to define the physico-chemical properties governing N-/C-terminal and N-terminal/coregulator interactions.

We mapped three motifs in BUS critical for AF3 function (3): two leucine-rich NR boxes, L1 (54LxxLL) and L2 (115LxxLL), plus a tryptophan residue (140W). Mutation of each, individually reduces transcriptional activity of a constitutive BUS-DBD construct, or of full-length PR-B, by more than 80%. Simultaneous mutation of all three to yield a dL140 mutant completely inactivates autonomous AF3, and full-length PR-B, function. No other residues in the PR-B N terminus possess this level of regulatory control. Here, for the first time, we use the dL140 mutant to inactivate AF3 with minimal sequence perturbation to define mechanisms by which the BUS region regulates PR-B activity. We show that AF3 synergizes with AF1 and AF2 only on multiple PRE-containing promoters, and this synergism correlates with SRC-1/p160 complex recruitment. Importantly, AF3 inactivation does not convert PR-B to the functional equivalent of PR-A as assessed by gene expression profiling; a finding consistent with the fact that BUS is not structurally and functionally independent, but controls PR-B-dependent transcription through allosteric modification of downstream N- and C-terminal regions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PR-BdL140 Mutant and Predicted Impact on BUS Structure
Constructs used in the present studies are shown in Fig. 1Go and described in Materials and Methods. We previously showed that combined PR-BdL140 mutation of 55LxxLL, 115LxxLL, and 140W in BUS, severely impairs or completely destroys AF3 function and PR-B activity (3). Figure 2Go shows the predicted impact of the combined mutation on PR-B structure using the Predictor of Natural Disordered Regions (PONDR) algorithm (30). A schematic of wild-type PR-B and the PR-BdL140 mutations is shown at the top, with their PONDR scores aligned below. Regions predicted to be disordered are defined by runs of at least 40 amino acids, and are indicated by the solid black bars labeled a–e. In wild-type PR-B, only the N terminus (amino acids 1–556) contains regions predicted to be disordered; the DBD and LBD are highly ordered. Consistent with our experimentally based conclusions (15), BUS is completely disordered (a and b) except for two small peaks indicated by the arrows. Three other extensively disordered regions are predicted for the N terminus: one downstream of BUS (c) and two (d and e) proximal to the DBD at the presumed location of AF1. PONDR analysis predicts that the two ordered peaks in BUS are obliterated by the dL140 mutation (compare arrows in the PR-B vs. PR-BdL140 plots). Note also enlargement of segment b.


Figure 1
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  		Fig. 1. PR Constructs Used in the Present Studies

Full-length PR-A, PR-B, BUS-DBD, N-terminal constructs and BUS-AR and BUS-ER or the respective BUS mutants were cloned into mammalian expression vectors. VP16 and Gal4 fusion constructs were used in mammalian two-hybrid interactions studies. For details, see Materials and Methods.

 

Figure 2
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  		Fig. 2. Predicted Conformational Properties of Wild-Type PR-B and PR-BdL140 Mutants

Shown are major structural elements of PR-B, aligned with PONDR plots of wild-type PR-B and PR-BdL140. PR-B are 933 amino acids in length, and location of key BUS residues and their mutations are shown. Disordered regions represented by a PONDR score above 0.5 were calculated from primary amino acid sequence data and are predicted to lack fixed tertiary structure, making them partially or fully unfolded. Larger regions of disorder, defined by runs of at least 40 amino acids, are indicated by the solid black bars, and labeled a–e. The arrows highlight ordered subregions in wild-type BUS. The predictor VL_XT algorithm used for the plots was developed by P. Romero, A. K. Dunker, X. Li, and Z. Obradovic (30 ). Further information and background can be found online at http://www.pondr.com/.

 
AF3 Synergism with Downstream AFs Is Prevented by the dL140 Mutant and Is Dependent on Promoter Architecture
The functional consequences of this structural loss are shown in Fig. 3Go. When the dL140 mutation is introduced into BUS-DBD or full-length PR-B, the receptors retain DNA binding activity (not shown), but their transcriptional activity assayed from a PRE2 luciferase reporter is reduced to a level comparable to PR-A, which is weakly active on this promoter. Note that the mutant and wild-type constructs are expressed at similar levels (2, 3). The transcriptional defects are most clearly observed at low, physiologically relevant receptor concentrations (0.5–5 ng), where the activity of low dose PR-B, containing AF3, AF1, and AF2, greatly exceeds the sum of transcriptional activities generated independently from equivalent doses of PR-A (expressing AF1 + AF2 activities) and BUS-DBD (expressing AF3 activity). We conclude that in PR-B, AF3 exerts synergistic influence on the activities of AF1 and AF2. In the PR-BdL140 mutant, AF1 and AF2 cannot synergize with AF3 and express only the autonomous activity characteristic of PR-A.


Figure 3
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  		Fig. 3. AF3-Related Transcriptional Synergism in PR-B

HeLa cells were transfected with the constructs shown, the PRE2-luciferase reporter (2 µg), and treated with 10 nM R5020 if a LBD was present. Other constructs are constitutively active. Transcriptional activity was normalized to a Renilla luciferase control (see Materials and Methods). Values shown are the mean ± SD from three different experiments. For the BUSdL140-DBD mutant, a single luciferase activity value generated from 25 ng of transfected cDNA is shown. Note that the plot of values for luciferase activity is discontinuous.

 
Synergism among the AFs of PR-B is entirely dependent on promoter structure (Fig. 4AGo). On a single PRE, the activities of PR-B, PR-BdL140 and PR-A are similar. On PRE2- and PRE3-driven promoters, PR-B activity is 5- to 10-fold greater than that of PR-A or PR-BdL140. These promoter-specific effects were also observed over a broad range (10–1000 ng) of input cDNA (not shown). The PRE data suggest that AF3-dependent synergism results from cooperative protein-protein interactions among two or more receptor dimers occupying adjacent palindromic PREs rather than within receptor monomers bound at a single PRE.


Figure 4
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  		Fig. 4. Transcriptional Synergism Regulated by BUS Requires at Least Two Hormone Response Elements and Is Transferable to Other Receptors

A, Expression vectors for PR-B, PR-BdL140 and PR-A (25 ng each) were transiently cotransfected into HeLa cells with 2 µg of a luciferase reporter plasmid driven by one, two or three PREs. Luciferase activities were expressed as the fold induction (mean ± SD) of 10 nM R5020 treatment over the no hormone control from three different experiments. B, Wild-type BUS or BUSdL140 were cloned in front of ER and AR. Expression vectors for wild-type or modified ER and AR, at the concentrations shown, were transfected into HeLa cells together with an ERE2-luciferase reporter (for ER) or the PRE2-luciferase reporter (for AR), treated with 10 nM estradiol or 100 nM dihydrotestosterone, and transcriptional activity was normalized to a Renilla luciferase control. Shown are the luciferase activities expressed as fold induction (mean ± SD) of hormone treatment over the no hormone controls from three different experiments.

 
We therefore asked whether this BUS-associated synergism is transferable to other steroid receptors, and cloned wild-type BUS or dL140 mutant BUS in front of full-length ER or AR. Figure 4BGo shows that wild-type BUS increases the transcriptional activity of ER from a tandem estrogen response element (ERE2), or of AR from a PRE2 response element, 3- to 14-fold. This property cannot be conferred by BUSdL140. Expression levels of wild-type ER and AR and their respective BUS fusion proteins were similar (see Fig. 9Go).


Figure 9
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  		Fig. 9. BUS Controls Ligand-Dependent Down-Regulation of PR-B

A, PR immunoblots were performed on extracts from T47D-Y breast cancer cells stably expressing PR-B, PR-A or the PR-BdL140 mutant (see Materials and Methods), and treated with vehicle or 10 nM R5020 for 8 or 18 h. Cell extracts were probed with the PgR1294 monoclonal antibody. B, T47D cells stably expressing PR-B, PR-BdL140, or PR-B containing a Serine294Alanine (S294A) mutation, were treated with ethanol or 10 nM R5020 for 1.5 h. Cell extracts were probed with the anti-PR PgR1294 or anti-phosphoserine294 (pS294) monoclonal antibodies, as indicated. C, HeLa cells were transiently transfected with constructs expressing wild-type ER, BUS-ER or BUSdL140-ER and treated with vehicle or 10 nM estradiol for 8 or 21 h. Immunoblots of cell extracts were probed with anti-ER Ab15. D, HeLa cells were transiently transfected with expression vectors encoding wild-type AR, BUS-AR or BUSdL140-AR and treated with vehicle or 100 nM dihydrotestosterone for 24 h. Cell extracts were immunoblotted with anti-AR PG-21.

 
SRC-1 Assembles on and Coactivates PR-B by an AF3-Dependent Mechanism
SRC-1 coactivation is dependent on a high-affinity interaction between its NR box motif and a ligand-induced hydrophobic groove in AF2 of the LBD (31). Additionally, on PR, SRC-1 makes secondary, lower affinity contacts with N-terminal regions common to both isoforms (23). The role, if any, of SRC-1 in AF3-related function is unknown. Figure 5Go shows the effect of SRC-1 on transcriptional activity of liganded PR-B or the PR-BdL140 mutant using promoters containing single or multiple PREs. SRC-1 has no coactivating properties on the low levels of wild-type PR-B-dependent transcription from a single PRE. To our knowledge, studies of SRC-1 coactivation never use a single PRE. On two or three PREs, SRC-1 is a strong coactivator of PR-B, but it cannot coactivate PR-BdL140 despite the receptor’s retention of an intact N terminus downstream of BUS (i.e. the entire N terminus of PR-A) containing a presumed SRC-1 binding site (23).


Figure 5
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  		Fig. 5. PR-B Coactivation by SRC-1 Requires an Intact BUS and at Least Two PREs

HeLa cells were cotransfected with PR-B, PR-BdL140 or PR-A expression vectors (25 ng), without or with SRC-1 (0.5 µg) or an empty vector, and treated with 10 nM R5020 or ethanol vehicle. Transcription was measured from a luciferase reporter driven by one, two or three PREs. The numbers above the bars represent the fold activation over the no hormone control. Values shown are the mean ± SD from three different experiments.

 
Because SRC-1 coactivates PR-A 3-fold, and assembles with PR-A on a stably integrated PRE2 promoter as determined by chromatin immunoprecipitation (ChIP) assay (not shown), we speculated that unlike for PR-A, for PR-B, SRC-1 coactivation of AF1 or AF2 requires BUS. This is confirmed by ChIP assay using HeLa cells transiently cotransfected with the PRE2 promoter, SRC-1 and wild-type PR-B or PR-BdL140 (Fig. 6Go). SRC-1 assembles with R5020 liganded PR-B but fails to load onto PR-BdL140. Thus, in contrast to the AFs of PR-A, the downstream AFs of PR-B cannot recruit SRC-1 when AF3 is inactivated. We propose that PR-B and -A assemble coregulatory proteins by fundamentally different mechanisms with AF-3 exerting regulatory control over this property for PR-B.


Figure 6
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  		Fig. 6. SRC-1 Assembles on Wild-Type PR-B But Not on the PR-BdL140 Mutant

HeLa cells were transiently transfected with 2 µg PR-B or PR-BdL140 and SRC-1 or empty vectors, and 2 µg PRE2-luciferase reporter plasmid. Cells were treated 1 h. with 10 nM R5020 or ethanol vehicle, fixed with formaldehyde, sonicated, and cross-linked proteins were immunoprecipitated with anti-PR or anti-SRC-1 antibodies. Isolated DNA was subjected to PCR using primers bracketing the PREs (see Materials and Methods). The reporter plasmid was used as a positive control. Fold increases are calculated from no hormone controls. Band densities were quantitated using Scion Image (Scion Corp., Frederick, MD). The data shown are representative of two different experiments.

 
AF3 Enhances Intramolecular PR N-/C-Terminal Interactions and Intermolecular PR N-Terminal/SRC-1 Interactions
The mammalian two-hybrid assay was used to evaluate AF3-regulated intra- and intermolecular communications. The ligand-dependent intramolecular interaction between the LBD and PR-B N terminus (NT-B) in the absence of SRC-1 is shown in Fig. 7AGo. SRC-1 increases the activity generated from this interaction 3- to 4-fold. NT-BdL140 exhibits neither the ligand-dependent LBD/NT-B interaction nor its enhancement by SRC-1. Importantly, Fig. 7BGo shows an NTB/SRC-1 interaction that is reduced in strength by the dL140 mutations to the weaker state observed with NT-A. This raises the possibility that BUS and SRC-1 interact directly. Figure 8Go further demonstrates that an SRC-1-anchored complex is required for full expression of PR-B activity on the PRE2-driven promoter. The ligand-dependent activity of PR-B is inhibited in a dose-dependent manner by the adenoviral oncoprotein E1A, which has previously been shown to disrupt assembly of the PR/SRC-1/CBP complex (32). The E1A N-terminal deletion mutant (33), which cannot bind CBP, has no effect on PR-B activity. Neither wild-type E1A nor the E1A mutant alters basal transcription. Together, these results indicate that for PR-B, recruitment of an SRC-1-anchored p160 complex to a multisite promoter involves a network of BUS-dependent interactions among surfaces on the N and C termini of PR, SRC-1, and other coregulatory proteins.


Figure 7
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  		Fig. 7. SRC-1 Binds Strongly to the PR-B N terminus Containing Wild-Type BUS But Not if BUSdL140 Is Present

A, The PR LBD was fused to the Gal4 DBD (G4); and NT-B or NT-BdL140 were fused to the VP16 activation domain. The mammalian two-hybrid expression vectors (2 µg each) were cotransfected into HeLa cells with SRC-1 (1 µg) or empty vector and treated 24 h. with 10 nM R5020 or ethanol vehicle. The numbers above the bars represent fold activation over the no hormone control. B, SRC-1 was fused to G4, and NT-B, NT-BdL140, or NT-A were fused to the VP16 activation domain. Mammalian two-hybrid expression vectors (2 µg each) were cotransfected as above and activity measured. The numbers above the bars represent fold activation over the G4-SRC-1 control. Values shown are the mean ± SD from three different experiments.

 

Figure 8
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  		Fig. 8. E1A Suppresses PR-B Transcription from a PRE2 Promoter

HeLa cells were transfected with 25 ng of the PR-B expression vector alone or together with an expression vector for the viral oncoprotein E1A (12.5 or 25 ng) or the N-terminal deletion mutant E1Am (25 ng), with a PRE2-luciferase reporter, and treated without or with 10 nM R5020. Relative luciferase activity was measured and normalized to the ligand-dependent activity of PR-B set at 100%. Values shown are the mean ± SD from three different experiments.

 
AF3 Is Required for Ligand-Dependent Down-Regulation of PR-B
Both PR-B and -A undergo ligand-dependent down-regulation; a step required for transcriptional activity. Down-regulation is mediated by ubiquitination and 26S proteasome degradation, after phosphorylation of serine (S) 294 in the N terminus common to both receptors (34). Surprisingly then, liganded PR-BdL140, unlike PR-B or PR-A, are completely resistant to down-regulation (Fig. 9AGo) even after 18 h of hormone treatment. Yet, PR-BdL140 are S294 phosphorylated (Fig. 9BGo). This is shown using cell lines stably expressing PR-B, PR-BdL140, or the phosphorylation-deficient PR-B S294A mutant, each treated with R5020 for 1.5 h. Immunoblotting for total PR-B shows similar expression levels of all three receptors. Their molecular weights are upshifted by ligand due to an overall increase in phosphorylation state. Probing PR with an S294 phospho-specific antibody confirms that in contrast to the S294A mutant, both wild-type PR-B and PR-BdL140 retain S294 phosphorylation. We speculate that BUS does not control S294 phosphorylation. Instead, BUS controls more downstream events required for down-regulation that cannot be activated in the dL140 mutant.

This putative modulatory effect of BUS is supported by its ability to impart significant down-regulatory control of other steroid receptors when it is fused to their N termini. ER undergo modest ligand-dependent down-regulation when transiently transfected (35). Figure 9CGo shows that presence of wild-type BUS at the ER N terminus greatly accelerates down-regulation, but the dL140 BUS mutant cannot confer this property. Unlike PR and ER, AR are paradoxically up-regulated by ligand (36). Figure 9CGo shows that wild-type BUS prevents this increase, but the dL140 mutant cannot. Interestingly, BUS fused to ER and AR resulted in a synergistic increase in their ligand-dependent transcriptional activity as compared with the wild-type receptors, whereas activity of the BUSdL140 fusion receptors more closely resembled wild-type ER and AR (not shown). These findings are consistent with recent reports documenting the close association between efficient transcriptional activity and 26S proteosome-mediated degradation of nuclear receptors and other transcription factors (37, 38).

BUS Selectively Controls the Global Endogenous Transcriptional Properties of PR-B
The studies above suggested that the PR-BdL140 mutant was not the equivalent of PR-A, but transcription studies using PRE-luciferase constructs were inadequate to document this. Endogenous gene expression profiling was therefore used to compare PR-B, PR-BdL140, and PR-A. Each construct was stably transfected into PR-negative T47D-Y human breast cancer cells. Cells were treated 6 h with or without progesterone and RNA expression was profiled using gene chips. Venn diagrams quantifying up- and down-regulated genes for the three receptors are shown in Fig. 10AGo. Complete gene lists are published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org. As we previously showed (1), wild-type PR-B and -A regulate substantially different sets of genes; overlap between them is 8% for up-regulated genes and 6% for down-regulated genes. Mutation of BUS eliminates 95% of PR-B up-regulated and 92% of PR-B down-regulated genes. Importantly, the mutant does not adopt a PR-A-like profile, with only 3% overlap between genes up-regulated by the mutant receptors and PR-A, and 5% overlap between their respective down-regulated genes. Rather, PRBdL140 is a different receptor that resembles neither PR isoform but has unique gene regulatory properties of its own. This is qualitatively evident from the dendrograms in Fig. 10BGo, which also show that substantial groups of genes up-regulated by wild-type PR-B are down-regulated by the PR-BdL140 mutant. The PR-BdL140 mutant also does not cause a general loss of activity because we know from transient transfection studies using the progestin-responsive region of the BCL-XL (1) promoter, that the PR-BdL140 mutant has greater activity than both PR-B and -A isoforms. We conclude that mutation of AF3 does not convert PR-B into PR-A, and that in the context of wild-type PR-B, AF3 uniquely controls the transcriptional properties of this isoform. Twelve genes, including p21 (see supplemental data), that are up-regulated by both PR-B and PR-BdL140, demonstrate that in limited cases, PR-B can activate genes even with AF3 inactivated. Interestingly, we had previously reported that a progestin-responsive p21 promoter fragment is activated by PR in a DNA-binding-independent manner through protein-protein interaction with Sp1 (39). This suggests that BUS may be dispensable when such nonclassical transcriptional pathways are activated.


Figure 10
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  		Fig. 10. Expression Profiling of Genes Regulated by Hormone-Treated PR-B, PR-A, and PR-BdL140; Mutation of BUS Does Not Convert PR-B to PR-A

T47D-Y cells stably expressing PR-B, PR-A or PR-BdL140 were treated with 10 nM progesterone or ethanol for 6 h in triplicate, time-separated experiments. Progesterone-regulated genes were assessed by expression profiling using Affymetrix HuFL-U95Av2 gene chips as described in Materials and Methods. A, Venn diagrams, showing the number of genes up- or down-regulated at least 1.5-fold by progesterone in a statistically significant manner, and the overlap among the groups. Complete gene lists are available as supplemental data. B, Dendrograms were constructed of the 75 most highly PR-B up- or down-regulated genes and compared with the regulation of the same genes by PR-BdL140 or PR-A, setting control levels at 1.0. In the presence of progesterone, the endogenous transcriptional activity of the PR-B BUS mutant resembles neither of the wild-type receptors.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
BUS Is a Strong Isoform-Specific Modulatory Domain that Lacks Ordered Structure
The nuclear receptor superfamily shares a common domain organization consisting of an N-terminal region, a central DBD and a C-terminal LBD. The DBDs and LBDs possess considerable secondary structure making them amenable to high resolution crystallographic analysis (40, 41, 42). This, combined with mutagenesis data, has resulted in a detailed structural/functional understanding of the isolated domains (43, 44). In contrast, nuclear receptor N termini differ extensively from one another in size and amino acid sequence, lack a compact globular structure (15, 45, 46), and no high resolution structural information is available. The uniqueness of PRs rests in the fact, that with the exception of BUS in PR-B, their primary amino acid sequence is identical including approximately 70% of their N termini. This has led to the assumption that their N termini must be mechanistically similar.

Here we show that in PR-B, mutation of key BUS residues not only completely inactivates autonomous AF3 activity but also inactivates AF3 modulation of downstream AFs. The mutant thus reveals a novel synergistic property for BUS, defined by its ability to cooperatively control the activities of downstream AFs. This mechanistically differentiates the N terminus of PR-B from that of PR-A. Interestingly, these properties of AF3 are seen only on promoters containing at least two palindromic PREs. This suggests that the strong transcriptional activity of PR-B is derived from AF3-dependent cooperative interactions between receptor dimers binding to adjacent palindromes rather than between monomers bound at a single palindrome. Consistent with this, Bain and co-workers (47), using quantitative DNAse footprinting, find that highly purified full-length PR-B bind cooperatively to a double PRE but noncooperatively to a single PRE.

The mechanisms by which BUS mediates its autonomous vs. synergistic properties are likely to be complex. We previously showed (15, 45) and PONDR analysis (Fig. 2Go) supports, that BUS is highly disordered. The prevalence of unstructured domains in many proteins has only recently been appreciated, and their biophysical properties are under intense study. Spectroscopic and proteolytic mapping of PR, GR, and AR N-terminal peptides show them to be unstructured but that secondary structure can be induced by binding to coregulators or to DNA (15, 26, 48, 49, 50). In fact, regions lacking ordered structure possess thermodynamic advantages that allow binding to multiple molecular targets without sacrificing specificity (51). We speculate that BUS binds to, or allosterically communicates with, downstream receptor regions generating a complex set of protein-protein interaction surfaces unique to full-length PR-B. Additionally, the possibility exists that BUS itself forms interaction surfaces for coregulators, though to date no such molecules have been identified. We propose that these physical-chemical properties imparted by BUS/AF3 to full-length PR-B make these receptors mechanistically very different from PR-A.

Intramolecular Interactions Are Responsible for BUS Modulation of PR-B Activity
Protein interaction studies using purified fragments of AR, PR, and other steroid receptors show that N and C termini interact in vitro (20, 29, 52). Consistent with these findings, FRET analysis demonstrates that compared with unliganded ER or AR, ligands increase the proximity between N and C termini (53). The role of BUS in PR N-/C-terminal interactions is unclear. Tetel et al. (20), using purified N- and C-terminal fragments for in vitro pull-down and in vivo mammalian two-hybrid assays, showed that N termini of both PR-B and -A interact with the LBD, but that PR-B bind with higher affinity. They concluded that the primary LBD contact sites lie in the N terminus common to both PR, and that BUS modulates but is not essential for this interaction. In contrast, Dong et al. (21) using isolated BUS rather than an intact N-terminal-DBD fragment in similar assays, showed that mutation of the 55LxxLL and 115LxxLL motifs within BUS abrogates BUS/LBD binding, implicating BUS as a direct interactor. We show here, using intact N-terminal-DBD fragments, that in vivo, N-/C-terminal interactions within PR-B require a functionally intact BUS and are abolished by the dL140 mutation (Fig. 7Go). This suggests that in PR-B, BUS does more than modulate binding affinity. Rather, the entire N-terminal region of PR-B assumes a structure that is different from that of PR-A while still permitting communication between N and C termini, in agreement with studies indicating that the N termini of the two receptors adopt different conformations (15, 45). Whether BUS within the context of full-length PR-B interacts directly with the LBD remains unclear due to the apparent AF3-dependent allostery within the N-terminal region.

The mechanisms by which intramolecular communication occurs within receptors is complicated by the fact that coregulators like SRC-1 also interact with both C and N termini (23). Like the PR LBD, SRC-1 binds the N termini of both PR-B and -A, but its binding affinity is higher for PR-B (23). We show here that for PR-B, interaction and promoter assembly with SRC-1 are entirely dependent on a functionally intact BUS (Figs. 5–7GoGoGo). Whether BUS interacts directly with the LBD and/or SRC-1, whether there exist bridging BUS binding proteins, or whether BUS modifies downstream N-terminal structures allowing SRC-1 binding downstream, is still unclear, but the BUS requirement for SRC-1 interaction with PR-B argues for a mechanism that differs from that of PR-A. We also find that SRC-1 interacts with itself (not shown) raising the possibility that PR-B-related synergy on multisite promoters involves assembly of multimers of the SRC-1 anchored p160 complex (17, 22, 54).

Finally, using expression profiling we show that PR-B and -A regulate different promoters in vivo, and that when BUS is mutated the resultant PR-BdL140 receptor does not resemble PR-A. This is entirely consistent with our findings for N-terminal functions, like down-regulation, where PR-A, like PR-B, are degraded in a ligand-dependent manner, whereas PR-BdL140 are resistant to degradation. We speculate that a transcriptionally active AF3 domain could promote degradation of PR-B because: 1) BUS contains PEST(proline/glutamic acid/serine/threonine)-like sequences and may therefore possess degron signals within or adjacent to AF3 (56); 2) it is unstructured and may provide an initiation site for proteasome degradation (57); and 3) it is highly phosphorylated—a characteristic of regions regulating proteasome-mediated degradation. Furthermore, as reported in Fig. 9BGo, a transcriptionally competent BUS appears to regulate degradation events downstream of serine 294 phosphorylation. Taken together, our results suggest that compared with PR-A, BUS induces unique conformers of the PR-B N terminus, that confer PR-B-specific properties on these receptors. Even subtle conformational changes during multiprotein complex assembly at promoters can dictate dramatic shifts in transcriptional activity, as has been reported for GR (58).

Transcriptional Synergy, Cooperativity, and Multisite PREs
Both acidic (like AF3) and nonacidic AFs can elicit synergistic transcriptional effects when their response elements are multimerized. Using purified viral activators (ZEBRA and GAL4-VP16), general transcription factors (TFIID, TFIIA, TFIIB), and defined promoter templates, Carey et al. (17, 54) showed that activation from multisite but not single DNA response elements produces transcriptional synergy through TFIIB-mediated stabilization of a TFIID/TFIIA complex. These studies provide functional and biophysical evidence for a cooperative network of protein-protein interactions, where binding of one or more activators to general transcription factors enhances recruitment, assembly and stabilization of the resulting complex. We find, for PR-B as compared with PR-A, that transcriptional synergism from multisite PREs is dependent on a functionally intact AF3. Because purified PR bind cooperatively to tandem PREs (47, 59), we postulate that cooperative interactions between PR-B dimers, and/or between PR-B and multiple surfaces on a p160 complex anchored by SRC-1, occur in an AF3-dependent manner. This cooperativity could be mediated by direct and/or indirect PR-PR contacts, and like assembly at the ZEBRA-responsive enhanceosome (60), could be influenced by high mobility group protein-1-induced DNA bending, which is augmented by PR-B compared with PR-A (61).

Based on the present observations, we hypothesize that on a single PRE, PR-B, PRBdL140, and PR-A are all unable to efficiently recruit SRC-1, or a full complement of general transcription factors. At multisite PREs, PR-B dimers cooperatively bind each other and the p160 coactivator complex, leading to efficient stabilization of a multiprotein complex. We propose that BUS allosterically induces ligand-dependent conformational changes throughout downstream N-, and possibly C-terminal, regions of PR-B that facilitate preinitiation complex assembly. For PR-A or when AF3 is inactivated (PRBdL140), this complex may be less stable or not optimally conformed for efficient preinitiation complex assembly, more closely resembling that seen on a single PRE.

These conclusions are based on data obtained from synthetic multimerized PREs. However, a survey of proximal promoter regions of endogenous genes regulated by PR shows a conspicuous lack of tandem palindromic PREs and a surfeit of PRE half-sites (B. Jacobsen, personal communication). This suggests additional mechanisms for transcriptional synergism dictated by promoter architecture. Synergistic transcriptional responses induced by PR-B and GR occur on the mouse mammary tumor virus promoter where an array of PRE half-sites exist in tandem with a single palindrome (62). Similarly, synergism occurs among ER half-sites widely spaced in the ovalbumin gene promoter (63). For GR, composite response elements consisting of a single hormone response element in tandem with heterologous binding sites leads to synergy between GR and a variety of factors, including Sp1, NF1, CACCC-box, and AP1 (58, 64, 65, 66, 67). Thus, it is likely that natural promoters adopt multiple strategies to enhance transcriptional signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Recombinant Expression Vectors and Reporter Plasmids
pSG5-PRB, pSG5-PRA, and pSG5-ER were gifts from P. Chambon (Institute of Genetics and Molecular and Cellular Biology, Strasbourg, France) (9, 68), pCMV5-AR was a gift from E. Wilson (University of North Carolina, Durham, NC) (69). pBind SRC-1e and pCR3.1-SRC-1e were gifts from B. O’Malley (Baylor College of Medicine, Houston, TX) (23). p{Delta}TAT1-Luc, p{Delta}TAT2-Luc and p{Delta}TAT3-Luc reporter plasmids were gifts from J. Iniguez-Lluhi (University of Michigan, Ann Arbor, MI) (67). PRE2-TATA-luciferase has been described previously (3). The E1A and E1Am ({Delta}4–25) plasmids were gifts from Y. Nakatani (National Institutes of Health, Bethesda, MD). Shown in Fig. 1Go are schematics of the plasmid constructs used. PR-B contains BUS with two LXXLL (NR) box motifs and tryptophan 140. These were mutated to alanine in PRBdL140 as described previously (3). PR-A lack BUS, whereas BUS-DBD and BUSdL140-DBD contain the respective wild-type or mutant BUS linked to the PR DBD. The BUS-AR fusion protein was constructed by ligating three fragments: a BglII fragment encoding BUS (residues 1–164 of PR); a BglII-SfoI fragment encoding residues 1–52 of AR; and a BglII-SfoI vector-containing fragment of pCMV5-AR. The BUS-ER fusion protein was constructed by ligating three fragments: an XhoI-EcoRV fragment encoding BUS (residues 1–164 of PR); an EcoRV-BlpI fragment encoding residues 1–323 of ER; and an XhoI-BlpI vector-containing fragment of pcDNA-ER (subcloned from pSG5-ER). Plasmids used in mammalian two-hybrid studies include the N-terminal fragments of PR-B (NTB), PR-BdL140 (NTBdL140), and PR-A (NTA) (3) fused to the activation domain of VP16. EcoRI-BamHI fragments encoding NTB (residues 1–645), NTBdL140 (1–645) and NTA (165–645) were cloned into the pVP16 vector (CLONTECH, Palo Alto, CA). The PR LBD fusion protein was constructed by cloning an EcoRI-HindIII fragment encoding residues 642–933 of PR into the pM vector (CLONTECH) that expresses the Gal4 DBD as an N-terminal fusion protein. pBind-SRC-1e expresses the Gal4 DBD as an N-terminal fusion protein of SRC-1e (residues 1–1440) and was a gift from B. O’Malley.

Transcription Assays
HeLa cells were plated in MEM containing 5% fetal bovine serum (twice charcoal-stripped for experiments containing full-length PR-B constructs) at a density of 1.2 x 105 cells per 60-mm dish, 1 d before transfection. Cells were transfected by calcium phosphate coprecipitation (3) using concentrations of expression vectors indicated in the figures. Reporter plasmids were added at 2 µg/dish. Simian virus 40-Renilla luciferase was added as an internal control vector at 20 ng/dish. Twenty-four hours later, the cells were washed and grown for an additional 24 h before collection during which time the hormone was added to dishes containing receptor constructs containing an LBD at a final concentration indicated in the figures. The N-terminal constructs lacking the LBD are constitutively active. Cells were collected in 150 µl lysis buffer (Promega, Madison, WI), and 50 µl were analyzed on a dual luminometer. Results were normalized to Renilla luciferase activity and expressed as indicated in the figures. Replicate experiments were done in duplicate.

Mammalian Two-Hybrid Assay
Interactions between PR N-terminal fragments and the PR LBD or SRC-1 were measured using the Mammalian Matchmaker 2-hybrid system (CLONTECH) and constructs shown in Fig. 1Go. The luciferase reporter gene contained five copies of the Gal-4 response element (5x Gal4-Luc). HeLa cells were cotransfected with the pM and pVP16 fusion constructs and the 5x Gal4-Luc reporter using calcium phosphate coprecipitation, as described above. Briefly, triplicate transfections used 2 µg of 5x Gal4-Luc reporter, 2 µg of VP16 fusion constructs, 2 µg of Gal4 fusion constructs, and 20 ng of Renilla-luciferase vector for normalization, in a 60-mm dish.

ChIP assay
HeLa cells were plated in MEM containing 5% twice charcoal-stripped fetal bovine serum at a density of 3 x 105 cells per 100-mm dish, 1 d before transfection. Cells were transfected by calcium phosphate coprecipitation (3) using 2 µg expression vectors and the PRE2-TATA-luciferase reporter plasmid. Twenty-four hours after DNA addition, the cells were washed and grown an additional 24 h, with 10 nM R5020 added 1 h before collection. ChIP was a modification of a previously described procedure (70). Briefly, cells were fixed in 1% formaldehyde for 10 min and the reaction terminated with 125 mM glycine. Cells were harvested in PBS (pH 7.4), washed and the cell pellets were stored at –70 C. 1–2 x 106 thawed cells were sonicated in 100 µl lysis buffer, microfuged and the supernatant was immunoprecipitated with anti-PR antibody (H-190; Santa Cruz Biotechnology, Santa Cruz, CA) using normal rabbit IgG as a control. Protease inhibitor cocktail (P8340; Sigma, St. Louis, MO) was present throughout cell and extract manipulations. Antibody-bound PR were captured on protein A Sepharose (Upstate Biotechnology, Lake Placid, NY), washed, eluted, treated with ribonuclease, and subjected to heat-induced cross-link reversal. Samples were then treated with proteinase K, phenol-chloroform extracted, ethanol precipitated, resuspended in water, and analyzed by PCR using the following primer pair: sense: 5'-atccccggtcgactctag; antisense: 5'-gcctttctttatgtttttggcg; which produced a 200-bp fragment. Band intensity was normalized to band intensity obtained from the input DNA. Both input bands and bands generated from amplification of the internal control glyceraldehyde-3-phosphate dehydrogenase gene were comparable. PCR samples were subjected to 27, 29, and 31 cycles and results from the optimal cycle number were reported.

Immunoblotting and Down-Regulation
Approximately equivalent protein expression levels and DNA binding activity of full-length and mutant PR constructs has been demonstrated previously (3). Immunoblots of PR wild-type and mutant constructs were probed with monoclonal antibodies, PgR1294 (DakoCytomation, Carpinteria, CA) and antiphosphoserine 294 (Zymed, South San Francisco, CA). Expression of PR fusion constructs was confirmed by immunoblot analysis of HeLa cell extracts as described previously (3) using monoclonal antibodies, Ab15 (Neomarker, Fremont, CA) for ER fusion constructs and PG21 (Upstate) for AR fusion constructs; monoclonal antibody PgR1294 (Dako-Cytomation) for VP16-NTB, VP16-NTBdL140, and VP16-NTA; and anti-Gal4 (CLONTECH) for G4-LBD and G4-SRC-1e. Briefly, 3 x 105 cells per 100-mm dish were plated 1 d before transfection. Two micrograms of expression plasmid were transiently transfected as described above. Cells were collected and lysed in buffer containing protease inhibitor cocktail. Equal amounts of extract protein were resolved by SDS-PAGE and probed with the appropriate antibody. Bands were detected by chemiluminescence. In down-regulation studies. With T47Dco cells stably expressing PR constructs, 5 x 105 cells were plated and allowed to grow for 24 h after which 10 nM R5020 were added successively as described for the transiently transfected constructs. After collecting the cells, extracts were prepared for immunoblotting as described (3).

Microarray Analysis
T47D-Y cells, a PR-negative, clonal derivative of T47Dco breast cancer cells have been previously described (55). They were stably transfected with PR-B, PR-A, or PRBdL140 as described (55). Briefly, for expression profiling, 5 x 105 cells were plated in 100-mm dishes and grown 24 h in antibiotic-free, Eagle’s MEM containing 5% charcoal-stripped fetal calf serum. The cells were then treated with either vehicle (ethanol) or 10 nM progesterone for 6 h, and total RNA was isolated. RNA was prepared from three independent experiments using each of the three cell lines. Poly(A)+ RNA was prepared; samples were labeled; and microarray analysis was performed with Affymetrix gene chips (HuFL-U95Av2) interrogating >12,000 transcripts. Student’s t tests (P < 0.05) were performed on triplicate minus vs. plus hormone samples to quantify statistical significance of transcript levels that changed by at least 1.5-fold in the PR-B, PR-A, or PR-BdL140 mutant containing cells as previously described (1). Each gene on each chip was normalized to the 50th percentile of all the measurements (hybridization intensity signals) taken from that chip. This normalizes for any global variations from chip to chip. Venn diagrams were created from these quantitative data. Complete gene lists are available as supplemental data. Dendrograms based on 75 genes with the highest fold up- or down-regulation based on PR-B were created using GeneSpring version 4.0 (Silicon Genetics, Redwood City, CA), then compared with the other two receptors. For this, expression levels of each gene were normalized by dividing the mean level in the presence of hormone by its mean level in the absence of hormone, effectively setting control levels to 1.0. Among other things, this allows qualitative assessments of relatedness for each gene among treatment groups.


   ACKNOWLEDGMENTS
 
We thank colleagues for gifts of reagents described in Materials and Methods, Drs. David Bain and Britta Jacobsen for communicating prepublication results, and Ted Shade for initial compilation of the microarray data.


   FOOTNOTES
 
This work was supported by grants from the National Cancer Institute (CA 26869), the Avon Foundation, the National Foundation for Cancer Research, and the Breast Cancer Research Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 8, 2006

Abbreviations: AF, Activation function; AR, androgen receptors; BUS, PR-B-upstream segment; CBP, cAMP response element binding protein (CREB)-binding protein; ChIP, chromatin immunoprecipitation; DBD, DNA binding domain; ER, estrogen receptors; ERE, estrogen response element; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptors; LBD, ligand binding domain; NR, nuclear receptor; NT-A, N-terminal region of PR-A; NT-B, N-terminal region of PR-B; NTBdL140, N-terminal region of PR-BdL140; PR, progesterone receptors; PRE, progesterone response element; PONDR, Predictor of Natural Disordered Regions; SRC, steroid receptor coactivator.

Received for publication March 3, 2006. Accepted for publication May 30, 2006.


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NURSA Molecule Pages Link:

Nuclear Receptors:   PR  |  AR
Coregulators:   SRC-1
Ligands:   17β-Estradiol  |  Dihydrotestosterone  |  Progesterone  |  R5020



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