| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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 |
|---|
|
|
|---|
-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 |
|---|
|
|
|---|
| RESULTS |
|---|
|
|
|---|
|
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 (HBO340611) 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 PR1930 or PR1663 constitutive mutant (Fig. 1A
) with the Gal4 transactivating domain (GalAD) were coexpressed with each HBO1 hybrid. PR1663, but not PR1930, strongly interacted with both HBO1 derivatives in the absence of ligand (Fig. 1D
). In contrast, the addition of R5020 induced the interaction of PR1930 with HBO1 and HBO340611. Therefore, the full-length HBO1, as well as its N-terminally truncated fragment, interacted with the N-terminal region of PR (aa 1663) when the LBD was either absent or liganded.
Together these data indicate that the full-length HBO1 targets the PR1663 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).
|
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-PR1663), 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 PR547930 (DBD-LBD) or PR663930 (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.
|
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 1340 (HBO-N), 340611 (HBO-C), and 465611 (HBO-C
Z). 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 1547), the DBD-hinge (aa 547663), and the LBD (aa 663930). The HBO-C mutant interacted with all PR fragments except the LBD. The further deletion of a region containing the MYST zinc finger (HBO1-C
Z) 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.
|
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 HBO1340 mutant instead of the wild-type HBO1 provoked a similar squelching of GalDBD-HBO1/VP-SRC-1-productive association, whereas the HBO340611 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.
|
Z) was coexpressed with a SRC-1 hybrid with Gal4-AD (AD). A two-hybrid signal was detected when the full-length partners were coexpressed (Fig. 5B
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-11987 mutant and was further abolished by deleting aa 782987. Note that the interaction of HBO1 with an SRC-17821208 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 PR547930 (DBD-LBD) or PR1663 (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 PR1930, PR1663, and PR547933, 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 (PR1663) abolished almost completely this effect. In contrast, when the NTD was deleted (PR547930), 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.
|
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 PR1930, PR547930 (DBD-LBD), PR1663 (NTD-DBD), or PR663930 fused to a constitutive SV40 NLS (LBDNLS). 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 LBDNLS 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 HBO11340 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.
|
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 HBO340611 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 LBDNLS 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 PR1663 and LBDNLS 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 1164 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.
|
6-integrin (36, 37), the 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) (37, 38), and the amphiregulin (35, 39) genes, the expression of which was increased by 3-fold after a 2-h hormone treatment of these cells (Fig. 8B
6-integrin transcript could be detected with SRC-1 or HBO1 alone. However, coexpression of both cofactors significantly enhanced
6-integrin expression 2-fold (P < 0.0001). The same effect of HBO1 and SRC-1 was also potentialized in 11ß-HSD2 expression. Conversely, whereas the amphiregulin gene transcript was induced by SRC-1 in the same cells (P < 0.0030), HBO1 was found to inhibit its effect (P < 0.0017). These results support that SRC-1 potentialization by HBO1 is physiologically relevant in PR-mediated transcriptional events. Interestingly, our data also indicate that HBO1 differentially modulates endogenous gene expression depending on the promoter context.
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.
|
| DISCUSSION |
|---|
|
|
|---|
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 689930 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, 3739, 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 11p58 (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 |
|---|
|
|
|---|
Z (aa 465611) 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)-TATAE1b-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-PR1592 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-PR1592 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 (PR1930, PR1663, PR663930). 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-PR1930 and hormone, or of pSos-PR1663, in ligand-free medium were selected. The corresponding inserts were identified by DNA sequencing as overlapped HBO1 cDNA fragments (HBO331611 and HBO223611).
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
Monkeys 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 2448 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 (112 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-PR1663 or pM-PR1663 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 OD420 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 (1025 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 50150 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 Students t test for two-tailed unpaired comparison. The threshold for statistical significance was set to P < 0.05.
| ACKNOWLEDGMENTS |
|---|
| FOOTNOTES |
|---|
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.
| REFERENCES |
|---|
|
|
|---|
6 integrin subunit gene. Biochem Biophys Res Commun 241:258263[CrossRef][Medline]NURSA Molecule Pages Link:
This article has been cited by other articles:
![]() |
I. Petit-Topin, N. Turque, J. Fagart, M. Fay, A. Ulmann, E. Gainer, and M.-E. Rafestin-Oblin Met909 Plays a Key Role in the Activation of the Progesterone Receptor and Also in the High Potency of 13-Ethyl Progestins Mol. Pharmacol., June 1, 2009; 75(6): 1317 - 1324. [Abstract] [Full Text] [PDF] |
||||
![]() |
X. Hu, H. M. Stern, L. Ge, C. O'Brien, L. Haydu, C. D. Honchell, P. M. Haverty, B. A. Peters, T. D. Wu, L. C. Amler, et al. Genetic Alterations and Oncogenic Pathways Associated with Breast Cancer Subtypes Mol. Cancer Res., April 1, 2009; 7(4): 511 - 522. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. L. Foy, I. Y. Song, V. C. Chitalia, H. T. Cohen, N. Saksouk, C. Cayrou, C. Vaziri, J. Cote, and M. V. Panchenko Role of Jade-1 in the Histone Acetyltransferase (HAT) HBO1 Complex J. Biol. Chem., October 24, 2008; 283(43): 28817 - 28826. [Abstract] [Full Text] [PDF] |
||||
![]() |
B. Miotto and K. Struhl HBO1 histone acetylase is a coactivator of the replication licensing factor Cdt1 Genes & Dev., October 1, 2008; 22(19): 2633 - 2638. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Hattori, F. Coustry, S. Stephens, H. Eberspaecher, M. Takigawa, H. Yasuda, and B. de Crombrugghe Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5 Nucleic Acids Res., May 1, 2008; 36(9): 3011 - 3024. [Abstract] [Full Text] [PDF] |
||||
![]() |
O. van Beekum, A. B. Brenkman, L. Grontved, N. Hamers, N. J. F. van den Broek, R. Berger, S. Mandrup, and E. Kalkhoven The Adipogenic Acetyltransferase Tip60 Targets Activation Function 1 of Peroxisome Proliferator-Activated Receptor {gamma} Endocrinology, April 1, 2008; 149(4): 1840 - 1849. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Iizuka, O. F. Sarmento, T. Sekiya, H. Scrable, C. D. Allis, and M. M. Smith Hbo1 Links p53-Dependent Stress Signaling to DNA Replication Licensing Mol. Cell. Biol., January 1, 2008; 28(1): 140 - 153. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Chen, M. R. Sowers, F. M. Moran, D. S. McConnell, N. A. Gee, G. A. Greendale, C. Whitehead, S. E. Kasim-Karakas, and B. L. Lasley Circulating Bioactive Androgens in Midlife Women J. Clin. Endocrinol. Metab., November 1, 2006; 91(11): 4387 - 4394. [Abstract] [Full Text] [PDF] |
||||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |