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Originally published In Press as doi:10.1074/jbc.M305626200 on September 11, 2003

J. Biol. Chem., Vol. 278, Issue 47, 46523-46532, November 21, 2003
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The T-box Factor Tpit Recruits SRC/p160 Co-activators and Mediates Hormone Action*

Mario Maira{ddagger}, Catherine Couture, Gwendal Le Martelot, Anne-Marie Pulichino, Steve Bilodeau, and Jacques Drouin§

From the Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, 110, avenue des Pins Ouest, Montréal, Québec H2W 1R7, Canada and the Université de Montréal, Département de Biochimie, C.P. 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada

Received for publication, May 29, 2003 , and in revised form, September 10, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tpit (Tbx19) is a transcription factor belonging to the T-box family, and it is essential for late differentiation of pituitary pro-opiomelanocortin (POMC)-expressing corticotroph and melanotroph cells. Tpit is also required, both in humans and mice, for cell-specific expression of the POMC gene in cooperation with the homeoprotein Pitx1. Despite their important roles as developmental regulators, the molecular mechanisms underpinning the functions of T-box factors in general, and of Tpit in particular, are still poorly defined. We now report that Tpit functions as an activator of transcription by recruiting SRC/p160 co-activators to its cognate DNA target in the POMC promoter, the Tpit/Pitx-RE. We also show that Tpit is a mediator of hormone signaling and that the Tpit/Pitx-RE is responsive to signals elicited by hypothalamic corticotropin-releasing hormone. These signals are mediated by the cAMP-dependent protein kinase and mitogen-activated protein kinase pathways, and activation of cAMP-dependent protein kinase also enhances Tpit and SRC-dependent transcription. We have previously shown that corticotropin-releasing hormone action is also exerted at the POMC promoter through the orphan nuclear receptor NGFI-B and its recruitment of SRC co-activators. Given that Tpit exhibits transcriptional synergy with NGFI-B, our results suggest that Tpit, along with NGFI-B and SRC-2, is part of a transcription regulatory complex assembled on the POMC promoter in response to hormonal stimulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tpit (also known as Tbx19) is a member of the T-box family of transcription factors. These transcriptional regulators play critical roles in development, and the prototypical and founding member of the family is Brachyury or T (1–3). Within the family, Tpit is most similar to T and exhibits a highly restricted pattern of expression; indeed, it is solely expressed in a subset of pituitary lineages, the POMC-expressing corticotrophs and melanotrophs. Its expression starts at embryonic day 12.5 and is maintained throughout adulthood (4). Tpit was shown to be crucial for cell-specific expression of the pro-opiomelanocortin (POMC)1 gene, in cooperation with the homeoprotein Pitx1 (4). Gain-of-function experiments showed that Tpit could induce uncommitted pituitary cells into POMC-expressing cells (4). Gene inactivation studies revealed a failure of prospective corticotrophs and melanotrophs to reach terminal differentiation (as defined by POMC expression) but no effect on their precursors: thus, Tpit is required for late differentiation but not for lineage commitment (5). Tpit is also a negative regulator of gonadotroph differentiation, as inactivation of the Tpit gene switches cells to the gonadotroph fate (5). As predicted by the exquisite association between Tpit and POMC-expressing cells, mutations of the TPIT gene are associated at high frequency (73%) with early onset isolated adrencorticotropin deficiency in humans (6).

Tpit is a 446-amino acid protein containing a sequence-specific DNA-binding domain, the T-box, which is highly homologous to Brachyury. Protein sequence analysis reveals 84% homology with Brachyury in the T-box compared with 58–64% with other T-boxes (4). T-box transcription factors can bind DNA either as dimers on a palindromic DNA element (7, 8) or as monomers on T element half-sites (4, 9). Tpit activates POMC gene transcription in cooperation with the homeoprotein Pitx1, and both transcription factors bind DNA as monomers at contiguous sites on the POMC promoter, within the Tpit/Pitx regulatory element (Tpit/Pitx-RE) (4). Tpit DNA binding activity is important for transcriptional activation, as highlighted by the fact that mutation in the Tpit binding site greatly reduces POMC promoter activity (4). Moreover, three TPIT mutations that abolish DNA binding have been identified in isolated adrenocorticotropin deficiency patients and no longer activate POMC transcription (6). Tpit was also involved in transcriptional repression, which likely accounts for its role as a negative regulator of gonadotroph differentiation. Tpit and the nuclear receptor SF-1 mutually antagonize each other's transcriptional activity by a mechanism of trans-repression that does not require Tpit DNA binding activity (5).

POMC expression becomes hormonally regulated by the hypothalamo-pituitary-adrenal axis at around embryonic day 15 in mice (10). Proteolytic processing of POMC in anterior pituitary corticotrophs produces the hormone adrenocorticotropin that controls adrenal glucocorticoid synthesis, and these steroids exert a negative feedback on the upper levels of the axis (11). POMC gene expression is itself under the positive control of hypothalamic corticotropin-releasing hormone (CRH). Extensive studies have characterized the early steps of corticotrophs response to CRH, which include activation of the cAMP/PKA pathway, MAPK activation and accumulation of intracellular Ca2+ (12–15). We have demonstrated that nuclear receptors (NRs) of the NGFI-B subfamily (Nur factors) are important mediators of CRH action on POMC, and at least part of the CRH effects are mediated through their binding element in the promoter, the NurRE (16–18). Nur factors are also implicated at the other levels of the hypothalamo-pituitary-adrenal axis, in regulation of cell death and in a variety of developmental events (reviewed in Ref. 18). In addition, an ever-increasing body of work has demonstrated that the transcriptional activity of NGFI-B is closely regulated by phosphorylation events. In basal conditions, the N terminus and DNA-binding domain of NGFI-B are phosphorylated, with key DNA-binding domain serines such as Ser316 preventing DNA binding (19, 20). Depending on the system and stimulus, the phosphorylation status of NGFI-B is modified, resulting in differential regulation of its transcriptional activity. For example, in adrenal-derived Y1 cells adrenocorticotropin treatment leads to dephosphorylation of Ser316 and unmasking of DNA binding activity, with a concomitant hyperphosphorylation of its N terminus that results in increased transcriptional activity (21, 22). We have also demonstrated that CRH treatment of AtT-20 cells leads to dephosphorylation of Ser316, thus playing a permissive role for subsequent NGFI-B dimer binding and co-activator recruitment (18).

We have also shown that SRC/p160 co-activators are important mediators of CRH action, because they are recruited to Nur dimers in response to CRH (18). The SRC/p160 family includes three homologous members, namely SRC-1, SRC-2 (also known as TIF2 or GRIP1), and SRC-3 (also known as p/CIP, RAC3, ACTR, AIB1, or TRAM-1) (23, 24). SRC/p160 co-activators were first cloned as transcriptional partners for NRs, but they were also shown to function with other transcriptional regulators, such as the bZIP factor c-Jun (25), the basic helix-loop-helix factor myogenin, and the MADS box factor MEF2c (26). SRC/p160 co-activators enhance the activity of transcription factors in part because of their intrinsic histone acetyltransferase activity and also by recruitment of other transcriptional regulators. These include an array of chromatin remodelers such as CBP/p300 and p/CAF, the histone methyltransferase CARM-1, and BAF 57, a subunit of the SWI/SNF complex. In addition, SRCs interact with proteins of the general transcriptional machinery such as TFIIB and TBP (for review see Refs. 23 and 24). SRC-2 has also been shown to function as a co-repressor for the glucocorticoid receptor GR (27), and different domains of SRC-2 are implicated in co-activator versus co-repressor activities (28).

We now report that Tpit recruits SRC co-activators, resulting in enhancement of Tpit-dependent transcription. PKA activation also modulates Tpit activity, seemingly by modulating SRC recruitment. We also show that the Tpit/Pitx-RE of the POMC promoter is responsive to signals elicited by CRH and mediated by PKA and MAPK pathways. Tpit also exhibits transcriptional synergism with NGFI-B, thus suggesting that Tpit is part of a transcription regulatory complex assembled on the POMC promoter in response to hormonal stimulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Oligonucleotides—The various reporter plasmids containing the minimal (–34/+63) POMC promoter were described before; the –480 POMC promoter in Ref. 29, the NurRE in Ref. 16, and the Tpit/Pitx-RE (previously known as CE3) and its various mutants, as well as the POMC promoter containing mutations in either the Tpit or Pitx site in Ref. 4. The reporter plasmid containing the Tpit/Pitx-RE with the minimal POMC promoter lacking the AP-1 site (–36/+26) was constructed by inserting three copies of the Tpit/Pitx-RE at the BamHI site of the pXP1 vector as described before (30). The NGFI-B (18), Pitx1 (30) as well as the Tpit, Brachyury, and Tbx1 (4) expression vectors were described elsewhere. The Myc-Tpit and FLAG-Pitx1 expression plasmids were constructed by PCR amplification of the complete cDNA sequences for mouse proteins cloned into the RSV-driven expression vectors previously used for Pitx1 (30); in both cases the 5' primer contained the coding sequence for the respective epitope. Tpit deletion mutants were constructed by PCR amplification in the same expression vector as wild-type Tpit (4). Tpit {Delta}N mutant lacks the first 30 amino acids, and the {Delta}C mutants are deletions that end at the amino acids indicated in Fig. 4C. The T-box expression construct encodes for a protein containing amino acids 47–224 of Tpit. Full-length SRC-1, SRC-2, and SRC-3 were described in Ref. 18. The various SRC-2 (TIF2) mutants were described in Ref. 31.



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  		FIG. 4.
Mechanisms of PKA/SRC-2 enhancement of Tpit activity. A, PKA potentiates SRC-2 enhancement of Tpit but not Pitx1 activity. CV-1 cells were transfected with expression plasmids for PKA (100 ng) and SRC-2 (250 ng), either alone or in combination, to assess their effect on Pitx- and Tpit-dependent transcription. The results are presented relative to the basal activity of the Tpit/Pitx-RE reporter. B, synergy between SRC-2 and PKA requires the glutamine-rich domain of SRC-2. Expression plasmids for Tpit (100 ng) and PKA (100 ng) were transfected in CV-1 cells either in absence (control) or presence of one of the SRC-2 constructs (wild-type or mutant) (250 ng), using the Tpit/Pitx-RE-luc as a reporter. The results are shown as the relative effect of PKA on the activity of Tpit. C, the Tpit N terminus is required for PKA/SRC-2 enhancement of transcriptional activity. Wild-type Tpit and a series of deletion mutants were transfected in CV-1 cells along with PKA (100 ng) and SRC-2 (250 ng), using the Tpit/Pitx-RE-luc as a reporter. All of the Tpit mutants are able to bind DNA, and the amount of plasmid transfected was adjusted so that equivalent levels of protein were expressed (data not shown). The results are presented as the relative effects of PKA+SRC-2. D, PKA does not affect Tpit DNA binding ability. 293 cells were transfected with PKA and Tpit expression vectors either alone or in combination. The cells were harvested 48 h later, and the nuclear extracts were submitted to electrophoretic mobility shift assay analysis using a T-box palindromic probe. E, PKA does not enhance Tpit-Pitx1 interaction. 293 cells were transfected with Myc-Tpit, FLAG-Pitx1, or both, in the absence or presence of PKA expression vectors as indicated. The nuclear extracts were incubated with an anti-FLAG M2 antibody covalently linked to agarose beads (Sigma). Immunoprecipitated complexes were subjected to SDS-PAGE, and the presence of Myc-Tpit was revealed using a polyclonal anti-Myc antibody (Santa-Cruz A-14). F, the Tpit N terminus interacts with SRC-2. The Tpit {Delta}N mutant is expressed as efficiently as Tpit (WT) and binds the Tpit/Pitx-RE equally well as shown by Western blot using Tpit antibody and gel mobility shift, respectively. Pull-down experiments using either MBP-Tpit or MBP-Tpit {Delta}N reveal similar interaction with NGFI-B but loss of interactions with in vitro translated SRC-2 for the {Delta}N mutant. MBP-LacZ was used as negative control and Input lanes represent 20% of the sample used. Ctl, control. n.s., non specific. WB, Western blot. IP, immunoprecipitation.

 
Cell Culture and Transfections—CV-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and maintained at 37 °C in 5% CO2. GH3 and AtT-20 D16v cells were grown in the same conditions, except when indicated otherwise in the legends to the figures. CV-1 cells were transfected using the calcium phosphate co-precipitation method, whereas GH3 and AtT-20 cells were transfected by lipofection using LipofectAMINE (Invitrogen), as previously described (32). Where indicated, hormone treatment was performed 4 h prior to harvesting and luciferase assay. The results are presented as the means of three to ten experiments performed in duplicate. Cytomegalovirus-{beta}-Gal was used as an internal control for transfection efficiency.

Electrophoretic Mobility Shift Assay—The electrophoretic mobility shift assays were performed as described (18). Binding reactions were performed in 20 µl containing gel shift binding buffer (18), 500 ng of poly(dI-dC), and 5 µg of nuclear extracts. We used 50,000 cpm/reaction (~20 fmol) of double-stranded probe (5'-GATCCAATTTCACACCTAGGTGTGAAATTG-3') corresponding to the Brachyury palindrome. The samples were separated by electrophoresis using 5% polyacrylamide gels in 0.5x Tris-borate (45 mM)/EDTA (1 mM) at 25 °C for 2.5 h.

Preparation of Nuclear Extracts—Nuclear extracts for electrophoretic mobility shift assay, and co-immunoprecipitation assay were performed essentially as described (18). When indicated, AtT-20 cells were treated with 10–7 M CRH (Sigma), 10–5 M forskolin (Sigma), or 50 µM PD 98059 (Calbiochem) prior to harvesting.

Western Blotting—Western blotting of 25 µg of nuclear extract was performed using anti-p42/44 MAPK or anti-phospho-p42/44 MAPK antibody (Cell Signaling) (see Fig. 3A), anti-Myc A-14 (Santa-Cruz) (see Fig. 4E), and anti-rabbit (or mouse) IgG horseradish peroxidase conjugate (Sigma). Revelation was performed by chemiluminescence as described by the manufacturer (ECL+plus; Amersham Biosciences).



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  		FIG. 3.
CRH responsiveness by Tpit requires MAPK signaling. A, CRH induces MAPK signaling. AtT-20 cells were cultured in low serum (1%) for 12 h and then treated with 10–7 M CRH for the indicated times prior to harvesting and subjected to SDS-PAGE. Activation of the MAPK pathway was detected by Western blot using antibodies directed against either the total (upper panel) or the active (phosphorylated) form of Erk1/2 (p-Erk1/2, bottom panel). B–D, CRH responsiveness requires the MAPK pathway. As reported previously (15), CRH- and forskolin-induced NurRE-dependent transcription requires MAPK signaling (B). The dependence on MAPK signaling for CRH (C) or forskolin (D) stimulation of POMC- and Tpit/Pitx-RE-dependent transcription was examined using the specific MEK1 inhibitor PD 98059 (PD) (50 µM) or the specific p38 inhibitor SB 203580 (SB) (20 µM) on AtT-20 cells transfected with the corresponding reporter plasmid. The cells were cultured in 1% serum for 12 h and then pretreated with the MAPK inhibitors for 1 h prior to a 4-h stimulation with CRH (10–7 M). The results are shown relative to the basal activity of each reporter. E, Western blot analysis of Tpit protein levels in AtT-20 cells treated as described above. Ctl, control. WB, Western blot.

 
Pull-down Assays—All MBP fusion proteins were produced, and 35S-labeled proteins were synthesized in vitro as described (33). Binding reactions were performed as described in Ref. 5. Bound proteins were resolved on SDS-PAGE, stained with Coomassie Blue to ensure that similar amounts of fusion proteins were recovered, and then autoradiographed.

Co-immunoprecipitation Assays—293 cells (10-cm plates) were transfected with 24 µg of total DNA, and nuclear extracts were prepared 48 h later. We used 250 µg of nuclear extract in 100 µl of buffer B (18) by assay. The total volume was brought to 1 ml by adding 167 µl of binding buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100) and 733 µl of binding buffer with 150 mM NaCl. Co-immunoprecipitation assays were performed using anti-FLAG affinity gel with the monoclonal antibody covalently attached to agarose beads (Sigma A2220). 40 µl of affinity gel (washed twice with 0.5 ml of Tris-buffered saline) was added and immunoprecipitation was performed at 4 °C for 4 h on a roller shaker. Immunoprecipitates were washed thrice in 1 ml of Tris-buffered saline for 5 min and eluted in Laemmli buffer without dithiothreitol. The samples were then subjected to SDS-PAGE and Western blot analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SRCs as Co-activators of Tpit—The T-box transcription factor Tpit was cloned as a cell-specific activator of the pituitary POMC gene in cooperation with the homeoprotein Pitx1 (4). To assess transcriptional partners of Tpit, we tested for recruitment of transcriptional co-activators previously implicated in POMC transcription, the SRC/p160 family (18).2 To investigate the ability of SRCs to modulate Tpit transcriptional activity, we performed transient co-transfections in CV-1 cells. As reported previously (4), transfection of either Tpit or Pitx1 alone had little effect on a luciferase reporter plasmid driven by a Tpit/Pitx target sequence, but together they exhibited transcriptional synergism (Fig. 1A). Co-transfection of an SRC-2 expression plasmid did not enhance Pitx1-dependent transcription, but in contrast enhanced both the transcriptional activity of Tpit alone as well as the synergistic activities of Tpit and Pitx1.



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  		FIG. 1.
SRC co-activators are recruited by Tpit to enhance its transcriptional activity. A, SRC-2 enhances Tpit transcriptional activity. The effect of the co-activator SRC-2 (cytomegalovirus-SRC-2, 500 ng) on Tpit-dependent (RSV-Tpit, 100 ng), Pitx1-dependent (RSV-Pitx1, 250 ng), and Tpit+Pitx1-dependent transcription was assessed by co-transfection in CV-1 cells of a luciferase reporter containing the minimal POMC promoter (–34/+63 bp) driven by three copies of the Tpit/Pitx-RE (4). The results are shown relative to basal activity of the reporter (means ± S.E. of three to five experiments performed in duplicate). B, SRC-2 interacts with Tpit but not with Pitx1. The ability of SRC-2 to interact with Tpit or Pitx1 was examined in a pull-down assay using MBP-Tpit, MBP-Pitx1, and MBP-{beta}-Gal fusion proteins and radiolabeled in vitro translated SRC-2. The position of SRC-2 (160 kDa) is indicated with an arrow. C, all three SRCs are Tpit co-activators. The ability of SRC family members (500 ng) to enhance Tpit-dependent (100 ng) activity was assessed by co-transfection in CV-1 cells using the Tpit/Pitx-RE-luc reporter. Inset, Western blot analysis shows similar Tpit expression with or without SRC-2. D, The glutamine-rich domain of SRC-2 is required for Tpit co-activation. The capacity of SRC-2 and its mutants to enhance Tpit-dependent activity was assessed by co-transfection as above. E, the glutamine-rich domain of SRC-2 is required for interaction with Tpit. The ability of SRC-2 and its various mutants to interact with Tpit was investigated by pull-down assay. Radiolabeled in vitro translated SRC-2 or mutants were tested for interaction with a MBP-Tpit fusion protein or with MBP-{beta}-Gal as a negative control. The arrow indicates the position of full-length SRC-2 (160 kDa). Ctl, control.

 
These results suggested that SRC-2 specifically enhances activity through interaction with Tpit. To test the respective abilities of Tpit and Pitx1 to physically recruit SRC-2, we performed in vitro pull-down assays using MBP fusion proteins for either Tpit, Pitx1, or {beta}-Gal chimeras and 35S-labeled SRC-2 proteins. Only MBP-Tpit effectively retained SRC-2 protein (Fig. 1B), which correlates with the transcriptional specificity observed with Tpit and Pitx1 (Fig. 1A). We also tested the ability of the other members of the p160 family to enhance Tpit-dependent activity. All three SRCs similarly enhanced Tpit transcriptional activity on a Tpit/Pitx target sequence (Fig. 1C). This enhancement cannot be attributed to effects on Tpit expression as indicated by Western blot analysis of Tpit (Fig. 1C, inset).

To define the domains of SRC-2 required for Tpit co-activation, we used a series of SRC-2 mutants known to disrupt different interactions with NRs. These included the SRC-2m123 mutant that contains mutations in all three LXXLL motifs essentials for stimulation of NR AF-2-dependent activity, a deletion mutant lacking the glutamine-rich domain necessary for co-activation of the NR AF-1-dependent activity (SRC-2{Delta}Q), as well as a double mutant SRC-2m123{Delta}Q. In co-transfection experiments, the SRC-2m123 mutant was as efficient as wild-type SRC-2, whereas the mutant SRC-2{Delta}Q abrogated SRC-2-mediated co-activation of Tpit transcriptional activity (Fig. 1D), as did the double mutant SRC-2m123{Delta}Q. The SRC-2 {Delta}Q mutant was previously shown to be appropriately expressed and to retain NR AF-2-dependent activity (31). To determine whether this was a consequence of impaired interaction with Tpit, we performed pull-down assays using wild-type and mutant in vitro translated SRC-2. The MBP-Tpit column bound only wild-type and SRC-2m123 mutant, whereas mutants SRC-2{Delta}Q and SRC-2m123{Delta}Q were not retained (Fig. 1E). Taken together, these results identify SRC-2 as a co-activator of Tpit and clearly implicate the glutamine-rich domain of SRC-2 in Tpit interaction and co-activation.

Tpit as a Target for Signaling Pathways—We have recently demonstrated that SRC-2 is recruited to the POMC promoter in response to cAMP/PKA signaling elicited by CRH, a pathway that targets the nuclear receptor NGFI-B in corticotroph cells (18).2 Given our observation that SRC-2 is also a co-activator of Tpit, we tested whether Tpit would also be a target for PKA signaling pathway. To test whether PKA would modulate Tpit transcriptional activity, a Tpit/Pitx-reporter plasmid was co-transfected in CV-1 cells with expression plasmids for Tpit and/or the catalytic subunit of PKA. Although the transcriptional response to Tpit is modest, co-expression of PKA greatly enhanced Tpit activity (Fig. 2A). PKA also enhanced the transcriptional activity of Brachyury, but not of Tbx1, another T-box transcription factor present in POMC-expressing AtT-20 cells (4).



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  		FIG. 2.
Tpit is a target for signaling pathways. A, PKA enhances the activity of Tpit and Brachyury. The effect of PKA on Tpit-, Tbx1-, and Brachyury-dependent transcription was assessed in CV-1 cells by co-transfecting their expression vectors (150 ng) with a plasmid coding for the constitutively active catalytic subunit of PKA (125 ng) and the Tpit/Pitx-RE-luc reporter. The results are shown relative to basal activity of the reporter. B, the Tpit and Pitx sites are required for CRH responsiveness of the Tpit/Pitx-RE. Wild-type and mutant Tpit/Pitx-RE reporters were used to examine the effect of each target site on CRH (10–7 M) responsiveness. AtT-20 cells were transfected by lipofection and stimulated for 4 h prior to harvesting. The basal activity of the wild-type Tpit/Pitx-RE (sequence of POMC –322 to –287 bp shown at top) was set to 100%. C, CRH responsiveness is conferred by other POMC regulatory elements in addition to Tpit/Pitx-RE. The requirement for the AP-1 site in the minimal POMC promoter of the Tpit/Pitx-RE reporter as well as the Tpit and Pitx sites within the POMC promoter was assessed by lipofection of various reporter constructs in AtT-20 cells. The cells were stimulated with 10–7 M CRH for 4 h prior to harvesting. As shown previously (29), the various POMC promoter mutants (constructs 6–11) retain about 10–20% of the activity of the intact (–480 bp) promoter (construct 5). Ctl, control.

 
We next tested whether the Tpit/Pitx-RE conferred responsiveness to activation by the CRH/cAMP/PKA pathway in AtT-20 cells. CRH treatment of AtT-20 cells resulted in activation of the transfected Tpit/Pitx-RE driven reporter plasmid, whereas CRH treatment had no effect on the minimal POMC promoter (Fig. 2, B and C). To confirm that the Tpit- and/or Pitx-binding sites were necessary, we scanned the entire Tpit/Pitx element with a set of 3-bp mutants and assessed them for CRH responsiveness. As expected, mutants M5 and M6, which destroy the Tpit half-site, were minimally active and were not enhanced upon CRH treatment (Fig. 2B). Interestingly, mutants M8 and M9, which destroy the Pitx-binding site, were also nonresponsive to CRH, confirming the interdependence of Tpit and Pitx activity in POMC-expressing cells (4).

To confirm that an AP-1 site present in the minimal POMC promoter, which had been implicated in CRH responsiveness (35), is not implicated in Tpit/Pitx-dependent CRH response, we used a reporter lacking this sequence, and this construct responded equally well to CRH treatment (Fig. 2C, construct 3). It is noteworthy that the AP-1 site in the minimal promoter reporter confers a slight but reproducible CRH response (compare constructs 2 and 4). Finally, to determine whether the Tpit/Pitx target sequence is essential for CRH responsiveness of the POMC promoter, we used reporter plasmids containing mutations in either Tpit (construct 6) or Pitx (construct 7) cognate sequence in the context of the intact promoter. Both mutant reporters were responsive to CRH (Fig. 2C), indicating that the Tpit/Pitx target sequence, although able to confer CRH responsiveness, is not essential for hormonal response within the context of the full promoter. Other promoter sequences, such as the NurRE (see below and Fig. 3B), may also contribute to CRH responsiveness in this context. In agreement with a role of the Tpit/Pitx element in CRH response of the intact POMC promoter (construct 5), we showed that deletion of distal sequences containing the NurRE (construct 8) do not prevent responsiveness but that further deletion of the region containing the Tpit/Pitx element (construct 9) abrogates responsiveness. Complementary reporter constructs containing either the distal NurRE (construct 11) or the central Tpit/Pitx element (construct 10) were both sufficient for CRH responsiveness.

Tpit as a Downstream Effector of a PKA and MAPK Pathways—We next set out to understand the signaling pathway downstream of PKA that leads to activation of POMC transcription. In a variety of endocrine systems, activation of cAMP signaling linked to G protein-coupled receptors results in rapid activation of the MAPK pathway. To determine whether it was the case in response to CRH stimulation, we harvested control and CRH-treated AtT-20 cells either 5 or 30 min after stimulation and subjected nuclear extracts to Western blot analysis. Activation of MAPK signaling was assessed using a phospho-specific antibody directed against the activated (phosphorylated) form of Erk1/2, which revealed a strong and transient activation of Erk1/2 within 5 min of CRH treatment (Fig. 3A, bottom panel). However, total amounts of Erk1/2 remained constant within this period (Fig. 3A, top panel). Similar experiments using anti-phopho-p38 failed to reveal activation of p38, another downstream MAPK (data not shown).

While we were preparing this manuscript, it was reported that NGFI-B is a target of MAPK signaling following CRH stimulation (15). Indeed, we also observed that pretreatment of AtT-20 cells with the MEK1 inhibitor PD 98059 abrogated most of the response of the NurRE reporter to both CRH and forskolin treatment (Fig. 3B). We then tested whether a Tpit/Pitx reporter would be similarly affected. Indeed, PD 98059 not only greatly diminished basal reporter activity but also completely blunted transcriptional enhancement in response to CRH treatment on both the Tpit/Pitx and POMC reporter plasmids (Fig. 3C). Similar results were obtained using UO126, another MEK1 inhibitor (data not shown). In contrast, the p38 inhibitor SB 203580 had no effect on the Tpit/Pitx reporter and only a minimal one on the POMC promoter (Fig. 3C). To ensure that MAPK activation was a direct consequence of cAMP signaling, we performed similar experiments using the adenylate cyclase activator forskolin, which also induced a transcriptional response from Tpit/Pitx and POMC reporters, and PD 98059 also blocked this activation (Fig. 3D). Throughout these experiments, the levels of Tpit protein remained constant as revealed by Western blotting (Fig. 3E). Altogether, these results show that Tpit can be a mediator of CRH signaling and a downstream effector of PKA/MAPK signaling pathways in corticotrophs.

Requirements for PKA Enhancement of Tpit Activity—Because SRC-2 was implicated in the CRH/PKA-dependent activation of NGFI-B (18), we tested whether it also favored PKA enhancement of Tpit activity. Indeed, co-transfection in CV-1 cells of limiting amounts (corresponding to about 20–40% of maximal effect; for example, compare PKA effect in Fig. 4A with Fig. 2A) of SRC-2 and PKA synergistically enhanced Tpit activity on a Tpit/Pitx reporter (Fig. 4A), suggesting that they are part of the same signaling cascade. In contrast, Pitx1 activity was only minimally enhanced by PKA and SRC-2 (Fig. 4A). Furthermore, this synergistic enhancement of Tpit activity by PKA/SRC-2 was not observed with the SRC-2{Delta}Q and SRC-2m123{Delta}Q mutants (Fig. 4B), presumably because of their impaired ability to interact with Tpit (Fig. 1E).

We next used a series of Tpit truncation mutants in transfection experiments to determine the domains required for PKA+SRC-2 potentiation of Tpit transcriptional activity. Progressive deletion of the Tpit C terminus up to amino acid 252 did not affect PKA+SRC-2-dependent transcriptional potentiation (Fig. 4C). However, truncation of the first 30 amino acids abrogated potentiation by PKA+SRC-2, suggesting a crucial role for the N terminus of Tpit in PKA/SRC-2 responsiveness. To gain further insight into the molecular mechanisms regulating PKA enhancement of Tpit transcriptional activity, we performed electrophoretic mobility shift assays. Co-expression of PKA did not increase the intrinsic affinity of Tpit for DNA (Fig. 4D). We also performed co-immunoprecipitation experiments using epitope-tagged Tpit and Pitx1, and PKA did not enhance Tpit-Pitx interaction in 293 cells (Fig. 4E). The Tpit {Delta}N mutant was expressed as efficiently as wild-type Tpit (Fig. 4F, WB), and its DNA binding activity was also similar to Tpit (Fig. 4F, EMSA). In agreement with the Tpit deletion analysis (Fig. 4C), pull-down experiments confirmed an in vitro interaction between SRC-2 and the Tpit N terminus that is lost in the Tpit {Delta}N mutant (Fig. 4F). Thus, these results indicate that the Tpit N terminus is a site of SRC-2 recruitment, and this activity is increased by PKA treatment. Altogether, these results suggest that PKA enhancement of Tpit transcriptional activity is primarily a result of increased co-activator recruitment to the Tpit/Pitx-RE by Tpit in response to intracellular signaling.

Tpit and NGFI-B Synergism in Presence of PKA and SRC-2—These studies have revealed mechanistic similarities between the T-box factor Tpit and the nuclear receptor NGFI-B. Indeed, both are transcriptional activators of the POMC gene, both are targeted by the CRH/PKA/MAPK pathway, and both recruit SRC/p160 co-activators. Given these similarities and the proximity of both sites on the POMC promoter, we investigated the possibility of transcriptional cooperativity between these two factors. Tpit, NGFI-B, or a combination of both were co-expressed with an intact POMC promoter-driven reporter in GH3 cells (that contain endogenous Pitx1 but not Tpit). Although there was no activation by factors alone, addition of either PKA or SRC-2 resulted in strong transcriptional synergy (Fig. 5A). Tpit and NGFI-B behaved slightly differently on a Tpit/Pitx reporter. Indeed, co-transfection of both resulted in strong transcriptional synergy independently of added SRC-2 or PKA (Fig. 5B). Because NGFI-B does not bind the Tpit/Pitx target sequence (data not shown), these results suggest direct interaction between Tpit and NGFI-B. In vitro pull-down assays using MBP-Tpit fusion proteins and radiolabeled NGFI-B confirmed that both factors could physically interact (Fig. 5C). Taken together, these results show that Tpit and NGFI-B are end point effectors of PKA and SRC-2 signaling, resulting in transcriptional cooperativity on the POMC promoter.



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  		FIG. 5.
Tpit and NGFI-B synergism in the presence of PKA and SRC-2. GH3 cells (containing endogenous Pitx factors) were transfected with Tpit (50 ng) and NGFI-B (50 ng), either alone or in combination, in the absence or presence of SRC-2 (250 ng) or PKA (100 ng) expression vectors as indicated. Using the POMC promoter reporter, synergism between Tpit and NGFI-B is only observed in presence of PKA or SRC-2 (A), whereas it is independent of these signals using the Tpit/Pitx reporter (B). C, Tpit and NGFI-B physically interact. The protein-protein interaction between Tpit and NGFI-B was assessed by pull-down assay. Radiolabeled in vitro translated NGFI-B was tested for interaction with a MBP-Tpit fusion protein or with MBP-{beta}-Gal as a negative control. Ctl, control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Despite their essential roles as developmental regulators, still little is known about the underlying transcriptional mechanisms of T-box factors. Tpit is one of the relatively few T-box factor for which target genes, such as POMC and {alpha}-GSU, have been identified and for which a role as either activator or repressor of transcription has been characterized (4, 5). The current paradigm dictates that transcription factors recruit co-factors to target genes, which in turn either recruit chromatin remodelers or stabilize the basic transcriptional machinery to exert their transcriptional functions. Whereas a plethora of such co-factors are known to modulate NR activity, currently only one case of co-factor recruitment by a T-box factor has been reported, the recruitment by Tbr-1 of the cytoskeleton-associated guanylate kinase CASK/LIN-2 (36).

The aim of this study was to further our understanding of the molecular mechanisms governing Tpit functions. We now report that Tpit is able to recruit SRC/p160 co-activators to modulate its transcriptional activity. Indeed, SRC-2 enhances the intrinsic activity of Tpit on its cognate POMC target as well as the synergistic activity with Pitx1, apparently by a mechanism where SRC-2 interacts only with Tpit (Fig. 1). Although the activity of Tpit on the Tpit/Pitx-RE requires a functional binding site for Pitx factors (Ref. 4 and Fig. 2), co-activation by SRC-2 does not, because Tpit-dependent activity on a palindromic Tail element is also enhanced by SRC-2 (data not shown). This suggests that Tpit activity on promoters other than POMC might not require the presence of Pitx factors but possibly of other transcriptional partners.

Future investigations will address the physiological significance of co-activator recruitment by T-box transcription factors, because this could be important in a number of developmental events. For example, recruitment of SRC-2 by myogenin and MEF2c is essential for proper skeletal muscle differentiation (26). The finding that SRC-2 is recruited by a T-box might also shed a new light on functional cross-talk between this subfamily and other transcription factors using common co-regulators. The antagonistic relationship between Tpit and SF-1 is of particular interest in the pituitary. SF-1 is a developmental regulator and a transcriptional modulator of cAMP-induced genes (37). In addition, MAPK-induced phosphorylation of SF-1 modulates its ability to recruit SRC-2 (38). Thus, the transcriptional antagonism observed between Tpit and SF-1 may involve competition for mutual co-activators. Another intriguing possibility is that SRC-2 might function as a co-repressor (rather than as a co-activator) of Tpit or SF-1 in the context of trans-repression. Indeed, such a role was recently shown in the case of trans-repression between the glucocorticoid receptor GR and AP-1 (27).

Our results indicate that the cAMP/PKA pathway regulates Tpit-dependent transcription. To our knowledge, this is the first example of regulation of a T-box transcription factor by a major signaling pathway. Indeed, stimulation of PKA signaling strongly potentiates the transcriptional activity not only of Tpit but also of Brachyury. Regulation of transcription factor activity by phosphorylation is a common occurrence, because it provides a means to rapidly fine-tune the transcriptional response of a target cell to environmental signals. PKA has been shown to modulate the activity of transcription factors at many levels, such as subcellular localization, DNA binding, interaction with other transcription factors, and co-factor recruitment. In the case of Tpit, the potentiation effect of PKA does not appear to be mediated by increased interaction with its known partner Pitx1 or by a change in affinity for DNA (Fig. 4). Moreover, the amount of Tpit protein is not affected by activation of cAMP signaling in AtT-20 cells (Fig. 3E) nor in overexpression experiments (Fig. 4E). Regulation of the subcellular distribution of Tpit is unlikely, given that it is present in nuclear extracts (Figs. 3 and 4) and that it is constitutively nuclear (4). Instead, PKA potentiation may be mediated through co-activator recruitment or alternatively through enhancement of co-factor (such as SRC-2) activity. Indeed, coexpression of SRC-2 synergistically enhanced the effects of PKA on Tpit activity, and this effect was not observed with an SRC-2 mutant unable to interact with Tpit (Fig. 4). The Tpit N terminus appears to be the target for SRC-2 interaction (Fig. 4, C and F). Phosphorylation-dependent recruitment of SRC-2 appears to be a widespread mechanism, because we and others have demonstrated that interaction of SRC-2 with both NGFI-B (18) and SF-1 (38) is modulated by the cAMP/PKA/MAPK, whereas recruitment of SRC-2 by MEF2c is regulated by the cyclin D-cdk complex (39). Further experimentation will be needed to determine which of Tpit or SRC-2 (or both) is the direct target of phosphorylation events. Tpit has a consensus MAPK site in the T-box. Both SRC-1 and SRC-3 have been shown to be directly phosphorylated by MAPK, and these modifications regulate transcriptional activity (40, 41). Alternatively, phosphorylation of SRC-2 might be controlling its cellular compartmentalization; such a regulation by MAPKs has been demonstrated for the co-repressor SMRT (42). Moreover, both SRC-2 (43) and SRC-3 (44) have been shown to be present in the cytoplasm in different systems.

We also show that Tpit is a mediator of CRH response at the POMC promoter. Indeed, we report that the Tpit/Pitx-RE is responsive to CRH, and mutation in either the Tpit or the Pitx target sites abolished responsiveness (Fig. 2). The requirement for the Pitx site, even though Pitx is only marginally responsive to PKA and does not recruit SRC-2, is in accordance with our previous observation that the activity of Tpit on the Tpit/Pitx-RE is totally dependent on the presence of a functional Pitx site (4). Signals elicited by CRH appear to involve MAPKs because pharmacological blockade of MAPK activity prevents CRH responsiveness of POMC and Tpit/Pitx-RE. The rapid and transient activation of MAPK signaling (Fig. 3A) correlates well with the rapid induction of POMC transcription, which is maximal within 15 min of CRH treatment (45). At first sight, the role of Tpit as a modulator of hormonal response may seem at odds with the traditional role ascribed to T-box factors as developmental regulators. However, this may explain why Tpit expression is maintained throughout adulthood. Indeed, we suggest that Tpit plays a dual role in the pituitary: first it functions as a factor essential for late differentiation of POMC-expressing corticotrophs and melanotrophs, and later it is recruited to participate in hormonal response of the POMC promoter. A precedent exists for pituitary Pit-1, a POU homeodomain factor essential for development of the somatotrophs, lactotrophs, and thyrotrophs lineages. Indeed, Pit-1 participates in hormonal response in each of these lineages as it modulates PKA-dependent induction of GH, prolactin, and thyrotropin-{beta} genes (46).

This study also revealed functional similarities between Tpit and NGFI-B as well as transcriptional cooperativity on the POMC promoter. Indeed, Tpit and NGFI-B physically interact and synergistically activate POMC transcription (Fig. 5). Thus, we propose in Fig. 6 a general model for CRH activation of POMC transcription integrating our findings for Tpit (this study) and NGFI-B (15, 17, 18). In basal conditions, Tpit/Pitx are present at the promoter, whereas NGFI-B is both in the cytoplasm and in the nucleus but in a minimal amount at the NurRE (18). CRH binding to its receptor initiates cAMP signaling, resulting in activation of PKA. Presumably because of the presence of Rap1, PKA signaling leads to activation of MAPKs and intracellular calcium entry (Ref. 15 and Fig. 3). This results in dephosphorylation of NGFI-B DNA-binding domain, thus allowing DNA binding (18). CRH-elicited signals enhance the ability of both Tpit and NGFI-B to recruit SRC-2, thus resulting in the assembly of an active transcriptional complex at the promoter. Further experimentation will be required to assess the existence of a tripartite complex in response to CRH, as suggested by the fact that Tpit and NGFI-B directly interact (Fig. 5) and that they bind the same region of SRC-2 (the glutamine-rich domain) (Fig. 1 and Ref. 18). Alternatively, they could be binding dimers of SRC-2 (or SRC heterodimers), given that SRCs possess a HLH-PAS domain and a known dimerization interface and that SRC hetero- and homo-multimeric complexes have been documented (34).



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  		FIG. 6.
Working model for action of CRH and PKA/MAPK pathway on POMC promoter. In basal conditions, Tpit appears to be nuclear and present at the Tpit/Pitx-RE, contributing together with Pitx1 to basal POMC transcription. NGFI-B is found both in the cytoplasm and nucleus, but very little is present at the NurRE. Upon CRH stimulation, accumulation of cAMP induces PKA activation, which activates Erk1/2 in corticotroph cells. This leads to NGFI-B DNA-binding domain dephosphorylation, thus allowing DNA binding of Nur dimers. Activation of PKA and MAPK signaling also enhances recruitment of SRC-2 to Tpit and NGFI-B cognate targets, presumably in part because of phosphorylation of the NGFI-B N-terminal AF-1 domain.

 
In summary, we have shown a novel mechanism for transcriptional activation by T-box factors such as Tpit, where intracellular signaling modulates SRC co-activator recruitment. In corticotroph cells, Tpit is a mediator of hormonal signaling, and its action may occur in parallel with the proposed mechanism for Nur-mediated CRH response. Given that the distal elements of the POMC promoter and their cognate transcription factors act in a highly synergistic manner (29), we postulate that CRH-elicited signaling leads to the assembly of a transcriptional regulatory complex that includes DNA-binding transcription factors such as NGFI-B and Tpit. This complex would also contain common transcriptional co-factors such as the SRCs, and the multiple co-activator interactions are consistent with their recruitment by different DNA-bound factors.


   FOOTNOTES
 
* This work was funded by the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a doctoral research award from the Canadian Institutes of Health Research. Back

§ To whom correspondence should be addressed: Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, 110, avenue des Pins Ouest, Montréal, PQ H2W 1R7, Canada. Tel.: 514-987-5680; Fax: 514-987-5575; E-mail: drouinj{at}ircm.qc.ca.

1 The abbreviations used are: POMC, pro-opiomelanocortin; CRH, corticotropin-releasing hormone; GH, growth hormone; PKA, cAMP-dependent protein kinase; MAPK, mitogen-activated protein kinase; RSV, Rous sarcoma virus; {beta}-Gal, {beta}-galactosidase; MBP, maltose-binding protein. Back

2 E. Batsche, J. Desroches, S. Bilodeau, Y. Gauthier, and J. Drouin, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Pierre Chambon, who generously provided the expression plasmids for SRC-2 (TIF-2) and mutant constructs, as did Dr. S. McKnight for PKAc. We thank Aurelio Balsalobre and colleagues of the laboratory for critical comments on the manuscript. We also thank Lise Laroche for expert secretarial assistance.



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 DISCUSSION
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