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Hormone Response of Rodent Phenylalanine Hydroxylase Requires HNF1 and the Glucocorticoid Receptor
Biochemical and Biophysical Research Communications 287, 852– 858 (2001) doi:10.1006/bbrc.2001.5673, available online at http://www.idealibrary.com on
Hormone Response of Rodent Phenylalanine Hydroxylase Requires HNF1 and the Glucocorticoid Receptor Anne Bristeau, Anne-Marie Catherin, Mary C. Weiss, 1 and Daniela M. Faust Unite´ de Ge´ne´tique de la Diffe´renciation, FRE 2364, Centre National de la Recherche Scientifique, De´partement de Biologie Mole´culaire, Institut Pasteur, Paris, France
Received August 14, 2001
Expression of the rodent phenylalanine hydroxylase (PAH) gene is dependent upon hormones. Induction by glucocorticoids and cAMP occurs slowly and maximal stimulation is obtained by a synergistic effect of the two compounds. Hormone responsiveness is conferred by the tissue-specific HSIII enhancer and involves (i) protein kinase A mediating the cAMP response, even though a consensus sequence for binding of the cAMP response element binding protein is not present; (ii) other serine/threonine kinases as deduced from inhibitor studies; (iii) glucocorticoid receptor protein bound to glucocorticoid response element half sites; and (iv) binding of the liver-enriched transcription factor hepatocyte nuclear factor 1 (HNF1) to sites in the enhancer. Glucocorticoid receptor and HNF1, bound to their cognate sites, cooperatively increase the glucocorticoid response of the PAH gene, this response being synergistically enhanced by cAMP after long-term treatment. © 2001 Academic Press Key Words: DNaseI hypersensitive sites; hepatoma cell; cAMP; protein kinase A.
Hydroxylation, an obligatory step in phenylalanine breakdown and in tyrosine biosynthesis, is mediated by phenylalanine hydroxylase (PAH) in the presence of oxygen and the cofactor tetrahydrobiopterin. Mutations causing deficiency of human PAH are responsible for phenylketonuria, leading in the most severe cases to mental retardation. In human as well as in rodents, PAH is present in liver and kidney and is detectable early in development, even though the rodent PAH gene was formerly considered as a neonatal function (1). PAH transcripts show a marked augmentation at birth (2) in parallel with elevation of glucocorticoids and PKA (cAMP-dependent protein kinase) activity. 1 To whom correspondence and reprint requests should be addressed at Unite´ de Ge´ne´tique de la Diffe´renciation, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France. Fax: ⫹33 1 40 61 32 31. E-mail: [email protected].
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Although rodent PAH is strictly dependent upon hormones for expression, the human gene is only marginally inducible. The rodent gene is strongly induced by dexamethasone (dex), weakly by cAMP, and a synergistic effect is observed with both compounds (3). Furthermore, hepatic expression of the mouse PAH gene absolutely requires the liver-enriched transcription factor hepatocyte nuclear factor 1␣ (HNF1␣): in HNF1␣ ⫺/⫺ mice, the PAH gene is totally silent, even in the presence of hormones, and the chromatin structure of the regulatory region is closed (2). As PAH, hepatic carbamoylphosphate synthetase I (CPS) is induced by cAMP and by glucocorticoids, and the two compounds provoke a synergistic effect (4). CPS gene induction involves interactions among different signal transduction pathways, and while the elements implicated in the individual inductions and in synergy were defined, understanding of the mechanisms involved remains elusive. Many adult hepatic functions such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK) (5, 6) are induced by cAMP and glucocorticoids. Expression of these genes is rapidly modulated by cAMP via stimulation of PKA that phosphorylates cAMP response element binding protein (CREB) whose capacity for transcriptional activation is thereby increased (7). Glucocorticoids induce TAT and PEPCK expression rapidly via activation of the glucocorticoid receptor, which binds to glucocorticoid response elements (GRE), and activates transcription (8). Dex and cAMP together lead to only additive effects on gene transcription. The genes coding for TAT and PEPCK possess in their regulatory regions glucocorticoid response units (GRU) which are composed of several GRE associated with binding sites for ubiquitous and liver-enriched transcription factors and confer tissue-specific glucocorticoid responsiveness (5). Two classes of GRUs have been described: they are either located within glucocorticoid-inducible DNaseI hypersensitive regions indicating that glucocorticoids induce rearrange-
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ments of the nucleosomal structure (9), or they are present in regions with a constitutively open chromatin structure (10, 11). By transient transfection assays, we characterized in the regulatory region of the mouse PAH gene a tissuespecific and hormone-responsive enhancer, HSIII (12). It contains neither cAMP response elements (CRE) nor complete GRE. It does possess GRE half sites that are able to bind the glucocorticoid receptor and sites for ubiquitous (AP1, NF1) and liver-enriched transcription factors [HNF1 and CCAAT/enhancer binding protein (C/EBP)]. Mutations of the C/EBP and HNF1 sites show that they contribute to basal activity and to hormone response. Here we have dissected the hormone response of the rodent PAH gene. We show that the kinetics of PAH induction are slow and involve cross-talk among signal transduction pathways. Moreover, the hormone response of the PAH gene requires both HNF1 and the glucocorticoid receptor. MATERIALS AND METHODS Cell lines and culture conditions. FGC4 and Fao, welldifferentiated rat hepatoma cells derived from H4IIEC3 (13) and stable transfectant Fao-m P11 (3), were cultured in modified Ham’s F-12 medium supplemented with 5% fetal calf serum in 7% CO 2 at 37°C. For Mirza cells (14) dialyzed instead of normal serum was used. For RNA preparations, cells were treated or not with 1 M dex and/or 100 M 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) for 24 h unless otherwise specified. Inhibitor experiments were carried out by pre-incubation for 2 h, with 1 or 2.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1 M okadaic acid or 1 M GF109203X (compounds purchased from Biomol Signal Transduction, Plymouth, PA) before addition of hormones and further incubation for 24 h. Transiently transfected cells were treated with 1 M dex for 48 h and/or 100 M 8-CPT-cAMP for 24 h. Sp-8-Br-cAMPS and Rp-8-Br-cAMPS (Biolog, Bremen) were added for the last 24 h. RNA analysis. Northern blots and quantifications were performed as in (3). Probes labeled by random priming correspond to cDNA fragments of PAH, TAT, and 28S rRNA (14).
FIG. 1. Hormone treatment causes a slow increase in rat PAH transcripts. PAH and TAT transcript accumulation was analyzed in FGC4 cells. The bars represent the factors of increase in the amounts of transcripts compared to the levels in the absence of hormones set to 1. The effects of hormones on PAH were reproduced in several independent experiments. Cells were treated with (A) 1 M dex (B) 100 M 8-CPT-cAMP for the indicated times, and (C) 1 M dex, 100 M 8-CPT-cAMP, or 1 M dex with 100 M 8-CPT-cAMP added 4 h after dex. Values were calculated after normalization for amounts of RNA loaded by hybridization with a 28S RNA probe.
Transfections, transient expression assays, and DNaseI hypersensitive site analysis. Experiments were performed as detailed in Ref. (3). Plasmids. P11-CAT6, an 11-kb fragment of the mouse PAH regulatory region in pBLCAT6 and HSIII-CAT5 (positions ⫺3283 to ⫺2873) are described (3). Mutations in the glucocorticoid receptor binding sites of the HSIII enhancer were introduced using the Chameleon kit (Stratagene, The Netherlands). Oligonucleotides used for mutation of GR1, 5⬘-CTTATATACACAGGAGCTCCTATCTCCTCAAACAGGGCAG-3⬘; of GR2, 5⬘-GCAGTGAAGGTACCAAACTGCTTACTCTATCTTGAG-3⬘; and of GR3, 5⬘-GATAAGAGGGAAAACTGCAAGCGCGAAAGAAAAGTGAC-3⬘ carry mutations that abolish activity of the TAT GRE and correspond to mut 1,4 of GRE-A (15). Both mutations f3mut and f5mut (12) were introduced into HSIIICAT5 or P11-CAT6 to abolish HNF1 binding.
RESULTS AND DISCUSSION Hormone-dependent accumulation of rat PAH transcripts occurs slowly. FGC4 rat hepatoma cells, which express the liver-enriched transcription factors and
functions of adult hepatocytes, were used to analyze the accumulation of PAH, TAT (Fig. 1) and PEPCK (data not shown) transcripts with time upon hormone treatment. Unlike the rapid response of TAT and PEPCK, an effect of dex (Fig. 1A) on PAH transcript levels was detected only after 4 h of treatment. cAMP induced PAH weakly and reproducibly (Fig. 1B) but only after 2 h. As expected, for TAT and PEPCK, the effect of cAMP was rapid and strong. When cells were treated with a combination of dex and cAMP (Fig. 1C), an 8-h treatment led to an additive effect on PAH transcript accumulation whereas after 24 h, the transcript levels were twice as high as for dex alone. Hence, PAH expression is synergistically increased by dex and cAMP but only after long term treatment. In contrast,
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the transcript levels of TAT and PEPCK showed at best additive effects of dex and cAMP. The slow hormone response of PAH could reflect a mechanism requiring de novo protein synthesis, in which case induction would be inhibited by cycloheximide. After the 4 h incubation necessary to obtain a hormone response of PAH, transcript levels of TAT and PAH were reduced by 50% in the presence of cycloheximide (data not shown). Maintenance of basal and cAMP-induced transcription of the TAT gene does require de novo protein synthesis (16). For the PAH gene, the cycloheximide-induced inhibition could be due to inhibition of the synthesis of a factor necessary either to initiate or to maintain the response. The glucocorticoid receptor binding sites of the HSIII enhancer are required for the dex response. The fragment of the HSIII enhancer required for hormone response contains in its 5⬘-portion three GRE half sites that bind glucocorticoid receptor protein in in vitro DNaseI footprinting experiments (12). Mutations were introduced into each of them within the HSIII enhancer fragment upstream of a heterologous promoter (HSIII-CAT5), individually or in all possible combinations and tested in transient transfections in FGC4 cells (Fig. 2A and data not shown). Mutations of GR1 and GR3 caused a reduction by half in the dex response and a drastic reduction of the synergy in the presence of the two hormones but not of basal activity. In contrast, mutation of GR2 had only a weaker effect. The GR1 seems to play the most important role since mutation of an additional GR does not produce a stronger reduction than mutation of GR1 alone. Nevertheless, integrity of the three half sites is necessary for maximal hormone response. While glucocorticoid receptor dimers do not bind GRE half sites, monomers can bind in vitro (17). In the HSIII enhancer, binding of monomers may be sufficient to mediate induction, or local conformation of the DNA may permit the contact of a receptor dimer or even a trimer (18) with several glucocorticoid receptor half sites. Glucocorticoid receptor mediates the glucocorticoid response by modulation of transcriptional activity, by remodeling chromatin structure or by interaction with other factors, such as AP1 (19) and NFB (20) to repress transcription, or STAT3 (21), STAT5 (22) and even CREB (23) to activate transcription. Recently, glucocorticoid receptor has been shown to recruit the chromatin remodeling complex SWI–SNF at a target promoter (24) and to interact with coactivators such as p300/CBP that possess histone acetylase activity (25, 26). In the TAT gene as well as in the mouse mammary tumor virus promoter, glucocorticoid receptor binds respectively to GRU or GRE and induces conformational changes of nucleosome structure, permitting the binding of other transcription factors (27, 28).
FIG. 2. Glucocorticoid receptor mediates dex induction of the mouse HSIII enhancer via GRE half sites and PKA mediates the cAMP response. (A) Mutations of glucocorticoid receptor binding sites (GR 1, 2, and 3) were introduced into HSIII-CAT5 and their effects on hormone responsiveness were analyzed in transiently transfected FGC4 cells. The bars represent factors of induction compared to the CAT activity measured in the absence of hormones. Error bars show standard deviations from the means of at least three experiments; for low values, the error bars are too small to be visible. (B) Reporter gene activity of HSIII-CAT5 transfected into FGC4 cells treated or not with dex, 8-CPT-cAMP, 25 M of the PKA-specific agonist Sp-8-Br-cAMPS (Sp) or 100 M of the antagonist Rp-8-BrcAMPS (Rp), alone or in combination with Sp is represented by the bars (fold induction).
PKA mediates the cAMP response of the PAH HSIII enhancer. The hormone responsive fragment of the HSIII enhancer is devoid of CRE. To verify that the cAMP response is indeed mediated by PKA, we used the PKA-specific agonist, Sp (Sp-8-Br-cAMPS), and its antagonist, Rp (Rp-8-Br-cAMPS) in transient transfections of HSIII-CAT5 in FGC4 cells (Fig. 2B and data not shown). Sp alone had the same weak effect as the more commonly used 8-CPT-cAMP and provokes the anticipated synergistic response in the presence of dex which is antagonized by Rp. All effects were dosedependent. Identical results were obtained from analysis of PAH transcripts (data not shown). Thus, PKA is indeed implicated in the cAMP response, but via a mechanism different from direct activation and binding of CREB to the HSIII sequences. Several genes in addition to PAH, like steroid-21hydroxylase (29, 30) and renin (31), exhibit a slow response to cAMP that is mediated by sequences lack-
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FIG. 3. Serine/threonine phosphorylation is involved in rat PAH expression. FGC4 cells were treated or not with the indicated compounds as described under Materials and Methods and RNA was analyzed by Northern blots, hybridized successively with PAH and TAT probes and normalized for the quantity of RNA loaded by hybridization with a 28S rRNA probe. Quantification gives, in the upper line, values of fold induction of basal level for the hormone treated samples (fold induction) and in the lower line, changes in the basal level upon inhibitor treatment compared to untreated control (fold control) (or DMSO treated for GF109203X). The values given were calculated after normalization, and these values should be given precedence over the apparent band intensities. Results were reproducibly obtained.
ing a CRE consensus site and the binding of proteins that are unrelated to the CREB family. For the PAH gene, the effect of cAMP is most clearly seen in synergy with dex. PKA modulates the effect of glucocorticoids at two levels: by increasing glucocorticoid receptor expression (32) or by controlling gene transcription through glucocorticoid receptor activity (33). Other serine/threonine kinases may be involved in rat PAH expression. Indirect mechanisms could be involved in PAH induction and cross-talk between pathways could underlie the slow synergistic effect of dex plus cAMP. The use of drugs interfering with wellcharacterized signal transduction pathways should indicate which among them could be involved. FGC4 cells were treated with drugs that modify activity of phosphatases or of serine/threonine kinases, to change equilibrium of the phosphorylation state of target proteins. Treated cells were induced as usual, and the effects of the drugs were evaluated by Northern blots of PAH and TAT (Fig. 3) or PEPCK (data not shown). Okadaic acid, an inhibitor of type 1 and 2A serine/ threonine phosphatases (34), stabilizes the phosphorylated state of substrate proteins. These events enhance PAH expression since in presence of the inhibitor, the basal transcript level was increased and cAMP provoked a 60% induction. In the presence of dex or dex plus cAMP, a 13-fold induction was observed, as though maximal induction had been achieved by dex alone. Maximal PAH transcript accumulation thus requires maintenance of serine/threonine phosphorylated substrates. In contrast, amounts of TAT basal transcripts were decreased. Phosphorylation of serine133 of CREB is required to maintain basal TAT activity (35) so okadaic acid treatment would be expected to increase basal transcript levels. Under the experimen-
tal conditions imposed, other serine/threonine phosphorylation events diminish the TAT transcript levels. IBMX nonspecifically inhibits phosphodiesterases (36), causing elevation of the intracellular cAMP concentration and increase of PKA activity. For PAH, the effect of IBMX depended upon its concentration. At 1 mM, the anticipated increase in basal and cAMPinduced transcript levels was observed with maximal accumulation when dex was added. In contrast, at 2.5 mM, IBMX caused a significant decrease of basal and cAMP-induced transcript levels, leaving dex induction but no synergy. Hence, IBMX acts on PAH expression as if an augmentation of cAMP leads to an increase in PAH transcript accumulation, but only to a certain point, beyond which the effect is reversed, becoming inhibitory. This paradoxical effect was not observed for the TAT (or the PEPCK) gene, for which both concentrations of the drug had the anticipated effects (37). These results indicate the participation of signal transduction pathways in the regulation of PAH expression that are not involved for TAT and PEPCK. GF109203X, an inhibitor of PKC (38) caused decrease of PAH transcripts under basal and cAMPtreated conditions whereas in both dex and dex plus cAMP, levels were similar to the control induced with dex alone. These results imply that PKC is involved in maintenance of basal PAH expression and in the cAMP response, but not in induction by dex. GF109203X reduced the TAT basal level but had no effect on induction by cAMP, suggesting that PKC-phosphorylated target proteins are necessary for basal expression but not for cAMP induction, in contrast to PAH. These data reveal that regulation of PAH involves combinations of different signal transduction pathways, including serine/threonine kinases other than PKA. Indeed, these kinases appear to participate in
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maintenance of the basal activity, in the cAMP effect and in the synergistic response to the two hormones, but not in the response to dex alone. The existence of two consensus AP1 sites overlapping with sites for either C/EBP or HNF1 in the HSIII enhancer imply that AP1 could be involved in modulation of PAH expression. Mutations introduced into the HSIII enhancer revealed that proteins binding to these sites are critically involved in maximal hormone response (12). However, due to the overlap, neither AP1 site can be inactivated by mutation without at the same time affecting binding of liver-enriched transcription factors. Since in HNF1-deficient cells, no induction of the full length or enhancer construct is observed (12), we assume that the binding of HNF1␣ and C/EBP and not of AP1 is critical for inducibility. The synergy obtained by dex and cAMP is only observed when the basal level of transcripts is high. PKC activity is implicated in maximal basal PAH expression and consequently appears to be involved in the synergistic response to the two hormones. A candidate to mediate this synergy is AP2, a transcription factor whose activity is modulated by PKA as well as by PKC (39). However, no AP2 consensus site has been found in the HSIII sequence. The HSIII enhancer corresponds to a DNaseI hypersensitive site whose presence does not depend upon dex. To test whether the DNaseI hypersensitive site HSSIII is constitutive or dex induced, we determined its presence in PAH-expressing cell lines that have been treated or not with glucocorticoids. In hepatoma cells, the latter provoke heritable modification in chromatin structure at the level of a GRU of the TAT gene (27). We analyzed Mirza cells maintained in medium with dialyzed serum and no dex for over 50 generations and stable transfectant clones of well-differentiated hepatoma cells expressing CAT under control of the 11-kb fragment (P11-CAT6). We have previously shown that this fragment drives correct reporter gene expression and develops the “open” chromatin structure without requiring dex treatment (3). The same pattern of DNaseI hypersensitive sites is observed for Mirza cells and for stable transfectant clones irrespective of the culture conditions (Fig. 4). In addition, the intensity of HSSIII is not increased in cells treated with dex. We conclude that in PAHexpressing hepatoma cells HSSIII is a constitutive DNaseI hypersensitive site. HNF1 is required for dex induction and synergy. The HNF1 binding sites of HSIII are involved in the dex response since their mutations provoke a drastic decrease of dex induction (12). To confirm the role of these sites, we introduced mutations of both HNF1 binding sites into HSIII-CAT5 as well as into P11CAT6 and tested their effects in transient transfection as well as in stable transfectant clones. The mutations
FIG. 4. DNaseI hypersensitive sites are not dependent upon dex. Cells of the stable transfectant clone Fao-m P11, containing 5–10 copies of the transgene, were cultivated with normal medium, medium plus dex and medium with dialyzed serum for 48 h whereas Mirza cells were permanently cultivated with dialyzed serum. Cultures were treated with increasing amounts of DNaseI. Genomic DNA was prepared and digested with appropriate restriction enzymes. DNaseI hypersensitive sites were detected by hybridization of Southern blots with a CAT probe for Fao-mP11 and a PAH probe for Mirza cells. The fragments created by limited DNaseI digestion are indicated by the brackets and the positions of the DNaseI hypersensitive sites are calculated relative to the sites of transcription initiation, given by the arrow. The black box represents transcribed sequences.
in the HSIII fragment provoke total abolition of the dex response and of synergy, whereas basal and the cAMPinduced activities are diminished by half (Fig. 5). In the context of the 11-kb fragment, the mutations cause total abolition of the basal activity and of hormonal response in transient transfection assays as well as in stable transfectants (data not shown). Thus, the HNF1 sites are absolutely necessary for the dex response and the synergy whether analyzed in the context of the enhancer alone or in that of the entire regulatory region, in both stable and transitory transfection. HNF1␣ is indispensable for hepatic activation of the mouse PAH gene and for the establishment/ maintenance of the appropriate chromatin structure of its regulatory region (2). Here we demonstrate that the HSIII enhancer is in a constitutive hypersensitive site and that its HNF1 binding sites are required for the dex response. Thus, in the regulation of PAH expression, HNF1 possesses a triple role: in basal enhancer activity and in the hormonal response as shown here, and in chromatin modeling as described in Ref. (2). The mechanism by which HNF1 and glucocorticoid receptor cooperatively increase PAH activity in the presence of dex is unknown. Possibilities include
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REFERENCES
FIG. 5. HNF1 binding sites are involved in the dex response. Transiently transfected FGC4 cells were treated or not (⫺) with dex (d), 8-CPT-cAMP (c) or dex plus 8-CPT-cAMP (d ⫹ c). Activity of HSIII and HSIII-HNF1 mut, carrying mutation of both HNF1 sites in the context of pBLCAT5, were tested. The activity of the tk minimal promoter in pBLCAT5 (CAT/-gal ⫽ 9.2) is set to 1 and values are expressed as fold activity of tk. The bar graph shows activity of the CAT reporter gene under control of either the 11 kb fragment of the mouse PAH regulatory region (P11-CAT6) or the fragment harboring the same mutations of the two HNF1 sites within the HSIII enhancer. The activity of the tk minimal promoter in pBLCAT5 (CAT/-gal ⫽ 14.6) was used for comparison and is set to 1; values are expressed as fold activity of tk. Error bars show standard deviations from the means of at least three experiments.
protein–protein interactions between HNF1 and the glucocorticoid receptor, or interaction of one or both factors with the basal transcription machinery or with coactivators. Like glucocorticoid receptor, HNF1 interacts with cofactors such as p300/CBP and P/CAF (40), potentially permitting HNF1 to regulate the chromatin dynamics of its target genes (41). Whereas HNF1 cooperates with the glucocorticoid receptor for the dex response of the insulin-like growth factor binding protein 1 gene (42), it does not influence the dex response of the -fibrinogen gene (43). Finally, in light of our evidence implicating other serine/threonine kinases coupled with the critical role of HNF1 in hormone response of the PAH gene, it may be relevant that: (i) the quantity of HNF1␣ protein is regulated by phosphorylation/dephosphorylation events (44) and (ii) a direct regulator of HNF1␣ expression, the orphan nuclear receptor HNF4␣, is itself modulated by kinases belonging to different signal transduction pathways (45). ACKNOWLEDGMENTS This work was supported by the Association pour la Recherche contre le Cancer. Anne Bristeau was supported by fellowships from the Ministe`re de l’Enseignement Supe´rieur, de la Recherche et de la Technologie and from l’Association pour la Recherche contre le Cancer.
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Hormone Response of Rodent Phenylalanine Hydroxylase Requires HNF1 and the Glucocorticoid Receptor Anne Bristeau, Anne-Marie Catherin, Mary C. Weiss, 1 and Daniela M. Faust Unite´ de Ge´ne´tique de la Diffe´renciation, FRE 2364, Centre National de la Recherche Scientifique, De´partement de Biologie Mole´culaire, Institut Pasteur, Paris, France
Received August 14, 2001
Expression of the rodent phenylalanine hydroxylase (PAH) gene is dependent upon hormones. Induction by glucocorticoids and cAMP occurs slowly and maximal stimulation is obtained by a synergistic effect of the two compounds. Hormone responsiveness is conferred by the tissue-specific HSIII enhancer and involves (i) protein kinase A mediating the cAMP response, even though a consensus sequence for binding of the cAMP response element binding protein is not present; (ii) other serine/threonine kinases as deduced from inhibitor studies; (iii) glucocorticoid receptor protein bound to glucocorticoid response element half sites; and (iv) binding of the liver-enriched transcription factor hepatocyte nuclear factor 1 (HNF1) to sites in the enhancer. Glucocorticoid receptor and HNF1, bound to their cognate sites, cooperatively increase the glucocorticoid response of the PAH gene, this response being synergistically enhanced by cAMP after long-term treatment. © 2001 Academic Press Key Words: DNaseI hypersensitive sites; hepatoma cell; cAMP; protein kinase A.
Hydroxylation, an obligatory step in phenylalanine breakdown and in tyrosine biosynthesis, is mediated by phenylalanine hydroxylase (PAH) in the presence of oxygen and the cofactor tetrahydrobiopterin. Mutations causing deficiency of human PAH are responsible for phenylketonuria, leading in the most severe cases to mental retardation. In human as well as in rodents, PAH is present in liver and kidney and is detectable early in development, even though the rodent PAH gene was formerly considered as a neonatal function (1). PAH transcripts show a marked augmentation at birth (2) in parallel with elevation of glucocorticoids and PKA (cAMP-dependent protein kinase) activity. 1 To whom correspondence and reprint requests should be addressed at Unite´ de Ge´ne´tique de la Diffe´renciation, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France. Fax: ⫹33 1 40 61 32 31. E-mail: [email protected].
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Although rodent PAH is strictly dependent upon hormones for expression, the human gene is only marginally inducible. The rodent gene is strongly induced by dexamethasone (dex), weakly by cAMP, and a synergistic effect is observed with both compounds (3). Furthermore, hepatic expression of the mouse PAH gene absolutely requires the liver-enriched transcription factor hepatocyte nuclear factor 1␣ (HNF1␣): in HNF1␣ ⫺/⫺ mice, the PAH gene is totally silent, even in the presence of hormones, and the chromatin structure of the regulatory region is closed (2). As PAH, hepatic carbamoylphosphate synthetase I (CPS) is induced by cAMP and by glucocorticoids, and the two compounds provoke a synergistic effect (4). CPS gene induction involves interactions among different signal transduction pathways, and while the elements implicated in the individual inductions and in synergy were defined, understanding of the mechanisms involved remains elusive. Many adult hepatic functions such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK) (5, 6) are induced by cAMP and glucocorticoids. Expression of these genes is rapidly modulated by cAMP via stimulation of PKA that phosphorylates cAMP response element binding protein (CREB) whose capacity for transcriptional activation is thereby increased (7). Glucocorticoids induce TAT and PEPCK expression rapidly via activation of the glucocorticoid receptor, which binds to glucocorticoid response elements (GRE), and activates transcription (8). Dex and cAMP together lead to only additive effects on gene transcription. The genes coding for TAT and PEPCK possess in their regulatory regions glucocorticoid response units (GRU) which are composed of several GRE associated with binding sites for ubiquitous and liver-enriched transcription factors and confer tissue-specific glucocorticoid responsiveness (5). Two classes of GRUs have been described: they are either located within glucocorticoid-inducible DNaseI hypersensitive regions indicating that glucocorticoids induce rearrange-
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ments of the nucleosomal structure (9), or they are present in regions with a constitutively open chromatin structure (10, 11). By transient transfection assays, we characterized in the regulatory region of the mouse PAH gene a tissuespecific and hormone-responsive enhancer, HSIII (12). It contains neither cAMP response elements (CRE) nor complete GRE. It does possess GRE half sites that are able to bind the glucocorticoid receptor and sites for ubiquitous (AP1, NF1) and liver-enriched transcription factors [HNF1 and CCAAT/enhancer binding protein (C/EBP)]. Mutations of the C/EBP and HNF1 sites show that they contribute to basal activity and to hormone response. Here we have dissected the hormone response of the rodent PAH gene. We show that the kinetics of PAH induction are slow and involve cross-talk among signal transduction pathways. Moreover, the hormone response of the PAH gene requires both HNF1 and the glucocorticoid receptor. MATERIALS AND METHODS Cell lines and culture conditions. FGC4 and Fao, welldifferentiated rat hepatoma cells derived from H4IIEC3 (13) and stable transfectant Fao-m P11 (3), were cultured in modified Ham’s F-12 medium supplemented with 5% fetal calf serum in 7% CO 2 at 37°C. For Mirza cells (14) dialyzed instead of normal serum was used. For RNA preparations, cells were treated or not with 1 M dex and/or 100 M 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) for 24 h unless otherwise specified. Inhibitor experiments were carried out by pre-incubation for 2 h, with 1 or 2.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1 M okadaic acid or 1 M GF109203X (compounds purchased from Biomol Signal Transduction, Plymouth, PA) before addition of hormones and further incubation for 24 h. Transiently transfected cells were treated with 1 M dex for 48 h and/or 100 M 8-CPT-cAMP for 24 h. Sp-8-Br-cAMPS and Rp-8-Br-cAMPS (Biolog, Bremen) were added for the last 24 h. RNA analysis. Northern blots and quantifications were performed as in (3). Probes labeled by random priming correspond to cDNA fragments of PAH, TAT, and 28S rRNA (14).
FIG. 1. Hormone treatment causes a slow increase in rat PAH transcripts. PAH and TAT transcript accumulation was analyzed in FGC4 cells. The bars represent the factors of increase in the amounts of transcripts compared to the levels in the absence of hormones set to 1. The effects of hormones on PAH were reproduced in several independent experiments. Cells were treated with (A) 1 M dex (B) 100 M 8-CPT-cAMP for the indicated times, and (C) 1 M dex, 100 M 8-CPT-cAMP, or 1 M dex with 100 M 8-CPT-cAMP added 4 h after dex. Values were calculated after normalization for amounts of RNA loaded by hybridization with a 28S RNA probe.
Transfections, transient expression assays, and DNaseI hypersensitive site analysis. Experiments were performed as detailed in Ref. (3). Plasmids. P11-CAT6, an 11-kb fragment of the mouse PAH regulatory region in pBLCAT6 and HSIII-CAT5 (positions ⫺3283 to ⫺2873) are described (3). Mutations in the glucocorticoid receptor binding sites of the HSIII enhancer were introduced using the Chameleon kit (Stratagene, The Netherlands). Oligonucleotides used for mutation of GR1, 5⬘-CTTATATACACAGGAGCTCCTATCTCCTCAAACAGGGCAG-3⬘; of GR2, 5⬘-GCAGTGAAGGTACCAAACTGCTTACTCTATCTTGAG-3⬘; and of GR3, 5⬘-GATAAGAGGGAAAACTGCAAGCGCGAAAGAAAAGTGAC-3⬘ carry mutations that abolish activity of the TAT GRE and correspond to mut 1,4 of GRE-A (15). Both mutations f3mut and f5mut (12) were introduced into HSIIICAT5 or P11-CAT6 to abolish HNF1 binding.
RESULTS AND DISCUSSION Hormone-dependent accumulation of rat PAH transcripts occurs slowly. FGC4 rat hepatoma cells, which express the liver-enriched transcription factors and
functions of adult hepatocytes, were used to analyze the accumulation of PAH, TAT (Fig. 1) and PEPCK (data not shown) transcripts with time upon hormone treatment. Unlike the rapid response of TAT and PEPCK, an effect of dex (Fig. 1A) on PAH transcript levels was detected only after 4 h of treatment. cAMP induced PAH weakly and reproducibly (Fig. 1B) but only after 2 h. As expected, for TAT and PEPCK, the effect of cAMP was rapid and strong. When cells were treated with a combination of dex and cAMP (Fig. 1C), an 8-h treatment led to an additive effect on PAH transcript accumulation whereas after 24 h, the transcript levels were twice as high as for dex alone. Hence, PAH expression is synergistically increased by dex and cAMP but only after long term treatment. In contrast,
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the transcript levels of TAT and PEPCK showed at best additive effects of dex and cAMP. The slow hormone response of PAH could reflect a mechanism requiring de novo protein synthesis, in which case induction would be inhibited by cycloheximide. After the 4 h incubation necessary to obtain a hormone response of PAH, transcript levels of TAT and PAH were reduced by 50% in the presence of cycloheximide (data not shown). Maintenance of basal and cAMP-induced transcription of the TAT gene does require de novo protein synthesis (16). For the PAH gene, the cycloheximide-induced inhibition could be due to inhibition of the synthesis of a factor necessary either to initiate or to maintain the response. The glucocorticoid receptor binding sites of the HSIII enhancer are required for the dex response. The fragment of the HSIII enhancer required for hormone response contains in its 5⬘-portion three GRE half sites that bind glucocorticoid receptor protein in in vitro DNaseI footprinting experiments (12). Mutations were introduced into each of them within the HSIII enhancer fragment upstream of a heterologous promoter (HSIII-CAT5), individually or in all possible combinations and tested in transient transfections in FGC4 cells (Fig. 2A and data not shown). Mutations of GR1 and GR3 caused a reduction by half in the dex response and a drastic reduction of the synergy in the presence of the two hormones but not of basal activity. In contrast, mutation of GR2 had only a weaker effect. The GR1 seems to play the most important role since mutation of an additional GR does not produce a stronger reduction than mutation of GR1 alone. Nevertheless, integrity of the three half sites is necessary for maximal hormone response. While glucocorticoid receptor dimers do not bind GRE half sites, monomers can bind in vitro (17). In the HSIII enhancer, binding of monomers may be sufficient to mediate induction, or local conformation of the DNA may permit the contact of a receptor dimer or even a trimer (18) with several glucocorticoid receptor half sites. Glucocorticoid receptor mediates the glucocorticoid response by modulation of transcriptional activity, by remodeling chromatin structure or by interaction with other factors, such as AP1 (19) and NFB (20) to repress transcription, or STAT3 (21), STAT5 (22) and even CREB (23) to activate transcription. Recently, glucocorticoid receptor has been shown to recruit the chromatin remodeling complex SWI–SNF at a target promoter (24) and to interact with coactivators such as p300/CBP that possess histone acetylase activity (25, 26). In the TAT gene as well as in the mouse mammary tumor virus promoter, glucocorticoid receptor binds respectively to GRU or GRE and induces conformational changes of nucleosome structure, permitting the binding of other transcription factors (27, 28).
FIG. 2. Glucocorticoid receptor mediates dex induction of the mouse HSIII enhancer via GRE half sites and PKA mediates the cAMP response. (A) Mutations of glucocorticoid receptor binding sites (GR 1, 2, and 3) were introduced into HSIII-CAT5 and their effects on hormone responsiveness were analyzed in transiently transfected FGC4 cells. The bars represent factors of induction compared to the CAT activity measured in the absence of hormones. Error bars show standard deviations from the means of at least three experiments; for low values, the error bars are too small to be visible. (B) Reporter gene activity of HSIII-CAT5 transfected into FGC4 cells treated or not with dex, 8-CPT-cAMP, 25 M of the PKA-specific agonist Sp-8-Br-cAMPS (Sp) or 100 M of the antagonist Rp-8-BrcAMPS (Rp), alone or in combination with Sp is represented by the bars (fold induction).
PKA mediates the cAMP response of the PAH HSIII enhancer. The hormone responsive fragment of the HSIII enhancer is devoid of CRE. To verify that the cAMP response is indeed mediated by PKA, we used the PKA-specific agonist, Sp (Sp-8-Br-cAMPS), and its antagonist, Rp (Rp-8-Br-cAMPS) in transient transfections of HSIII-CAT5 in FGC4 cells (Fig. 2B and data not shown). Sp alone had the same weak effect as the more commonly used 8-CPT-cAMP and provokes the anticipated synergistic response in the presence of dex which is antagonized by Rp. All effects were dosedependent. Identical results were obtained from analysis of PAH transcripts (data not shown). Thus, PKA is indeed implicated in the cAMP response, but via a mechanism different from direct activation and binding of CREB to the HSIII sequences. Several genes in addition to PAH, like steroid-21hydroxylase (29, 30) and renin (31), exhibit a slow response to cAMP that is mediated by sequences lack-
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FIG. 3. Serine/threonine phosphorylation is involved in rat PAH expression. FGC4 cells were treated or not with the indicated compounds as described under Materials and Methods and RNA was analyzed by Northern blots, hybridized successively with PAH and TAT probes and normalized for the quantity of RNA loaded by hybridization with a 28S rRNA probe. Quantification gives, in the upper line, values of fold induction of basal level for the hormone treated samples (fold induction) and in the lower line, changes in the basal level upon inhibitor treatment compared to untreated control (fold control) (or DMSO treated for GF109203X). The values given were calculated after normalization, and these values should be given precedence over the apparent band intensities. Results were reproducibly obtained.
ing a CRE consensus site and the binding of proteins that are unrelated to the CREB family. For the PAH gene, the effect of cAMP is most clearly seen in synergy with dex. PKA modulates the effect of glucocorticoids at two levels: by increasing glucocorticoid receptor expression (32) or by controlling gene transcription through glucocorticoid receptor activity (33). Other serine/threonine kinases may be involved in rat PAH expression. Indirect mechanisms could be involved in PAH induction and cross-talk between pathways could underlie the slow synergistic effect of dex plus cAMP. The use of drugs interfering with wellcharacterized signal transduction pathways should indicate which among them could be involved. FGC4 cells were treated with drugs that modify activity of phosphatases or of serine/threonine kinases, to change equilibrium of the phosphorylation state of target proteins. Treated cells were induced as usual, and the effects of the drugs were evaluated by Northern blots of PAH and TAT (Fig. 3) or PEPCK (data not shown). Okadaic acid, an inhibitor of type 1 and 2A serine/ threonine phosphatases (34), stabilizes the phosphorylated state of substrate proteins. These events enhance PAH expression since in presence of the inhibitor, the basal transcript level was increased and cAMP provoked a 60% induction. In the presence of dex or dex plus cAMP, a 13-fold induction was observed, as though maximal induction had been achieved by dex alone. Maximal PAH transcript accumulation thus requires maintenance of serine/threonine phosphorylated substrates. In contrast, amounts of TAT basal transcripts were decreased. Phosphorylation of serine133 of CREB is required to maintain basal TAT activity (35) so okadaic acid treatment would be expected to increase basal transcript levels. Under the experimen-
tal conditions imposed, other serine/threonine phosphorylation events diminish the TAT transcript levels. IBMX nonspecifically inhibits phosphodiesterases (36), causing elevation of the intracellular cAMP concentration and increase of PKA activity. For PAH, the effect of IBMX depended upon its concentration. At 1 mM, the anticipated increase in basal and cAMPinduced transcript levels was observed with maximal accumulation when dex was added. In contrast, at 2.5 mM, IBMX caused a significant decrease of basal and cAMP-induced transcript levels, leaving dex induction but no synergy. Hence, IBMX acts on PAH expression as if an augmentation of cAMP leads to an increase in PAH transcript accumulation, but only to a certain point, beyond which the effect is reversed, becoming inhibitory. This paradoxical effect was not observed for the TAT (or the PEPCK) gene, for which both concentrations of the drug had the anticipated effects (37). These results indicate the participation of signal transduction pathways in the regulation of PAH expression that are not involved for TAT and PEPCK. GF109203X, an inhibitor of PKC (38) caused decrease of PAH transcripts under basal and cAMPtreated conditions whereas in both dex and dex plus cAMP, levels were similar to the control induced with dex alone. These results imply that PKC is involved in maintenance of basal PAH expression and in the cAMP response, but not in induction by dex. GF109203X reduced the TAT basal level but had no effect on induction by cAMP, suggesting that PKC-phosphorylated target proteins are necessary for basal expression but not for cAMP induction, in contrast to PAH. These data reveal that regulation of PAH involves combinations of different signal transduction pathways, including serine/threonine kinases other than PKA. Indeed, these kinases appear to participate in
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maintenance of the basal activity, in the cAMP effect and in the synergistic response to the two hormones, but not in the response to dex alone. The existence of two consensus AP1 sites overlapping with sites for either C/EBP or HNF1 in the HSIII enhancer imply that AP1 could be involved in modulation of PAH expression. Mutations introduced into the HSIII enhancer revealed that proteins binding to these sites are critically involved in maximal hormone response (12). However, due to the overlap, neither AP1 site can be inactivated by mutation without at the same time affecting binding of liver-enriched transcription factors. Since in HNF1-deficient cells, no induction of the full length or enhancer construct is observed (12), we assume that the binding of HNF1␣ and C/EBP and not of AP1 is critical for inducibility. The synergy obtained by dex and cAMP is only observed when the basal level of transcripts is high. PKC activity is implicated in maximal basal PAH expression and consequently appears to be involved in the synergistic response to the two hormones. A candidate to mediate this synergy is AP2, a transcription factor whose activity is modulated by PKA as well as by PKC (39). However, no AP2 consensus site has been found in the HSIII sequence. The HSIII enhancer corresponds to a DNaseI hypersensitive site whose presence does not depend upon dex. To test whether the DNaseI hypersensitive site HSSIII is constitutive or dex induced, we determined its presence in PAH-expressing cell lines that have been treated or not with glucocorticoids. In hepatoma cells, the latter provoke heritable modification in chromatin structure at the level of a GRU of the TAT gene (27). We analyzed Mirza cells maintained in medium with dialyzed serum and no dex for over 50 generations and stable transfectant clones of well-differentiated hepatoma cells expressing CAT under control of the 11-kb fragment (P11-CAT6). We have previously shown that this fragment drives correct reporter gene expression and develops the “open” chromatin structure without requiring dex treatment (3). The same pattern of DNaseI hypersensitive sites is observed for Mirza cells and for stable transfectant clones irrespective of the culture conditions (Fig. 4). In addition, the intensity of HSSIII is not increased in cells treated with dex. We conclude that in PAHexpressing hepatoma cells HSSIII is a constitutive DNaseI hypersensitive site. HNF1 is required for dex induction and synergy. The HNF1 binding sites of HSIII are involved in the dex response since their mutations provoke a drastic decrease of dex induction (12). To confirm the role of these sites, we introduced mutations of both HNF1 binding sites into HSIII-CAT5 as well as into P11CAT6 and tested their effects in transient transfection as well as in stable transfectant clones. The mutations
FIG. 4. DNaseI hypersensitive sites are not dependent upon dex. Cells of the stable transfectant clone Fao-m P11, containing 5–10 copies of the transgene, were cultivated with normal medium, medium plus dex and medium with dialyzed serum for 48 h whereas Mirza cells were permanently cultivated with dialyzed serum. Cultures were treated with increasing amounts of DNaseI. Genomic DNA was prepared and digested with appropriate restriction enzymes. DNaseI hypersensitive sites were detected by hybridization of Southern blots with a CAT probe for Fao-mP11 and a PAH probe for Mirza cells. The fragments created by limited DNaseI digestion are indicated by the brackets and the positions of the DNaseI hypersensitive sites are calculated relative to the sites of transcription initiation, given by the arrow. The black box represents transcribed sequences.
in the HSIII fragment provoke total abolition of the dex response and of synergy, whereas basal and the cAMPinduced activities are diminished by half (Fig. 5). In the context of the 11-kb fragment, the mutations cause total abolition of the basal activity and of hormonal response in transient transfection assays as well as in stable transfectants (data not shown). Thus, the HNF1 sites are absolutely necessary for the dex response and the synergy whether analyzed in the context of the enhancer alone or in that of the entire regulatory region, in both stable and transitory transfection. HNF1␣ is indispensable for hepatic activation of the mouse PAH gene and for the establishment/ maintenance of the appropriate chromatin structure of its regulatory region (2). Here we demonstrate that the HSIII enhancer is in a constitutive hypersensitive site and that its HNF1 binding sites are required for the dex response. Thus, in the regulation of PAH expression, HNF1 possesses a triple role: in basal enhancer activity and in the hormonal response as shown here, and in chromatin modeling as described in Ref. (2). The mechanism by which HNF1 and glucocorticoid receptor cooperatively increase PAH activity in the presence of dex is unknown. Possibilities include
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REFERENCES
FIG. 5. HNF1 binding sites are involved in the dex response. Transiently transfected FGC4 cells were treated or not (⫺) with dex (d), 8-CPT-cAMP (c) or dex plus 8-CPT-cAMP (d ⫹ c). Activity of HSIII and HSIII-HNF1 mut, carrying mutation of both HNF1 sites in the context of pBLCAT5, were tested. The activity of the tk minimal promoter in pBLCAT5 (CAT/-gal ⫽ 9.2) is set to 1 and values are expressed as fold activity of tk. The bar graph shows activity of the CAT reporter gene under control of either the 11 kb fragment of the mouse PAH regulatory region (P11-CAT6) or the fragment harboring the same mutations of the two HNF1 sites within the HSIII enhancer. The activity of the tk minimal promoter in pBLCAT5 (CAT/-gal ⫽ 14.6) was used for comparison and is set to 1; values are expressed as fold activity of tk. Error bars show standard deviations from the means of at least three experiments.
protein–protein interactions between HNF1 and the glucocorticoid receptor, or interaction of one or both factors with the basal transcription machinery or with coactivators. Like glucocorticoid receptor, HNF1 interacts with cofactors such as p300/CBP and P/CAF (40), potentially permitting HNF1 to regulate the chromatin dynamics of its target genes (41). Whereas HNF1 cooperates with the glucocorticoid receptor for the dex response of the insulin-like growth factor binding protein 1 gene (42), it does not influence the dex response of the -fibrinogen gene (43). Finally, in light of our evidence implicating other serine/threonine kinases coupled with the critical role of HNF1 in hormone response of the PAH gene, it may be relevant that: (i) the quantity of HNF1␣ protein is regulated by phosphorylation/dephosphorylation events (44) and (ii) a direct regulator of HNF1␣ expression, the orphan nuclear receptor HNF4␣, is itself modulated by kinases belonging to different signal transduction pathways (45). ACKNOWLEDGMENTS This work was supported by the Association pour la Recherche contre le Cancer. Anne Bristeau was supported by fellowships from the Ministe`re de l’Enseignement Supe´rieur, de la Recherche et de la Technologie and from l’Association pour la Recherche contre le Cancer.
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