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Ligand-Controlled Interaction of Histone Acetyltransferase Binding to ORC-1 (HBO1) with the N-Terminal Transactivating Domain of Progesterone Receptor Induces Steroid Receptor Coactivator 1-Dependent Coactivation of Transcription

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 693 (A.G.-M., B.D., G.M., H.L., L.R., M.G., M.L., S.W.), Récepteurs Stéroïdiens, Physiopathologie Endocrinienne et Métabolique, Faculté de Médecine Paris-Sud, 94276 Le Kremlin-Bicêtre Cedex, France; Centre National de la Recherche Scientifique (CNRS), Unité Propre de Recherche 9079 (A.C.), Institut Andre Lwoff, 94800 Villejuif, France; Biochimie Hormonale (E.M.), Hôpital de Bicêtre, 94275 Le Kremlin-Bicêtre, France; Department of Molecular and Cellular Biology (L.A.), Baylor College of Medicine, Houston, Texas 77030; Unité de Médecine Vasculaire (N.C.-B.), Hôpital Tenon Assistance Publique-Hôpitaux de Paris, 75020 Paris, France; and Department of Reproduction and Development and Obstetrics and Gynaecology (L.J.B., C.W.B.), Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Hugues Loosfelt, Institut National de la Santé et de la Recherche Médicale, Unité 693, Faculté de Médecine Paris-Sud, 63 rue Gabriel Péri, 94276 Le Kremlin-Bicêtre Cedex, France. E-mail: hugues.loosfelt{at}kb.u-psud.fr.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Modulators of cofactor recruitment by nuclear receptors are expected to play an important role in the coordination of hormone-induced transactivation processes. To identify such factors interacting with the N-terminal domain (NTD) of the progesterone receptor (PR), we used this domain as bait in the yeast Sos-Ras two-hybrid system. cDNAs encoding the C-terminal MYST (MOZ-Ybf2/Sas3-Sas2-Tip60 acetyltransferases) domain of HBO1 [histone acetyltransferase binding to the origin recognition complex (ORC) 1 subunit], a member of the MYST acetylase family, were thus selected from a human testis cDNA library. In transiently transfected CV1 cells, the wild-type HBO1 [611 amino acids (aa)] enhanced transcription mediated by steroid receptors, notably PR, mineralocorticoid receptor, and glucocorticoid receptor, and strongly induced PR and estrogen receptor coactivation by steroid receptor coactivator 1a (SRC-1a). As assessed by two-hybrid and glutathione-S-transferase pull-down assays, the HBO1 MYST acetylase domain (aa 340–611) interacts mainly with the NTD, and also contacts the DNA-binding domain and the hinge domains of hormone-bound PR. The HBO1 N-terminal region (aa 1–340) associates additionally with PR ligand-binding domain (LBD). HBO1 was found also to interact through its NTD with SRC-1a in the absence of steroid receptor. The latter coassociation enhanced specifically activation function 2 activation function encompassed in the LBD. Conversely, the MYST acetylase domain specifically enhanced SRC-1 coupling with PR NTD, through a hormone-dependent mechanism. In human embryonic kidney 293 cells expressing human PRA or PRB, HBO1 raised selectively an SRC-1-dependent response of PRB but failed to regulate PRA activity. We show that HBO1 acts through modification of an LBD-controlled structure present in the N terminus of PRB leading to the modulation of SRC-1 functional coupling with activation function 3-mediated transcription. Importantly, real-time RT-PCR analysis also revealed that HBO1 enhanced SRC-1 coactivation of PR-dependent transcription of human endogenous genes such as -6 integrin and 11ß-hydroxydehydrogenase 2 but not that of amphiregulin. Immunofluorescence and confocal microscopy of human embryonic kidney-PRB cells demonstrated that the hormone induces the colocalization of HBO1 with PR-SRC-1 complex into nuclear speckles characteristic of PR-mediated chromatin remodeling. Our results suggest that HBO1 might play an important physiological role in human PR signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
PROGESTERONE RECEPTOR (PR) belongs to the type I nuclear receptor (NR) subfamily including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) (1). PR is a hormone-inducible transcription factor activated through a multistep mechanism. Hormone interaction with the C-terminal ligand-binding domain (LBD) induces a major conformational change that promotes receptor homodimerization and recognition via the DNA-binding domain (DBD) of specific hormone-responsive elements within promoter of target genes. Several activation functions (AFs) were characterized by cDNA mutagenesis. A low-level activation function AF2 is located in the LBD and is shared by other steroid receptors with high similarity. The N-terminal domain (NTD) of the longest human PR isoform (hPRB), contains two major activation functions (N-AFs), mapped as AF1 (2) and AF3 (3, 4), which are constitutively activated in mutant lacking the LBD. The shortest isoform (hPRA) corresponding to amino acids (aa) 165–933 of hPRB lacks AF3 and can form an heterodimer with hPRB or can compete with it as homodimer, resulting in transrepression of target gene expression (5). Transcriptional activity of agonist-bound hPRB results from a synergistic cooperativity between these N- and C-terminal functions, through a mechanism not yet clearly understood. The N-AFs are silenced by either unliganded or antagonist-bound LBD, in connection with a corepressor complex containing NR corepressor and silencing mediator of retinoid and thyroid hormone receptor (6). Upon binding of agonist ligand, the LBD promotes the recruitment of coactivators, the release of corepressors, and an ordered assembling of multiprotein complexes having either histone acetyltransferase (HAT) or non-HAT activity. The functional interaction of the NTD and the LBD has been shown to play an important role in the activation mechanism of PR (7, 8), ER (9), and AR (10), and to be facilitated by multiple associations of cofactors (7). Several coactivators are known to interact with PR, such as members of the p160/steroid receptor coactivator (SRC) family including SRC-1 (11), SRC-2 (12), and SRC-3 (13, 14, 15), or other proteins like TAT HIV interacting protein (Tip60) (16), CREB binding protein (CBP)-associated factor (pCAF) (17), the cointegrator p300/CBP (18), L7/switch protein for antagonists (19), steroid receptor RNA activator (20), E3 ubiquitin ligase Nedd4 (21), and Jun dimerization protein 2 (22). Identification and analysis of receptor-cofactor interactions are critical for studying the molecular mechanisms of ligand action in target tissues. During the past years, cloning of such molecules has been performed using transcriptional two-hybrid methods and NR LBD as bait proteins. The high transcriptional activity of the NTD of PR in yeast cells has impeded the use of similar techniques for cloning cofactors specific of this domain, resulting in the actual lack of data. To characterize NTD-specific cofactors involved in PR-dependent signaling, we used another technique, the Sos-Ras system (23), which allows the trapping of interactants in the yeast cytoplasmic membrane. We thus isolated HBO1 [histone acetyltransferase binding to the origin recognition complex (ORC) 1 subunit], also referenced as MYST2, which belongs to the MYST (MOZ, Ybf2/Sas3, Sas2, Tip60) acetyltransferase family (review in Refs.24, 25, 26), involved in transcriptional regulation, apoptosis, and DNA repair. HBO1 was initially cloned as interacting with ORC-1 subunit and with MCM2 (minichromosome maintenance protein 2 homolog) subunit of the prereplicative complex, both involved in initiation of DNA replication (27, 28). HBO1 interacts also with cyclin-dependent kinase 11^p58 (29), which is closely associated with transcriptional regulation and cell cycle progression. Recently, HBO1 has been isolated by the two-hybrid method as a hormone-dependent corepressor of AR (30), whereas Tip60 was found to coactivate steroid receptors (24). Our data show that HBO1 controls specifically the coupling of SRC-1 with AF3 of hPRB and with AF2, through a hormone-dependent mechanism involving the intramolecular association of PR LBD and NTD domains. HBO1 might be not only a new modulator of the progestative signaling but also might differentially regulate the other steroid receptors.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
HBO1 Interacts with the NTD of PR
To clone cofactors interacting with the NTD of PR, it was necessary to use other methods than conventional two-hybrid techniques, because this domain highly induced the transcription of reporter genes in yeast. We used the Sos-Ras trap technique, originally described by Aronheim et al. (23). Prey proteins encoded by a human testis cDNA library were targeted to membrane via a myristoylation domain encoded by a pMyr vector. As bait protein, we used a PR_1–592 fragment (Fig. 1A), devoid of nuclear localization signal (NLS), and fused to hSos carrier protein, a human ortholog of yeast Cdc25 gene product required in the yRas signaling pathway. Both library and bait vectors were used to transform cdc25H-2 yeast strain expressing a thermosensitive mutation, which can be complemented when Sos hybrids are conditionally trapped to the membrane via protein-protein interaction. A primary selection of 20 clones, growing at a nonpermissive temperature (37 C) when the bait synthesis was induced by galactose, was obtained among 5.10⁶ cotransformants tested. The corresponding library plasmids were then tested for a second-round selection in other yeast cells expressing Sos hybrids of either the full-length receptor (PR_1–930) or mutants corresponding either to the constitutive fragment (PR_1–663) or to the LBD (PR_663–930) in the absence or presence of hormone. Two clones were selected in cells growing conditionally when either the liganded Sos-PR_1–930 or the Sos-PR_1–663 hybrid was induced, but not in those producing Sos-PR_663–930 (Fig. 1B). Sequencing analysis has shown that the corresponding cDNAs encoded either aa 331–611 or aa 287–611 regions of the same protein, HBO1 (Fig. 1C), a member of the MYST acetylase family (GenBank accession no. AF074606).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Cloning of HBO1 MYST Domain A, Amino acid sequence alignment of PR deletion mutants used. The functional domains of PR are indicated: NTD, LBD, DBD, hinge (H) domains, NLS. AF1, AF2, and AF3 activation functions are shown. B, cDNAs encoding proteins interacting with PR1–592 fragment inserted into pSos were selected by the Sos-Ras trap technique as described in Materials and Methods. The second-round selection was managed using either PR1–930 or PR1–663 or PR663–930 fusion protein with Sos. The LBD-interacting protein JAB1 and a homodimerizable protein unrelated to PR (control) were used in parallel for control experiments. The cotransformants were dotted on selective agar plates containing either R5020 progestin (+) or vehicle alone (–), and either glucose repressor (Gluc) or galactose inducer (Gal) of bait biosynthesis. The plates were incubated either at permissive (25 C) or at nonpermissive (37 C) temperature and were scanned. The result obtained with one HBO1-encoding clone is shown (HBO331–611). C, Schematic alignment of HBO331–611 and HBO223–611 library peptides selected in panel B with HBO1 amino acid sequence. Putative functional domains are indicated as zinc fingers (ZF1 and ZF2), LAT, NLS, protein family signature (MYST), NTD (N-ter), and serine-rich subdomain (Ser-rich). D, pAS2 vector encoding either a HBO1 C-terminal fragment (HBO340–611) or the full-length HBO1 (HBO1–611) both fused to a GalDBD were used to transform a yeast strain containing a Galuas-LACZ reporter gene, and expressing the indicated PR fragments fused to a GalAD domain. Control experiments for background signals generated independently of the bait were done using the empty GalDBD and either the empty GalAD or GalAD-prey vectors. The cotransformants were grown either in absence (–) or presence (+) of 10–6 M R5020. ß-Gal activity per cell was measured as described in Materials and Methods. After subtracting back ground signal, results (means ± SEM from eight independent colonies) were calculated as fold increase of the bait-specific activity by setting the ß-Gal concentration produced by each corresponding GalDBD-bait vector with the empty GalAD vector to 1.0 (control).

To confirm the specificity of PR-HBO1 interaction, we used the classic yeast Gal4 two-hybrid technique, allowing the detection of protein-protein associations in the nucleus through induction of reporter gene activity. The full-length cDNA of HBO1 obtained by PCR and a deletion mutant coding the MYST domain (HBO_340–611) were coupled to the Gal4 DBD module (GalDBD). When both vectors were separately expressed in yeast cells containing a pGal1-LacZ reporter gene, the basal concentration of ß-galactosidase (ß-Gal) was enhanced by 5- and 8-fold, respectively (data not shown). Fusion proteins of either the full-length PR_1–930 or PR_1–663 constitutive mutant (Fig. 1A) with the Gal4 transactivating domain (GalAD) were coexpressed with each HBO1 hybrid. PR_1–663, but not PR_1–930, strongly interacted with both HBO1 derivatives in the absence of ligand (Fig. 1D). In contrast, the addition of R5020 induced the interaction of PR_1–930 with HBO1 and HBO_340–611. Therefore, the full-length HBO1, as well as its N-terminally truncated fragment, interacted with the N-terminal region of PR (aa 1–663) when the LBD was either absent or liganded.

Together these data indicate that the full-length HBO1 targets the PR_1–663 fragment via the MYST domain. Because the interaction is inhibited in the apo-receptor and is induced by the hormone in the absence of any other PR-specific cofactor, HBO1 is a candidate of special interest with which to study the hormonal control of cofactors coupling with the NTD.

HBO1 Enhances Transcription Mediated by Steroid Receptors and Interferes with Coactivator Functions
HBO1 has been found already to associate with the LBD but not the NTD of AR (30) and to inhibit transcriptional coactivation by the ARA70 cofactor in mammalian cells. Because progestins induced the coupling of the same protein with PR NTD, we wondered whether HBO1 could act through various mechanisms in type I NR-mediated transcription. Moreover, because SRC-1 is known to be a major coactivator of PR and ER (11) and to interact also with other steroid receptors, we wondered whether HBO1 could interfere with this cofactor. To investigate these questions, we cotransfected CV1 cells by HBO1 expression vector together with the individual steroid receptor and the corresponding reporter gene in the presence or absence of SRC-1a isoform. After 1-d treatment of transfected cells by the corresponding hormone, the reporter gene product [chloramphenicol acetyltransferase (CAT)] was measured by immunoassay, and we compared the various increases in receptor-mediated transcription provoked by HBO1 and/or SRC-1a coexpression over the threshold (set to 1.0 unit for each receptor) obtained in the absence of exogenous cofactor (Fig. 2A). After addition of HBO1 (white bars), ligand-induced activities of PR, GR, and MR were markedly increased (3-, 6-, and 7-fold signal, respectively; P < 0.001). In similar experimental conditions, ER and AR activities were also increased to a lesser extent (2-fold signal; P < 0.05). By comparison, SRC-1a (gray bars) increased PR-, ER-, and AR-specific transcription to a level similar to that obtained with HBO1 alone, whereas MR and GR activities remained unchanged. In contrast, the coexpression of HBO1 and SRC-1a (black bars) led to the cooperation of both proteins in PR- and ER-specific responses (7-fold signal; P < 0.001). HBO1-specific stimulations of GR and MR were decreased by SRC-1a coexpression, consistent with a possible inhibition of endogenous coactivators, which might be preferentially used by these receptors. Finally, HBO1 and SRC-1 did not cooperate in AR-mediated transcription. When the mouse mammary tumor virus-CAT reporter gene was used instead of progesterone response element (PRE)₂-TATA-CAT in similar experiences, a 10-fold excess of HBO1 vector over receptor decreased the hormone induction of AR activity by 20%, whereas PR-, MR-, and GR-driven transcriptions were still enhanced 2- to 6-fold (data not shown). PR and SRC-1 synthesis were not significantly modified by the coexpression of a 10-fold excess of HBO1 as controlled by Western blotting with anti-PR and anti-SRC-1 monoclonal antibodies (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 2. HBO1 Interferes with Steroid Receptor and Coactivator Functions A, CV1 cells were cotransfected by pSG5 vectors (1 µg/ml) expressing either PR, ER, GR, MR, or AR, and by either PRE2-TATA-CAT or ERE-TATA-CAT (for ER) reporter genes (3 µg/ml). Cells were further transfected as indicated by pSG5-HBO1 (8 µg/ml) and pSG5-SRC-1a (3 µg/ml) expression vectors and were incubated in the presence of 10 nM of the corresponding hormone: R5020, 17ß-estradiol, dexamethasone, aldosterone, or dihydrotestosterone. Relative CAT concentration was measured and normalized as described in Materials and Methods. The cofactor-specific stimulations (means ± SEM from three independent transfections) were calculated as fold increase of the corresponding basal hormonal signal obtained in the absence of cofactor (set to 1 for each receptor). B, CV1 cells were cotransfected by 1 µg/ml pSG5-PR and 3 µg/ml PRE-TATA-CAT reporter gene, in the absence (white squares) or presence (black squares) of pSG5-SRC-1a (3 µg/ml), together with increasing amounts (µg/ml) of pSG5-HBO1. Cells were incubated with 10 nM R5020 and CAT immunoassays and expression of results were as in panel A. C, As in panel B except that pSG5-ER vector (1 µg/ml) and ERE2-TATA-CAT reporter gene (3 µg/ml) were used and tested in the presence of 4 µg/ml pSG5-HBO1 and 10 nM 17ß-estradiol.

Our data show the ability of HBO1 to modulate transcription driven by steroid receptors through differential effects, depending on cofactor environment and on the receptor. HBO1-dependent stimulation could be functionally linked to SRC-1a coactivating functions in the case of PR and ER, whereas it could be putatively dependent of other cofactors in the case of GR, MR, and AR.

Multiple Hormone-Dependent Interactions of HBO1 with PR Domains
To determine whether the coactivating function of HBO1 was reliable with its direct recruitment by DNA-bound PR, we first examined the hormone dependence of both proteins’ association using the two-hybrid method in mammalian cells. We used plasmids encoding either wild-type PR or its deletion mutants fused to a GalDBD as baits (Fig. 3A), and the full-length HBO1 fused to a transactivating domain of VP16 (VP-HBO1) as preys. CV1 cells were cotransfected by the indicated plasmids and were grown in the absence or presence of R5020. In this system, because all GalDBD-PR hybrids could bind equally to the Gal4 reporter gene independently of the ligand, any change in their basal transcriptional activity (set to 1.0 for each hormone condition) reflected the recruitment of VP-HBO1 (Fig. 3B). The full-length VP-HBO1 bound to the wild-type receptor only when the hormone was present (P < 0.01). When the LBD was deleted, leading to the production of a constitutively activated mutant (GalDBD-PR_1–663), the recruitment of VP-HBO1 occurred independently of the ligand (Fig. 3B), as previously observed in yeast cells (Fig. 1C). In the absence of NTD, the hormone induced the recruitment of VP-HBO1 by either PR_547–930 (DBD-LBD) or PR_663–930 (LBD) mutants to similar levels (P < 0.01), whereas a lower but significant induction was also raised in the absence of hormone (P < 0.05). Therefore, HBO1 was recruited by both PR N- and C-terminal domains constitutively bound to DNA through a hormone-dependent mechanism in mammalian cells. Under hormone depletion conditions, the LBD inhibited the interaction of HBO1 with the N terminus of PR.

View larger version (28K): [in this window] [in a new window]   Fig. 3. HBO1 Associates with PR Subdomains in Mammalian Cells and in Cell-Free System A, Peptide sequence alignment of PR and mutants used for the experiments. B, CV1 cells containing a Galuas-TATA-CAT reporter gene were cotransfected by vectors expressing the indicated PR fragments fused with the GalDBD (1 µg/ml), and either the full-length HBO1 fused with the VP16 transactivating domain (VP-HBO1) (3 µg/ml) or empty VP vector. Control experiments for background signal generated independently of the bait were processed with the empty GalDBD and VP-HBO1 (or empty VP) vectors. The cells were grown in the absence (white bars) or presence (black bars) of 10 nM R5020 for 24 h. Relative CAT concentration was measured and normalized as detailed in Materials and Methods. After subtracting the corresponding background signal, results (means ± SEM from three independent experiments) were calculated as fold increase of the corresponding GalDBD hybrid activity (control set to 1.0 for the empty VP vector at each ligand concentration). C, GST pull-down assays were performed as described in Materials and Methods by incubating the indicated 35S-labeled PR peptides with GST-HBO1 hybrid or empty GST coupled to Sepharose beads. Aliquots of bound proteins were analyzed by SDS-PAGE and autoradiography and were compared with the corresponding labeled peptide not incubated with the matrix (Input). Definitions are as shown in Fig. 1.

Because the HBO1 MYST domain was found to associate in yeast cells with the NTD but not with the LBD of PR (Fig. 1B), we further mapped the region involved in these multiple contacts to construct a model of the molecular organization of the putative HBO1-PR complex. Various PR-overlapped domains fused to the GalAD module (Fig. 4A) were used as preys for yeast two-hybrid assays. Deletion mutants of HBO1 were produced as fusion proteins with the GalDBD (Fig. 4B), coding aa 1–340 (HBO-N), 340–611 (HBO-C), and 465–611 (HBO-CZ). All these hybrids were addressed to the nucleus via a NLS located in the corresponding Gal4 subdomain tag. As shown in Fig. 4C, PR domains interacting with the full-length HBO1 were mapped to the NTD (aa 1–547), the DBD-hinge (aa 547–663), and the LBD (aa 663–930). The HBO-C mutant interacted with all PR fragments except the LBD. The further deletion of a region containing the MYST zinc finger (HBO1-CZ) abolished the interaction with the NTD but retained that with the DBD-hinge region. Finally, the NTD of HBO1 (HBO-N) was responsible for the hormone-induced interaction with the LBD structure. In the absence of hormone, a background interaction of either HBO1 or HBO-N was raised by the deletion of the PR NTD, whereas it did not occur with the full-length PR.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Cross-Interactions of PR and HBO1 Subdomains A, Alignment of amino acid sequences of wild-type PR with its deletion mutants used for the experiment. B, Alignment of HBO1 and its deletion mutants. C, Yeast two-hybrid assays were performed using the HBO1 mutants shown in panel B fused to GalDBD, and PR mutants shown in A fused to GalAD. Control experiments, ß-Gal assays, and expression of data were as in Fig. 1D (means ± SEM from eight independent colonies). Control values obtained with the corresponding GalDBD hybrid and the empty GalAD vector were all set to 1.0 for each concentration of ligand. D, Molecular model of the PR-HBO1 complex (symbols are shown in panel A). N, N-terminal extremity; C, C-terminal extremity; other definitions are as shown in Fig. 1.

HBO1 Interacts with SRC-1a and Enhances Cofactor Recruitment by the Hormone-Bound LBD
We have shown that HBO1 enhanced the coactivating functions of SRC-1a on PR- and ER-mediated transcription. To analyze this mechanism, it was first necessary to determine whether HBO1 and SRC-1 might be directly coupled in the absence of receptor. We tested this hypothesis in mammalian cells by coexpressing HBO1 fused to a Gal4 DBD module, and SRC-1a together with a Gal4-CAT reporter gene. Whereas the GalDBD-HBO1 hybrid only slightly enhanced the basal transcription of the reporter gene (Fig. 5A), coexpression of SRC-1a increased this signal moderately, indicating that both factors might be functionally linked in the absence of exogenous receptor. A further increase in HBO1-dependent transcription was observed when a VP16 AD domain was fused to the N-terminal extremity of SRC-1a (VP-SRC-1). Such enhancement would not be detected if SRC-1a acted through transcription independently of HBO1 association, and therefore suggested that DNA-tethered HBO1 recruited the cofactor. Furthermore, coexpression of a 10-fold excess of wild-type HBO1 over GalDBD-HBO1 hybrid abolished the two-hybrid signal, showing that the association of VP-SRC-1 with DNA-bound HBO1 was squelchable (Fig. 5). Coexpression of an equal excess of HBO_1–340 mutant instead of the wild-type HBO1 provoked a similar squelching of GalDBD-HBO1/VP-SRC-1-productive association, whereas the HBO_340–611 mutant was only 20% as efficient (data not shown). These data strongly indicated that HBO1 and SRC-1 can interact independently of the receptor. However, transcription raised by the DNA-bound HBO1-SRC-1 complex in the absence of any heterologous transactivating domains was increased 15-fold by coexpressing the liganded PR in the same system (data not shown), enlightening the role of the hormonal control of HBO1 and SRC-1-coupled functions.

View larger version (29K): [in this window] [in a new window]   Fig. 5. HBO1 Interacts with SRC-1a A, A pM vector (1 µg/ml) encoding either HBO1 fused to GalDBD or GalDBD alone (empty) was coexpressed in CV1 cells with 3 µg/ml of a vector encoding either SRC-1a (wt) or SRC-1 fused with VP16 AD domain (VP) or the corresponding empty vector, and with 10 µg/ml of pSG5-[Flag]-HBO1 (HBO1wt) as indicated. Total DNA concentration was maintained constant using pSG5-[Flag] vector. CAT assays were performed, and values were normalized to total protein content as described in Materials and Methods (means ± SEM from three independent transfections). B, Yeast two-hybrid assays were performed using pAS2 vectors expressing GalDBD hybrids of either HBO1 or mutants (N, C, CZ) described in Fig. 4B, and GalAD-SRC-1 hybrid (AD). Control experiments for background signals, ß-Gal assays, and expression of results were as in Fig. 1D (means ± SEM from eight independent colonies). Control values obtained with the corresponding GalDBD-bait hybrid and the empty GalAD vector were all set to 1.0 (control for only GalDBD-HBO1 is shown). C, GST-pull-down assays were performed as described in Materials and Methods using either GST-HBO1 hybrid or empty GST coupled to Sepharose beads and 35S-labeled SRC-1a deletion mutants (A–E). The latter mutants are aligned (left panel) under the wild-type SRC-1a amino acid sequence including basic helix-loop-helix/periodicity/aryl hydrocarbon receptor nuclear translocator/single-minded domain (bHP), receptor interacting domain (black bars), coactivating domain (CD), and poly-Q region (Q). Interactions were analyzed by SDS-PAGE and autoradiography (middle panel), and by scintillation counting (right panel) of 35S-labeled proteins eluted from the matrix compared with the corresponding input. wt, Wild type.

Next, we performed in vitro GST pull-down assays to map the SRC-1 regions involved in the interaction in a cell-free environment. ³⁵S-labeled SRC-1 and deletion mutants were tested for their interaction with GST-HBO1. As shown in Fig. 5C (middle panel), HBO1 interacted with the aa 782-1208 region of SRC-1. This region encompasses a transactivating domain, the domain interacting with CBP and pCAF cofactors, several LXXLL elements for NR recognition (NR boxes), and the poly-Q domain. A weaker interaction was detected with the N-terminal SRC-1_1–987 mutant and was further abolished by deleting aa 782–987. Note that the interaction of HBO1 with an SRC-1_782–1208 fragment (40% of the total labeled input) was higher than with the full-length SRC-1 (15%). Furthermore, by increasing the salt concentration from 50 mM to 150 mM in the washing buffer, we observed a 60% decrease in the interaction of wild-type SRC-1 but not of its aa 782-1208 fragment. This observation might indicate that some domains of SRC-1 would partially mask the interacting domain in a cell-free system.

Dual interaction of HBO1 with both SRC-1 and PR could be the first step of the receptor potentiation mechanism. SRC-1 is recruited by the hormone-bound LBD, but it binds also, although more weakly, to the constitutive NTD of PR devoid of LBD (33). HBO1 interacts also with both PR domains but apparently with inverted affinities when compared with SRC-1. In an attempt to determine how association of HBO1 and PR would impact the assembling of SRC-1 with both PR domains, we performed a two-hybrid assay (Fig. 6) in CV1 cells transiently transfected by the Gal4 reporter gene together with either the full-length PR, or PR_547–930 (DBD-LBD) or PR_1–663 (NTD-DBD) subdomains, fused to a GalDBD module. VP-SRC-1 expression vector was added, and the cells were grown in the presence of hormone. In the absence of HBO1, the SRC-1-specific interaction resulted in 2-, 2-, and 4-fold stimulation of the reporter gene activity mediated by PR_1–930, PR_1–663, and PR_547–933, respectively, and were plotted as 100% values for each series. Recruitment of VP-SRC-1 by GalDBD-PR was stimulated in a dose-dependent manner by increasing amounts of wild-type HBO1 expression vector. Deletion of LBD (PR_1–663) abolished almost completely this effect. In contrast, when the NTD was deleted (PR_547–930), VP-SRC-1 recruitment remained highly dose dependent of HBO1 vector. Therefore, in this system, the enhancement of SRC-1 interaction with PR required the presence of the LBD, although HBO1 was potentially able to target both the NTD and SRC-1.

View larger version (24K): [in this window] [in a new window]   Fig. 6. HBO1 Enhances the LBD-Dependent Recruitment of SRC-1a by PR CV1 cells were cotransfected by Galuas-TATA-CAT reporter gene, by 1 µg/ml of either GalDBD-PR1–930 (white squares) or GalDBD-PR1–663 (white triangles) or GalDBD-PR547–930 (white circles), and by 3 µg/ml of either VP-SRC-1 or VP empty vector. Each series was also transfected by increasing amounts of pSG5-[Flag]-HBO1, while total DNA concentration was kept constant using Herring sperm DNA. Cells were grown for 24 h in the presence of 10 nM R5020, and SRC-1-specific interaction signals obtained in normalized CAT assays were plotted for each indicated series as percent of the basal CAT concentration raised in the absence of HBO1 (means ± SEM from three independent experiments). One control experiment using GalDBD-PR1–930 and pSG5-[Flag] mock vector instead of pSG5-[Flag]-HBO1 is superposed (black circles).

HBO1 Induces the Functional Coupling of SRC-1 with PR N-AFs
Hormone-induced activity of PR molecule involves SRC-1 recruitment by the ligand-activated LBD and the coupling of PR derepressed N-AFs with cofactors and transcriptional machinery (33). PR intramolecular interaction between the LBD and the NTD plays a critical role in this mechanism and is also promoted by SRC-1 (7, 8, 34). Because we found that HBO1 strengthened SRC-1 recruitment by the LBD and was tightly bound to the NTD, our data suggested that HBO1 might activate these steps. To test this hypothesis, we first compared the transcriptional effects of HBO1 on N- and C-terminal AFs of PR either by expressing the corresponding PR domains separately or by coexpressing both of them from distinct vectors in CV1 cells. We used vectors coding PR_1–930, PR_547–930 (DBD-LBD), PR_1–663 (NTD-DBD), or PR_663–930 fused to a constitutive SV40 NLS (LBD_NLS). As shown in Fig. 7A, HBO1 enhanced the basal hormone-induced activities of PR (lanes 1 and 2) and DBD-LBD (lanes 3 and 4). Deletion of the LBD produced a mutant (NTD-DBD) harboring a constitutive activity that was 50% lower than the hormone-induced PR transcriptional signal (lane 5). HBO1 further enhanced 2-fold these constitutive N-AFs (lane 6), independently of the presence of LBD and hormone. As previously shown in Fig. 6, this effect was not due to any enhancement of SRC-1 interaction with LBD-free NTD. Coexpression of LBD_NLS fragment together with the constitutive NTD-DBD (lane 7) increased stimulation of the N-AFs, consistent with hormone-dependent PR interdomain association and with the partial restoration of a synergistic mechanism. HBO1 further enhanced 3-fold the latter signal (lane 8). Therefore, HBO1 has the ability to stimulate both independent PR AFs and to also induce PR interdomain synergy. We also observed that a truncated HBO1_1–340 mutant used instead of HBO1 was unable to enhance significantly neither PR and NTD-DBD activities nor interdomain synergy, but was able to fully stimulate DBD-LBD fragment (data not shown). This further indicated that HBO1 MYST domain was not mandatory for AF2 response, whereas it was required for a major function acting through PR interdomain synergy.

View larger version (34K): [in this window] [in a new window]   Fig. 7. HBO1 Induces the Functional Coupling of SRC-1 with PR N-AFs The plasmids used for transfection experiments were pSG5 vector expressing either PR1–930 (PR) or PR547–930 (DBD-LBD), or PR1–663 (NTD-DBD) (1 µg/ml) together with or without a 3-fold excess of pSG5-[Flag-NLS]-PR663–930 plasmid expressing PR663–930 fused to a Flag-tagged SV40 NLS (LBDNLS). Total DNA concentration was kept constant in each situation using pSG5-[Flag] vector. CV1 cells were transfected with the indicated plasmids together with a PRE2-TATA-CAT reporter gene and were treated for 24 h by 10 nM R5020. CAT immunoassays were performed and normalized as described in Materials and Methods. A, HBO1 induces PR AFs and interdomain synergy. The cells received the indicated plasmids together with pSG5-HBO1 vector (HBO1) (5 µg/ml) or not. Results are expressed as percent of the hormone-dependent signal (100%) measured in cells expressing PR1–930 in the absence of any added cofactors (means ± SEM from three independent transfections). B, HBO1 main function acts through a mechanism independent of SRC-1 coupling with the LBD. The cells received the indicated PR-derived plamids supplemented by HBO1 vector (5 µg/ml) together with (black bars) or without (gray bars) pSG5-SRC-11199–1411 (SRC-DN) dominant-negative mutant (10 µg/ml). Results are expressed as percent of the activity of each corresponding PR combination of vectors (100%, white bars) in the absence of any added cofactor (means ± SEM from three independent transfections). C, The major function of HBO1 regulates the N-AFs through its MYST domain. Plasmids further used were 3 µg/ml pSG5-SRC-1a (SRC-1wt) and 5 µg/ml of either pSG5-[Flag]-HBO1 (HBO1wt) or pSG5-[Flag]-HBO1340–611 (HBO1-C) as indicated (black bars). Values are expressed as in panel B. D, HBO1 MYST domain induces hormone-dependent coupling of SRC-1 with PR NTD. Cells expressing the NTD-DBD together with the LBDNLS plasmid further received 3 µg/ml of a plasmid expressing either SRC-1 (black bars) fused to a VP16 AD (VP-SRC-1) or the empty VP domain (white bars) together with HBO1-C vector at the indicated concentrations (micrograms per ml). Each hormone-induced signal was subtracted for the corresponding signal obtained in the absence of hormone, and the results were expressed as percent of the basal level (100%) obtained in the absence of HBO-C (means ± SEM from three independent transfections). Wt, Wild type.

It was conceivable that HBO1 acted either directly by inducing the N-AFs or indirectly by facilitating PR interdomain association. Our data predicted that deleting the NTD of HBO1 would eliminate its SRC-1-dependent function linked to the LBD, as well as its putative PR interdomain bridging effect, and would allow direct testing of the MYST domain function. We therefore compared the HBO_340–611 deletion mutant (HBO-C) with the wild-type HBO1 in the presence of ectopically expressed SRC-1a and of either PR or its derivated mutants. As shown in Fig. 7C, HBO1 enhanced SRC-1-coactivated transcription mediated by association of PR (lane 2), DBD-LBD (lane 5), NTD-DBD (lane 8), and PR subdomains (lane 11). Importantly, although, as expected, HBO-C did not stimulate DBD-LBD fragment (lane 6), it retained the ability of HBO1 to stimulate PR (lane 3) but lost that for NTD-DBD (lane 9). This lost effect was recovered by the coexpression of LBD_NLS fragment to which HBO-C cannot bind (lane 12). Furthermore, we noted that the previous effects of HBO-C were highly decreased when SRC-1 was omitted (data not shown). We further controlled, by two-hybrid assay, that the MYST domain was not involved in any indirect PR subdomain association mechanism (data not shown). These experiments indicated that the HBO1 MYST domain acted through an SRC-1-dependent mechanism in PR interdomain synergy independently of any HBO1-specific connection with the LBD.

We next tested the hypothesis that the LBD was required to trigger a cooperative function of the MYST domain in hormone-dependent coactivation of PR N-AFs by SRC-1 (Fig. 7D). We transfected CV1 cells with various amounts of HBO-C vector and with either VP-SRC-1 fusion protein or VP domain together with PR_1–663 and LBD_NLS fragments. We compared the hormone-induced stimulations of PRE₂-TATA-CAT reporter gene obtained for both VP vectors. As shown in Fig. 7D, the VP-SRC-1-specific signal was clearly enhanced by increasing the synthesis of HBO-C. As neither PR interdomain association nor SRC-1 interaction with the LBD fragment was dependent of HBO-C, the hormonal control of the MYST function was directly addressed from the LBD to the NTD. This mechanism was also in agreement with the ability of HBO1 to relieve the inhibition of PR-mediated transcription by dominant-negative SRC-1 shown in Fig. 7B. One may therefore propose that the NTD-coupled MYST domain function is triggered by the interaction of both N and C-terminal domains of PR, and controls the interaction of SRC-1 with the NTD.

Our data together strongly indicate that HBO1 acts through two distinct mechanisms. The major function of HBO1 resides in its MYST domain, which potentiates PR interdomain synergy by enhancing the functional coupling of SRC-1 with the N-AFs. A minor function resides in its NTD consisting in SRC-1-dependent stimulation of AF2, probably by promoting contacts between the LBD and SRC-1.

HBO1 Is Physiologically Linked to Progestative Signaling in Living Cells
Because HBO1 function targeted mainly the N-AFs of PR, it was important to analyze its effect through expression of human PR isoforms, which differ in their NTDs. We produced two HEK293 cell lines stably expressing either hPRA or hPRB isoforms as controlled by Western blot (Fig. 8, top inset). These cells were then transfected by equal amounts of either HBO1 or SRC-1 or in combination with a PRE₂-Luciferase reporter gene. As shown in Fig. 8, HBO1 was unable, unexpectedly, to modify either the basal hormonal response or the SRC-1-dependent output signal of PRA (white bars). Conversely, it dramatically enhanced hPRB coactivation by SRC-1 (black bars). Note that the scaling of the y-axes is different because the transcriptional activity of hPRB is much higher than that of hPRA. Comparison of the activity of both isoforms as fold induction of the basal hormonal signal (Fig. 8, right) shows that HBO1 activates only the hPRB-SRC-1 complex (11-fold), although their respective responses to SRC-1 alone are similar (4-fold). As hPRA lacks only the 1–164 N-terminal region of hPRB containing notably AF3, these experiments indicate that PR specificity of HBO1 function targets the hormone-dependent coactivation of AF3 by SRC-1 and therefore is selective for the hPRB function.

View larger version (28K): [in this window] [in a new window]   Fig. 8. Differential Effects of HBO1 on PR-Mediated Transcription in Human Cells A, HBO1 modulates PRB but not PRA. HEK293 cell lines expressing stably either hPRB or hPRA isoform were obtained as described in Materials and Methods. Alignment of both isoform domains is shown in the top inset (ID: inhibiting domain; other symbols are as in Fig. 1A). Expression of the corresponding clonal cells was analyzed by Western blot using anti-PR monoclonal antibody (or anti-Actin antibody) and compared with nontransfected HEK (right top inset). HEK-hPRA and HEK-hPRB cell lines were transfected as indicated by pSG5-HBO1 (2 µg/ml), pSG5-SRC-1a (2 µg/ml) together with PRE2-luciferase reporter gene (2 µg/ml), and a ß-Gal vector. After 24 h with 10 nM R5020, luciferase assays were performed as described in Materials and Methods and normalized for total cellular protein content and ß-Gal activities. Results are expressed either as relative luciferase units (left) or as fold induction of the basal hormone-induced signal set to 1 for each PR isoform (right bottom inset). B, HBO1 differentially modulates human endogenous genes. An endometrial Ishikawa cell line stably expressing hPRB (endo PRB) was treated by either 10–8 M R5020 or vehicle for 2 h. Total RNA was extracted, reverse transcribed, and analyzed by real-time quantitative PCR as described in Materials and Methods. The 6-integrin, 11ß-HSD2, and amphiregulin mRNA levels are normalized to 18S RNA (relative mRNA), and results are expressed as femtomoles of specific mRNA per nanomole of 18S RNA (means ± SEM, n = 6). The parental Ishikawa cells were transfected by 0.3 µg/well of pSG5-PRB (transient PRB) and by 0.6 µg of either pSG5-SRC-1, or pSG5-HBO1, or both. The cells were all treated by 10–8 M R5020, and relative mRNA expression was analyzed by quantitative RT-PCR. Results are expressed as fold induction of normalized mRNA obtained in the presence of PRB alone. P values obtained by Student’s t test are indicated for each significant comparison.

Finally, we attempted to correlate our results with the hormone-dependent formation of a complex containing hPR-SRC-1 together with HBO1 in living cells. We used another experimental approach by analyzing the effect of hormonal treatment on the subcellular distribution of the three partners. PR and HBO1 are known to be mainly nuclear (30, 40), whereas SRC-1 can be detected in the cytoplasm as well as in the nucleus, depending on the balance between kinetics of neosynthesis and nuclear import and export mechanisms (41). We have shown previously that a SRC-1 mutant deleted of its NLS and fused to enhanced green fluorescent protein (EGFP) (EGFP-SRC-1_NLS) was exclusively cytoplasmic, and that it could be imported conditionally into the nuclei of COS-7 cells expressing liganded PR (41). As PR shuttles between the nucleus and the cytoplasm, this trafficking results from protein-protein interaction of both partners occurring temporarily in the cytoplasm. We used this SRC-1 mutant instead of wild-type SRC-1 as a dynamic fluorescent marker to facilitate detection of the hormone dependence of the three partners’ subcellular distribution (Fig. 9). Immunofluorescence detection of HBO1 (blue) and PR (red) was obtained by mouse anti-PR and rabbit anti-HBO1 antibodies and the corresponding species-specific-labeled anti-Ig. Control experiments were done to verify that EGFP-SRC-1_NLS was exclusively cytoplasmic in HEK293 cells by comparison with the main nuclear localization of EGFP-SRC-1 (Fig. 9, left top images). Although endogenous HBO1 was detected at low levels in the nucleus, no colocalization with EGFP-SRC-1_NLS could be detected in that condition (Fig. 9, left). When HBO1 was ectopically coexpressed with EGFP-SRC-1_NLS in HEK293 (Fig. 9A), the HBO1-specific immunofluorescence was detected in cytoplasmic speckles formed by EGFP-SRC-1_NLS, whereas EGFP fluorescence was also clearly emitted from some nuclear particles containing HBO1. This indicated that, in the absence of PR, HBO1 could partially import SRC-1_NLS in the nucleus and, conversely, that high cytoplasmic accumulation of SRC-1_NLS could retain a fraction of neosynthetized HBO1 outside of the nucleus, both effects being dependent on protein-protein interaction mechanisms. Next, if both proteins were coexpressed in HEK293-hPRB cells, the HBO1-SRC-1_NLS cytoplasmic speckles (cyan) were unchanged (Fig. 9B). No colocalization with the unliganded PR could be significantly detected in the nucleus because only random superposition of blue and red particles was observed. When the cells were treated by hormone for 4 h (Fig. 9C), a major fraction of SRC-1_NLS cytoplasmic speckles was clearly shifted to the nucleus, and an intranuclear rearrangement of PR distribution occurred with the formation of nuclear particles containing PR and either HBO1 (magenta) or SRC-1_NLS (yellow), or both (white). We verified that HBO1 could also be codetected with PR in similar nuclear speckles containing the wild-type SRC-1 fused to EGFP (data not shown). However, in the absence of PR, HBO1 did not clearly modify the nuclear distribution of SRC-1. Similar experiments were also performed with the PRA-expressing cell line. The colocalization of HBO1 with the PRA-SRC-1 complex was also observed as smaller and more diffuse speckles (data not shown). These combined results strongly support that a hPRB-SRC-1-HBO1 complex can exist in vivo and that HBO1 might participate physiologically in transcriptional progestative signaling.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Hormone-Induced Colocalization of HBO1 with hPRB and SRC-1 in HEK Cells HEK293 cells permanently expressing hPRB (PRB+) or not (PR–) were cotransfected as indicated by a vector coding EGFP tagged to either SRC-1 (EGFP-SRC-1) or a SRC-1 mutant deleted of its NLS (EGFP-SRC-1NLS) together with pSG5-HBO1. After a 4-h incubation with 10 nM R5020 (or vehicle alone), cells were processed for differential immunofluorescence labeling of PR (red channel) and HBO1 (blue channel). The green fluorescence is emitted by EGFP tag. The cells were analyzed by confocal microscopy as described in Materials and Methods. Image scaling in shown with black bars (5 µm). Controls: The control views on the left side show that, in PR(–) cells not transfected by HBO1 vector, SRC-1NLS (NLS) is exclusively detected in the cytoplasm by comparison with the main nuclear localization of SRC-1 (wt). Endogenous HBO1 (endo-HBO1) is detected at low levels in the nucleus. A, Ectopically expressed HBO1 is partially colocalized with SRC-1NLS in large cytoplasmic speckles and in nuclear microgranulations (cyan color) in PR(–) cells. B, The nuclear unliganded PR (PRB+ cells, no ligand) does not colocalize with any one of both cofactor partners. C, In PRB+ cells treated with R5020, the nuclear liganded PR interacts with HBO1 (nuclear magenta particles), whereas a major fraction of SRC-1NLS is imported in the nucleus through the nucleo-cytoplasmic shuttling of PR (nuclear yellow particles). HBO1 and SRC-1NLS coparticles are now mainly nuclear. The three interacting proteins are colocalized in the white and large nuclear speckles. These results provide evidence for in vivo interaction of HBO1 with PRB-SRC-1 complex in living cells in response to hormone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

The NTD of HBO1 is unique among the MYST acetylase family. It contains an additional canonical C2H2C zinc finger very similar to a motif found as clusters in various transcriptional factors such as the Myt1/NZF family (46) and postmitotic neural gene-1 (47) involved in neurogenesis. The N terminus of Tip60 does not share any sequence similarity with HBO1. Recently, Lemercier et al. (48) demonstrated that cyclin-dependent phosphorylation of two serines present in this region induced acetylation of free histones by the Tip60 MYST domain. The N-terminal serine-rich domain of HBO1 is also putatively phosphorylatable and thus might be targeted by a similar regulatory process. This region was found to associate with the LBD through the aa 689–930 region in a cell-free system, and also to be mainly involved in HBO1 interaction with SRC-1, as supported by the squelching experiment. SRC-1 associates with PR-liganded LBD mostly via its C-terminal LXXLL NR box, and with the NTD via a central domain containing three NR boxes (33). The 782-1139 region of SRC-1, which we found to mainly interact with HBO1, encompasses the coactivator domain involved notably in association with the CBP/p300 integrator (49) and pCAF histone acetylase (50) and the poly-Q region. It has been also reported to bind PR in intact cells but not in cell-free systems (33), suggesting that HBO1 might be one of the putative cellular components expected to bridge this region to PR NTD.

Those multiple contacts between HBO1, PR, and SRC-1 were synergistically correlated to the amplification of PR transcriptional activity. The major transcriptional function of SRC-1 is to simultaneously connect cofactors, such as CBP/p300 and pCAF, and NRs. HBO1 can enhance both N- and C-terminal activation functions of PR independently, whereas, in the absence of PR and SRC-1, it fails to activate transcription significantly when tethered to a reporter gene via a heterologous DBD. Furthermore, the HAT activity of HBO1 (27) is weak as for SRC-1 (50) and therefore is likely either to cooperate with other HAT cofactors or to regulate directly the activity of PR-SRC-1 complex by a factor acetyltransferase (FAT) activity. Such mechanism has been recently involved in HBO1-dependent formation of the prereplication complex required for DNA replication (51). HBO1 might also act through differential mechanisms regulating type I NRs other than PR, in particular MR and GR, and, to a lesser extent, ER and AR. The stimulation produced by the HBO1-SRC-1a tandem was only observed with ER and PR. Because the effect on PR-mediated transcription was also found in some other transfected cell lines, i.e. HEK293, Hela, and Ishikawa, the HBO1-SRC-1 coupled function was independent of cell context when ectopically expressed proteins were used. In contrast, coexpression of SRC-1a decreased the HBO1-dependent transactivation of GR and MR. This could be explained by the fact that these receptors recruit preferentially GR-interacting protein 1/transcriptional intermediary factor 2 or SRC-1e (12, 52, 53) and that coassociation of HBO1 with receptors and exogenous SRC-1a might result in a squelching of HBO1 coactivating functions linked to other more potent coactivators. This could also explain the discrepancies between our data and that of Sharma et al. (30), which might reflect some differential mechanisms involving competitive AR cofactors. Such discrepancies were also found in the case of Tip60 (16, 43, 54). Therefore, we suggest that HBO1 modulates differentially the association of NRs with the SRC family of cofactors.

Northern blot experiments showed that HBO1 is ubiquitously expressed in tissues and in various cell types (27, 30). However, overexpression of HBO1 was found in ovaries (27) and testis (30). We have studied by immunohistochemical method the expression of HBO1 in female genital tract (data not shown). We found that HBO1 and PR are coexpressed in smooth muscle cells of myometrium and fallopian tubes, in luteal cells of corpus luteum, and breast epithelial cells. In endometrium, HBO1 was detected in stromal cells but not in epithelial cells. HBO1 expression is therefore likely to be subjected to unknown hormonal regulation and might play a specific role in reproductive functions.

Because the MYST acetylase function of HBO1 is not yet defined, we focused mainly on the HBO1-dependent mechanism of SRC-1 coupling with PR. Multiple interactions of HBO1 resulted in the enhancement of SRC-1 recruitment by PR through two distinct mechanisms. The main contact between the LBD-bound HBO1 NTD and SRC-1 enhanced AF2 activity independently of PR NTD and of HBO1 acetylating domain. However, this stimulation was only detected in the absence of the NTD and probably reflected a minor mechanism leading to increased specificity of the PR/SRC-1 tandem and to the stabilization of MYST domain interaction. In contrast, the NTD-interacting MYST domain strengthened conditionally the association of SRC-1 with PR NTD when the liganded LBD was present. These interactions resulted in a high enhancement of both PR-specific SRC-1 coactivating function and interdomain synergy, underlying probably a hormone-induced transconformation of PR molecule. Interaction between the NTD and the hinge-LBD domains has already been shown to be crucial in coactivator modulation of ER (55), AR (56), and PR (7, 8). Other reports showed that agonist-liganded LBD mediates a cis-repression of the N-AFs in cell-free transcription systems that could be relieved by the addition of cofactors (57). Derepression of the N-AFs is likely to be dependent not only on the structure of the ligand-bound LBD but also on the disruption of repressive interactions between the AF3 region and the DBD (3, 5, 58) and on the release of the inhibitory function present in the NTD (59). Significantly, HBO1 was able to relieve the inhibition of coactivator coupling with the NTD raised by a LBD-specific SRC-1 dominant-negative mutant. Dependent only of the N-terminally truncated MYST domain bound to the constitutive NTD-DBD-hinge region, this function was induced by the ligand-bound LBD but not by the apo-LBD. This argues against the possibility of a direct modification of SRC-1 itself by HBO1. It is instead consistent with a modification of structural elements present in the NTD-DBD-hinge domain and controlled by the hormone-dependent interaction of the LBD, leading to the enhancement of SRC-1 functional coupling. It remains unclear whether the neostructure of the NTD, coinduced by HBO1 and the LBD, directly involves a target site for SRC-1 or indirectly that of an unknown SRC-1 competitor. Moreover, our findings of differential effects on human PRA and PRB isoforms suggest a possible role of HBO1 as a down-regulator of PRB transrepression by PRA. Indeed, no HBO1-specific stimulation of PRA was observed, whereas the corecruitment of HBO1 and SRC-1 by the C terminus of PRA could potentially coactivate AF1 and AF2. The ability of HBO1 to amplify specifically the coupling of SRC-1 with AF3 might thereby allow the up-regulation of PRB transcriptional activity in competition with a PRA trans-dominant effect. SUMO-1-sumoylation of PR NTD has been described to release the intramolecular autoinhibition of both PR isoforms as well as transrepression mediated by the inhibiting domain present in the NTD (58). In contrast, our data indicate that the function of HBO1 may consist specifically in the derepression of the N terminus region of PRB by modifying contacts with cofactors. Selection of interacting coactivators is crucial for regulating PR target gene expression and is likely to be tissue specific (60) and to depend on the promoter context. The 6-integrin gene expression is induced by PRB (36, 37) through a PRE-binding site, and we found that HBO1 enhances its SRC-1-dependent coactivation in a endometrial Ishikawa cell line. No such binding site has been identified in the proximal region of amphiregulin and 11ß-HSD2 gene promoters (61, 62). PRB-specific induction of these genes (35, 37–39, 63) could therefore occur indirectly through interaction with other transcription factors as for the glycodelin gene (64, 65). The fact that HBO1 differentially modulates PR-SRC-1 activity either positively, by stimulating transcription of 6-integrin and 11ß-HSD2, or negatively, by repressing that of amphiregulin, strongly suggests that HBO1 regulates SRC-1 utilization, depending on the promoter context.

PRB-specific effects of HBO1 were also physiologically correlated with the colocalization of HBO1 with PRB-SRC-1 complex. HBO1 was shifted by the hormone in nuclear bodies formed by PR-SRC-1 with promyelocytic leukemia protein (66), allowing the visualization, by confocal microscopy, of the hormone-induced reorganization of subchromatin structures. This suggests that HBO1 could play an important role in PR-mediated transcription. However, the physiological conditions of HBO1 recruitment by PR remain unknown. Analysis of this mechanism will require new data on regulation of HBO1 expression in tissues, and studies of HBO1 functions in normal and cancer cell lines through PRA/PRB differential regulation of target genes. A possible link between ORC complex and cyclin-dependent kinase 11^p58 (27, 29) with steroid receptors might be also particularly relevant.

In conclusion, we propose that HBO1 acetylase might promote a LBD-dependent derepression of the N-terminal activation function AF3 of PRB, leading to the functional coupling with the coactivator complex and to an antagonistic action of PRA-specific transrepression. This hypothesis remains to be further tested using precise mutagenesis experiments and also employing biochemical approaches to specify the role of the putative acetylating function.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Plasmids and cDNAs
The parental plasmids used in the study were pMyr (coding a myristoylation domain, Ura marker) and pSos (coding the human guanyl exchange factor hSos, Leu marker) for two-hybrid screening (Cytotrap; Stratagene, La Jolla, CA), modified pAS2 (coding Gal4 DBD, Trp marker), pACT2 (coding Gal4 AD, Leu marker) for expression in yeast (CLONTECH Laboratories, Inc., Palo Alto, CA), pM (coding Gal4 DBD), and pVP16 (coding VP16 AD) for two-hybrid assays in mammalian cells (CLONTECH). pSG5 (Stratagene), pSG5-[Flag] (coding the Flag epitope tag), and pSG5-[Flag-NLS] (further coding the SV40 T antigen NLS) were used for expression in mammalian cells or in vitro translation, and pGEX (coding GST) was used for expression in Escherichia coli (Amersham Pharmacia Biotech, Arlington Heights, IL). The rabbit PR cDNA (67) was initially used to produce deletion mutants cloned in the indicated systemic vectors. The human PR, hPRB (B isoform), was cloned in pSG5, and the hPRA expression vector (A isoform) was obtained by exchanging the DNA fragment coding aa 1–372 of hPRB by a PCR-generated DNA fragment coding for aa 165–208 using tagged primers and pfu-DNA polymerase. HBO1 full-length cDNA was reconstituted by PCR using pfu-DNA polymerase (Stratagene), the original pMyr cDNA library as template, and tagged primers, and was inserted into the above systemic vectors as indicated in the figure legends. The HBO1 deletion mutants HBO-N (aa 1–340), HBO-C (aa 340–611), and HBO-CZ (aa 465–611) were obtained from pSG5-[Flag]-HBO1 using restriction endonucleases. Human SRC-1a cDNA mutants were cloned in pSG5 (32), pVP16, and pACT2. EGFP-SRC1 and EGFP-SRC1_NLS both cloned in pSG5 have been described previously (41). The PRE₂-TATA-CAT, estrogen response element (ERE)₂-TATA-CAT, Gal4uas(4x17mers)-TATA_E1b-CAT reporter genes, and vectors expressing human MR, GR, AR, and GR have been described previously (32), as well as PRE₂-luciferase reporter gene (68).

Yeast Two-Hybrid Screening
The yeast strain cdc25H-2 (Stratagene) containing pSos-PR_1–592 as bait vector was cotransformed by the Li-acetate/polyethylene glycol method (69), with a human testis cDNA library cloned into pMyr, and was processed as described by the manufacturer with some modifications. Briefly, 5.10⁶ potential cotransformants were spread over 150-mm agar plates containing synthetic complete (SC) medium with 1% glucose (gluc) and amino acid dropout without leucine and uracile (SC-LU+gluc). The plates were incubated for 2 d at a permissive temperature (25 C) until colonies began to appear. Replicas were produced using velvet pads over SC-LU agar medium supplemented with galactose and raffinose (SC-LU+gal) to induce biosynthesis of the Sos-PR_1–592 bait. After incubation at nonpermissive temperature (37 C) in a water-saturated atmosphere, the growing colonies were picked up each day for 4 d and were dispatched into 96-well master plates. First-round replicas were produced on either SC-LU+gluc or SC-LU+gal agar medium using a 96-pins replicator and were incubated at either 25 C or 37 C. Twenty clones growing at 37 C only in SC-LU+gal medium were selected, and the library plasmids were extracted from uracile auxotrophic colonies. For second-round selection, the latter plasmids were used to cotransform cdc25H-2 strain containing PR deletion mutants inserted in pSos (PR_1–930, PR_1–663, PR_663–930). JAB1 (CSN5) cDNA inserted in pSos was used as a positive control for detecting LBD-specific and hormone-dependent interaction (32). Cells expressing MAFB protein encoded by pSos and pMyr (Cytotrap) were used to control the kinetics of the two-hybrid selection through homodimerization. The resuspended cotransformants were dotted on SC-LU+gal or SC-LU+gluc agar plates containing either 1 µM promegestone (R5020) or vehicle and were grown at either 25 C or 37 C. Two plasmids displaying galactose-inducible growth at 37 C in the presence either of pSos-PR_1–930 and hormone, or of pSos-PR_1–663, in ligand-free medium were selected. The corresponding inserts were identified by DNA sequencing as overlapped HBO1 cDNA fragments (HBO_331–611 and HBO_223–611).

Gal4 Two-Hybrid Assays in Yeast Cells
A yeast strain derived from Y526 (leu2-, Trp-, Gal4-, Gal80-, Gal1p-LACZ) was transformed by the lithium acetate/polyethylene glycol method (69) with pAS2 and pACT2 linker-modified plasmids containing the inserts indicated in each figure. After selection on SC-Trp-Leu medium, eight colonies were picked up and processed as already described (32) for ß-Gal assays using ortho nitro phenyl galactopyranoside (ONPG). After subtracting the background signal given by GalAD hybrid in the presence of empty GalDBD, the protein-protein interaction signals were calculated as the factor increase of the basal concentration of ß-Gal obtained with the corresponding bait in the presence of empty GalAD vector.

Cell Cultures and Expression Assays in Mammalian Cells
Monkey’s kidney cells (CV-1) were transiently transfected using the calcium phosphate coprecipitation procedure as described previously (32). Briefly, 3.10⁵ cells per well were plated in six-well dishes with DMEM containing 10% charcoal-stripped serum, and after 24–48 h were transfected in triplicate by 0.3 µl calcium phosphate suspension containing the indicated reporter gene (3 µg/ml) and combinations of expression vectors (1–12 µg/ml). The total amount of plasmids was kept constant at 20 µg/ml by adding pSG5-[Flag] vector. To monitor transfection efficiency, pSV-ß-Gal vector (Promega Corp., Madison, WI) was added at 0.1 µg/ml. Interexperiment normalization was obtained by managing a standard sample using pSV-ßGal and either pSG5-PR_1–663 or pM-PR_1–663 vector depending on the corresponding reporter gene. After incubation for 24 h, the cells were washed with PBS and incubated in fresh medium containing 10 nM of the indicated steroid for 24 h. The cells were harvested and lysed to measure CAT concentration by ELISA (Roche Molecular Biochemicals, Indianapolis, IN), total protein content by BCA (Pierce Chemical Co., Rockford, IL), and ß-Gal concentration (Promega kit). Relative CAT concentration was calculated as OD_{420 nm} units per mg of total proteins, corrected for transfection efficiency and for between-assay variation of the standard sample activity. The HEK293 cell line expressing stably the hPRB and hPRA isoforms was produced by cotransfecting the corresponding cells by either pSG5-hPRB or pSG5-hPRA together with pSV-Neo. One clone of each neomycin-resistant series was selected for similar production of the corresponding PR isoform by Western blot and immunocytochemical assays using PGR 312/2 monoclonal antibody recognizing both isoforms (Novocastra Laboratories, Newcastle, UK). The hormone-induced transcriptional activity of both isoforms was measured by transfection with a PRE₂-luciferase reporter gene and pSV-ß-Gal vector. After 24 h of hormone treatment, the cells were lysed, and luciferase assays were performed using a luminometer. Results were normalized for total protein content and ß-Gal concentration as described above for CAT immunoassays.

In Vitro GST Pull-Down Assays
The BL21 E. coli strain expressing either pGEX or pGEX-HBO1 vectors was processed essentially as described by the manufacturer (Amersham). Briefly, the supernatant of lysed cells was incubated with glutathione matrix and, after extensive washing, the immobilized hybrids were refolded in buffer A [20 mM HEPES, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40, 10% glycerol (pH 8)]. ³⁵S-radiolabeled proteins were synthetized using the TNT T7-coupled reticulocyte lysate system (Promega) in the presence of [³⁵S]methionine as described by the manufacturer, using the indicated cDNA fragments inserted into pSG5 or pSG5-[Flag] vectors. Each in vitro translated lysate (10–25 µl) was incubated for 2 h at 4 C with the matrix containing either GST-HBO1 or empty GST in 250 µl of buffer A supplemented with 1 mM unlabeled methionine and 1 mM of the indicated steroid. The samples were washed with buffer A and a multistep 50–150 mM NaCl gradient. The ³⁵S-labeled SRC-1 that was retained on the resin was eluted by boiling with a gel-loading buffer containing sodium dodecyl sulfate, analyzed by scintillation counting, SDS-PAGE followed by Coomassie blue staining, and fluorography. Specific activity refolded in washed matrix was expressed as bound ³⁵S percent of total trichoroacetic acid-precipitable ³⁵S input.

Real-Time PCR Analysis
The Ishikawa cell line expressing PRB (35) or not was transfected by the indicated plasmids by Polyfect reagent (QIAGEN) in six-well plates (six wells per condition). After a 2 h-treatment by R5020, cells were washed and lysed by Trizol reagent (Life Technologies, Gaithersburg, MD). Total RNA was extracted as described by the manufacturer. One microgram of each sample was treated by DNase I and was reverse transcribed using random primers as previously described (70). PCR primers set for the human 6-integrin gene were 5'-TGCTGTTGGTTCCCTCTCAGA and 5'-TTTAACCTGGAGGCATATCCCA (150-bp fragment). PCR primers set for the 11ß-HSD2 gene were 5'-CTGGACTCCATGGGCTTCAC and 5'-TGAACTCTAGCACGCGGCTAA (150-bp fragment). Primers set for the amphiregulin gene were 5'-ACTCTGGGAAGCGTGAACCAT and 5'-TAGTCATAGTCGGCTCCCG-AG (150-bp fragment). Primers set for human 18S RNA gene were 5'-GTGCATGGCCGTTCTTAGTTG and 5'-CATGCCAGAGTCTCGTTCGTT (100-bp fragment). The three corresponding PCR fragments, which were used as internal standards for quantitative assays, were initially cloned in PGEM-T easy vector (Promega). Real-time quantitative PCR with Platinum SYBR Green I (Invitrogen) was then performed in 25-µl reactions in duplicate with 1:20 fraction of each cDNA sample and the corresponding primers, using a ABI Prism 7000 apparatus. For each sample, the mRNA concentration was interpolated from standard curve and averaged Ct value and was divided by that of the corresponding reverse-transcribed 18S RNA (relative mRNA).

Immunofluorescence and Confocal Microscopy
HEK and HEK-PRB cells were spread in glass slides and were grown in DMEM without phenol red supplemented with 10% charcoal-treated fetal calf serum. The cells were transfected as indicated by either pSG5-EGFP-SRC1 or pSG5-EGFP-SRC1_NLS, and pSG5-HBO1 at a ratio 1:1 by the Polyfect method (QIAGEN). After transfection (24 h) the HEK-PRB cells were treated or not by 10 nM R5020 for 4 h. The cells were then successively washed with PBS, fixed in 1% paraformaldehyde in PBS for 10 min at 4 C, washed in PBS, dried at 20 C, and frozen at –20 C for 30 min. After thawing at 20 C, cells were treated 10 min in 10% normal donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS and were sequentially incubated with 1 µg/ml anti-PRB Let126 mouse monoclonal IgG (71), and 1:400 anti-HBO1 rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) in donkey serum-PBS. After three washes with PBS, the cells were treated for 45 min by a mixture of fluorescent secondary antibodies at 1:400 dilution containing donkey CY3-conjugated antimouse IgG and donkey CY5-conjugated antirabbit IgG (Jackson ImmunoResearch). The subcellular localization was first scored in at least 40 cotransfected cells for each condition by fluorescent microcopy. At least three representative groups of fluorescent cells were scanned by confocal microscopy with 10 sections of 0.33 µm voxel depth (TCS SP2 Leica system; Leica Corp., Deerfield, IL) with 63x objective. Phase contrast views were obtained in parallel to control integrity of the cells. Because PR and HBO1 are expressed mainly in the cell nucleus, their labelings were taken as reference, instead of 4',6-diamidino-2-phenylindole revelation, for the identification of nuclear structures to avoid any interference in the three-colored detection.

Statistical Analysis
All experiments were performed at least in triplicate and repeated three times. Data are expressed as means ± SEM and were analyzed by a Student’s t test for two-tailed unpaired comparison. The threshold for statistical significance was set to P < 0.05.

	ACKNOWLEDGMENTS

 
We thank B. Aronheim for the generous gift of two-hybrid vectors, and P. Fontanges and P. Leclerc for assistance on confocal microscopy. We thank M. Marden for support and a critical reading of the manuscript.

	FOOTNOTES

 
This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM, U135, U473 and U693), the Faculté de Médecine Paris-Sud, the Ligue contre le Cancer, the Association pour la Recherche sur le Cancer and the Fondation pour la Recherche Médicale. M.G. was supported by a fellowship from foundation A. Onasis of Greece and Association pour la Recherche sur le Cancer.

Disclosure statement: The authors have nothing to disclose.

First Published April 27, 2006

Abbreviations: aa, Amino acids; AD, activation domain; AF, activation function; AR, androgen receptor; CAT, chloramphenicol acetyltransferase; CBP, CREB-binding protein; CREB, cAMP response element binding protein; DBD, DNA-binding domain; EGFP, enhanced green fluorescent protein; ER, estrogen receptor; ERE, estrogen response element; ß-Gal, ß-galactosidase; GR, glucocorticoid receptor; GST, glutathione S-transferase; HAT, histone acetyltransferase; HBO1, histone acetyltransferase binding to ORC-1; HEK, human embryonic kidney; hPRA, isoform A of human PR; hPRB, isoform B of human PR; 11ß-HSD2, 11ß-hydroxy dehydrogenase 2; LAT, lysine acetyltransferase; LBD, ligand-binding domain; MR, mineralocorticoid receptor; MYST, MOZ-Ybf2/Sas3-Sas2-Tip60 acetyltransferases; N-AFs, N-terminal activating functions of PR; NLS, nuclear localization signal; NR, nuclear receptor; NTD, N-terminal domain; ORC-1, origin recognition complex; pCAF, CBP interacting protein; PR, progesterone receptor; PRE, progesterone response element; SC, synthetic complete medium; SRC-1, steroid receptor coactivator 1; SRC-1a, an isoform of SRC-1; Tip60, TAT HIV-interacting protein.

Received for publication April 11, 2005. Accepted for publication April 18, 2006.

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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  GR  |  MR  |  PR

Coregulators:   Tip60  |  HBO1  |  SRC-1

Ligands:   Dexamethasone  |  17β-Estradiol  |  Dihydrotestosterone  |  Aldosterone  |  R5020

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