TIF1β/KAP-1 Is a Coactivator of the Orphan Nuclear Receptor NGFI-B/Nur77*

In efforts to define mechanisms of transcriptional activation by the orphan nuclear receptor NGFI-B (Nur77), we identified TIF1β by mass spectrometry within a nuclear protein complex containing NGFI-B. TIF1β, also known as KAP-1 (KRAB domain-associated protein) or KRIP-1, acts as a transcriptional corepressor for many transcription factors, in particular for the Krüppel-associated box domain-containing zinc finger transcription factors. TIF1β is also an intrinsic component of two chromatin remodeling and histone deacetylase complexes, the N-CoR1 and nucleosome remodeling and deacetylation complexes. In contrast to these activities, we report that TIF1β is a coactivator of NGFI-B and that it is as potent as the SRC coactivators in this context. Using pull-down assays and immunoprecipitation, we showed that TIF1β interacts directly with NGFI-B and with other Nur family members. NGFI-B is an important mediator of hypothalamic corticotropin-releasing hormone (CRH) activation of proopiomelanocortin (POMC) transcription, and TIF1β enhances transcription mediated through the NGFI-B target, the Nur response element (NurRE). The NurRE binds Nur factor dimers and is responsive to signaling pathways. In keeping with the role of NGFI-B as mediator of CRH signaling, we found that TIF1β is recruited to the POMC promoter following CRH stimulation and that TIF1β potentiates CRH and protein kinase A signaling through the NurRE; it acts synergistically with the SRC2 coactivator. However, the actions of TIF1β and SRC2 were mapped to different NGFI-B AF-1 subdomains. Taken together, these results indicate that TIF1β is an important coactivator of NGFI-B-dependent transcription.

In efforts to define mechanisms of transcriptional activation by the orphan nuclear receptor NGFI-B (Nur77), we identified TIF1␤ by mass spectrometry within a nuclear protein complex containing NGFI-B. TIF1␤, also known as KAP-1 (KRAB domain-associated protein) or KRIP-1, acts as a transcriptional corepressor for many transcription factors, in particular for the Krü ppel-associated box domain-containing zinc finger transcription factors. TIF1␤ is also an intrinsic component of two chromatin remodeling and histone deacetylase complexes, the N-CoR1 and nucleosome remodeling and deacetylation complexes. In contrast to these activities, we report that TIF1␤ is a coactivator of NGFI-B and that it is as potent as the SRC coactivators in this context. Using pull-down assays and immunoprecipitation, we showed that TIF1␤ interacts directly with NGFI-B and with other Nur family members. NGFI-B is an important mediator of hypothalamic corticotropin-releasing hormone (CRH) activation of proopiomelanocortin (POMC) transcription, and TIF1␤ enhances transcription mediated through the NGFI-B target, the Nur response element (NurRE). The NurRE binds Nur factor dimers and is responsive to signaling pathways. In keeping with the role of NGFI-B as mediator of CRH signaling, we found that TIF1␤ is recruited to the POMC promoter following CRH stimulation and that TIF1␤ potentiates CRH and protein kinase A signaling through the NurRE; it acts synergistically with the SRC2 coactivator. However, the actions of TIF1␤ and SRC2 were mapped to different NGFI-B AF-1 subdomains. Taken together, these results indicate that TIF1␤ is an important coactivator of NGFI-B-dependent transcription.
NGFI-B (also known as Nur77, TR3, and NAK-1) is a transcription factor belonging to the superfamily of nuclear receptors (NRs). 4 NGFI-B is closely related to Nurr1 (Nur-related factor 1) (RNR-1, TINUR, and HZF-3) and NOR-1 (neuronderived orphan receptor 1) (MINOR) (1)(2)(3), together forming a distinct subfamily, the Nur factors. NGFI-B and NOR-1 are constitutively expressed in some regions of the brain as well as in peripheral tissues (3)(4)(5). In contrast, the Nurr1 expression pattern is more restricted in the central nervous system. The Nur factors are immediate early response genes that share a well conserved DNA binding domain and ligand binding domain but a poorly conserved N-terminal A/B region (6). Nur subfamily members are important physiological regulators implicated at multiple levels of the hypothalamo-pituitary-adrenal axis. This axis mediates the stress response via secretion of adrenocorticotropic hormone (ACTH) and induction of adrenal glucocorticoid synthesis. ACTH is derived from the processing of the proopiomelanocortin (POMC) precursor, and it is under the control of hypothalamic corticotropin-releasing hormone (CRH). At the hypothalamic level, CRH-producing neurons exhibit induced Nur factors after stress (7,8), and these may in turn regulate CRH gene transcription (9). In pituitary corticotroph cells, CRH activates POMC gene transcription (10,11). Upon binding to its receptor CRHR-1 on corticotrophs (12), CRH induces a signaling cascade that ultimately leads to increased POMC gene transcription. CRH increases cAMP levels, followed by activation of the protein kinase A (PKA) and mitogen-activated protein kinase pathways (13)(14)(15). Nur factors regulate the POMC promoter via the Nur response element (NurRE) that binds homodimers or heterodimers of Nur factors containing at least NGFI-B (16,17). NGFI-B was shown to be an important mediator of CRH action on POMC transcription through the NurRE (17,18). The molecular events involved in CRH activation include dephosphorylation of Ser 316 of NGFI-B, which allows dimer binding to the NurRE and recruitment of SRC2 and Rb (18,19). SRC/p160 coactivators enhance transcription in part by their intrinsic histone acetyltransferase activity and by recruitment of CBP/p300 and coactivators that contain other enzymatic activities (20,21) such as the histone methyltransferase CARM-1.
Finally, glucocorticoids exert a negative feedback on POMC gene transcription. The NurRE activity is subject to glucocorticoid receptor (GR) trans-repression, involving direct interactions between NGFI-B and GR (17,22,23). In order to identify hormone responsive regulators that might be involved in CRH activation and glucocorticoid repression of POMC transcription through the NurRE, we investigated CRH-dependent NGFI-B-associated proteins that may serve a transcription co-regulatory function in POMC-expressing cells. We thus report the identification and the characterization of TIF1␤ as a novel CRH-dependent coactivator of NGFI-B.
TIF1␤, also known as KAP-1 (KRAB domain-associated protein) (31) or KRIP-1 (32), is proposed to act as the universal transcriptional corepressor for the Krüppel-associated box (KRAB) domain-containing zinc finger transcription factors, such as KOX1 (33), ZNF133, ZNF140 (31), and Kid-1 (32), through a conserved interaction between variants of the KRAB domain and TIF1␤ (34). For transcriptional repression, TIF1␤ recruits different enzymatic activities through direct proteinprotein interactions, including 1) the nucleosome remodeling and deacetylation complex (NuRD/Mi-2␣) (35) and 2) the histone methyltransferase SETDB1, which specifically methylates Lys 9 of histone H3 (36). TIF1␤ is also an intrinsic component of the N-CoR-1 complex containing both histone deacetylase and ATP-dependent SWI/SNF activities (37). Finally, TIF1␤ interacts directly with endogenous members of the HP1 (heterochromatin protein 1) family (27,38,39). These data support a role for TIF1␤ in the epigenetic regulation of transcription via the initiation and maintenance of heterochromatin structures. In agreement with this general repressor function, TIF1␤ was shown to be localized in heterochromatin; however, it was also observed in euchromatin (38). Consistent with this presence in active euchromatin, TIF1␤ was found to be a coactivator of CCAAT/enhancer-binding protein-␤ and GR for the induction of the ␣1-acid glycoprotein gene (40).
In this study, we show that TIF1␤ enhances POMC gene transcription through Nur orphan NRs. In particular, TIF1␤ is recruited to the POMC promoter under CRH stimulation, and it enhances Nur-dependent CRH and PKA responses. We further show that the action of TIF1␤ on NGFI-B-dependent transcription may involve synergistic activation with SRC2, a coactivator of a different structural class that has been implicated in the action of many NRs.
Cell Culture and Transfection-AtT-20 and HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin/streptomycin. AtT-20 cells were transfected by lipofection with Lipofectamine (Invitrogen), as previously described (17). Each transfection (in duplicates) was repeated at least three times.
Generation of Retrovirus-infected AtT-20 Cells-The Eco-Pack-2 293 cells (Clontech, Mountain View, CA) were plated (5 ϫ 10 6 ) in a 10-cm plate and transfected by the calcium phosphate coprecipitation method with 10 g of expression plasmid pLNCX 2 -3ϫFLAG-NGFI-B-V5-His for 8 h. 16 h later, the medium containing the retrovirus was filtered with a 0.22-m filter; diluted in Dulbecco's modified Eagle's medium, 10% fetal bovine serum with penicillin/streptomycin and 1 g/ml Polybrene (Sigma); and added to AtT-20 cells. This infection procedure was repeated three times/day for 2 days, and infected AtT-20 cells were cultured with 400 g/ml neomycin. The growing neomycin-resistant colonies were pooled together to generate a retrovirus-infected population of about 5,000 independent colonies.
Nuclear Extract Preparation and FLAG Purification Procedure-Nuclear extracts used for affinity FLAG purification of flagged proteins were prepared with 4 ϫ 10 9 cells AtT-20 cells. The cells were washed twice with cold phosphate-buffered saline and collected in a 50-ml tube. The cells were then centrifuged and resuspended in 10 volumes of buffer A (20 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, and the protease inhibitors leupeptin, aprotinin, and pepstatin (10 g/ml each)) and centrifuged immediately. The cytoplasmic extract was prepared by resuspending the cell pellet in 2 volumes of buffer A and leaving it at 4°C for 10 min with gentle mixing. The nuclei

TIF1␤/KAP-1 Is a Coactivator of NGFI-B/Nur77
were collected by centrifugation, washed once with 1 volume of buffer A, and extracted with the addition of 1 volume of buffer HS (20 mM Hepes, pH 7.9, 500 mM KCl, 10% glycerol, 3 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM PMSF, 1 mM DTT, and the protease inhibitors leupeptin, aprotinin, and pepstatin (10 g/ml each)). The nuclei were allowed to extract for 30 min at 4°C with continuous gentle mixing. The extract was centrifuged, and the protein concentration of the supernatant was determined by a Bradford assay (Bio-Rad). The nuclear extracts were precleared at 4°C using IgG-Sepharose 6 fast flow beads (Amersham Biosciences), A/G-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and agarose IgG beads (Sigma) for 1 h. The extracts were then diluted with 1 volume of IP 2ϫ buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 10% glycerol, 1.5 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM PMSF, 1 mM DTT, 0.2% Tween 20, and the protease inhibitors leupeptin, aprotinin, and pepstatin (10 g/ml each)), and the supernatant was immunoprecipitated for 3 h at 4°C with ␣-FLAG M2 or nonimmune IgG cross-linked to agarose beads (Sigma). Immunoprecipitates were washed twice with IP buffer (20 mM Hepes, pH 7.9, 125 mM KCl, 5% glycerol, 1.5 mM MgCl 2 , 0.1 mM EDTA, and 0.1% Tween 20) and twice with IP buffer containing 300 mM KCl instead of 125 mM KCl. The eluate was obtained by incubating the bead with 500 g/ml of 3ϫ FLAG peptide.
Mass Spectrometry Analysis-The eluate was resolved on an 8% SDS-polyacrylamide gel, and the gel was silver-stained. The protein bands were excised from the gel and subjected to reduction, alkylation, and in-gel tryptic digestion, as previously described (47). The resulting tryptic peptides were purified and identified by MALDI-TOF mass spectrometry. For the proteins indicated in Fig. 2, different peptides were identified, as indicated in supplemental Table 1.
Coimmunoprecipitation Assays and Western Blot Analysis-HEK293T cells (3 ϫ 10 6 ) were plated in a 10-cm plate and transfected by the calcium phosphate coprecipitation method with 5 g of expression plasmids for tagged polypeptides for 48 h. The cells were then washed twice with cold phosphatebuffered saline and then centrifuged and resuspended in 5 volumes of buffer A (20 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.5 mM PMSF, 1 mM DTT, and the protease inhibitors leupeptin, aprotinin, and pepstatin (10 g/ml each)) and centrifuged immediately. The cell pellet was resuspend in 2 volumes of buffer A, and cells were allowed to swell on ice for 10 min before the addition of 3 volumes of buffer B (20 mM Hepes, pH 7.9, 500 mM KCl, 10% glycerol, 3 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM PMSF, 1 mM DTT, and the protease inhibitors leupeptin, aprotinin, and pepstatin (10 g/ml each)). Nuclei were allowed to extract for 30 min at 4°C with continuous gentle mixing. The extract was centrifuged, and the protein concentration of the supernatant was determined by a Bradford assay (Bio-Rad). The extracts were precleared at 4°C using IgG Sepharose 6 fast flow beads (Amersham Biosciences), A/G-agarose beads (Santa Cruz Biotechnology), and agarose IgG beads (Sigma), and the supernatant was immunoprecipitated for 2 h at 4°C with ␣-FLAG M2 cross-linked to agarose beads (Sigma). Immunoprecipitates were washed twice with buffer C (20 mM Hepes, pH 7.9, 125 mM KCl, 5% glycerol, 1.5 mM MgCl 2 , 0.1 mM EDTA, and 0.1% Tween 20) and twice with buffer D (buffer C with 300 mM KCl). After SDS-PAGE, Western blots were revealed with antibodies against V5 (R960-25; Invitrogen), FLAG M2 (Sigma), hemagglutinin 12CA5 (ab16918; Abcam). Immunodetection was done with horseradish peroxidase-conjugated anti-rabbit (or mouse) IgG (Sigma), and revelation was performed with the ECLϩ reagent (Amersham Biosciences) as described by the manufacturer.

TIF1␤/KAP-1 Is a Coactivator of NGFI-B/Nur77
Recombinant Protein Production and Pull-down Assays-The TIF1␤-GST and empty-GST was provided by M. Aubry.
Recombinant protein production and pull-down assays were done as previously described (19).

Several Transcriptional Regulatory Proteins Interact with NGFI-B-
We have previously shown that SRC2 and Rb enhance the activity of the POMC promoter, and a part of this action was ascribed to the interactions with the orphan nuclear receptor NGFI-B (19). These coactivators are probably recruited as part of larger coactivator complexes. Thus, we used gel filtration of AtT-20 nuclear extracts on a Superose 6 column to test whether NGFI-B and Nurr1 are present within high molecular weight protein complexes (Fig. 1A). NGFI-B and Nurr1 were detected by Western blot in many fractions ranging in size from Ն1 MDa to free protein. A similar profile was obtained for Tpit, another target of CRH signaling (18). In contrast, other POMC transcription factors, NeuroD1 and Pitx1, exhibited different profiles. Indeed, NeuroD1 is exclusively present in a ϳ500-kDa protein complex, whereas Pitx1 is mainly found in its free form. The profiles were similar in CRH-treated AtT-20 cells (data not shown). These findings suggest that NGFI-B and Nurr1 are present in high molecular weight protein complexes, which supports the hypothesis of an association with multiple protein complexes in AtT-20 cells.
In order to identify such NGFI-B-associated proteins, we produced AtT-20 cells expressing a tagged version of NGFI-B at levels that are similar to endogenous NGFI-B ( Fig. 2A). NGFI-B-associated proteins were purified from AtT-20 nuclear extracts using a FLAG-M2 affinity column followed by elution with FLAG peptides (Fig. 2B). This eluate was resolved by SDS-PAGE, and the interacting proteins were revealed by silver staining and analyzed by mass spectrometry (Fig. 2C). A list of tryptic peptides identified by mass spectrometry is provided in supplemental Table 1. All bands identified on Fig. 2C were only present in the eluate from FLAG-NGFI-B-V5-His but not mock-transfected AtT-20 cells, except the three lower bands (EF-1␣, ␤-actin, and TMOD3) (data not shown). Different subunits of known multiprotein complexes were identified, such as BAF57 and BAF155 of the SWI/SNF complex and Chd4/Mi-2␤, MTA2, and p66␤ of the Mi-2␤/NuRD repression complex. These proteins were present at similar levels in control and forskolin-treated AtT-20 cells (data not shown). In contrast, some proteins appeared more abundant in treated compared with control cells (Fig. 2D): Mybbp1a, Pelp1 (MNAR), and the transcriptional coregulator TIF1␤ were among this group. We did not detect the previously characterized NGFI-B coactivators

TIF1␤/KAP-1 Is a Coactivator of NGFI-B/Nur77
SRC2 and Rb in these experiments. This may be due to low expression levels of these proteins in AtT-20 cells or to lower stability of their association with the NGFI-B complex. We confirmed these interactions by reverse coimmunoprecipitation in HEK293T cells that overexpressed the different FLAG-tagged polypeptides (FLAG-X) identified by mass spectrometry with V5-His-tagged NGFI-B. This experiment confirmed the interactions between NGFI-B-V5-His and the different proteins tested; in contrast, the basic helix-loop-helix factor E47 used as control did not co-immunoprecipitate with NGFI-B (Fig. 2E). The purification of these different proteins with NGFI-B suggests that they are part of large protein complexes similar to those described above (Fig. 1A). Indeed, we found that TIF1␤ has a similar molecular weight distribution as Nurr1 and NGFI-B in AtT-20 nuclear extracts (Fig. 1B) in agreement with the hypothesis that they may act together. It is also possible that NGFI-B is a part of protein complexes of different molecular weights, and that could relate to different profiles on Superose 6, such as the SRC2 profile (Fig. 1B).

NurRE-dependent Transcription Is Enhanced by TIF1␤-Previous
analyses of POMC promoter sequences responsive to both CRH and NGFI-B identified the NurRE as target (17). The dimer-binding NurRE is much more responsive than the monomer-binding NBRE to NGFI-B (16,18), particularly to signal-activated NGFI-B (17). The putative action of TIF1␤ on NGFI-B activity was tested directly using NurRE and NBRE reporters in AtT-20 cells (Fig. 3A). Expression vector for NGFI-B or empty vector was cotransfected in AtT-20 with increasing amounts of TIF1␤ expression plasmid using the NBRE-Luc (right) or NurRE-Luc (left) reporter. Without NGFI-B, TIF1␤ had no effect on NurRE and NBRE, but in its presence, we observed that TIF1␤ behaved as a strong coactivator of NGFI-B dimers acting on the NurRE compared with the NBRE (Fig. 3A).
TIF1␤ Enhances POMC Gene Transcription-Since the NurRE was previously shown to be important for POMC expression and responsiveness to NGFI-B (17), we tested whether TIF1␤ could regulate POMC promoter activity in AtT-20 cells (Fig. 3B). Whereas the minimal promoter was not affected (Fig. 3B, right), we observed that the intact POMC promoter was indeed activated by increasing amounts of TIF1␤ (left). Similar results were obtained in HEK293T and CV1 cells (data not shown). Mutagenesis of the POMC promoter NBRE-like sequence did not affect the ability of TIF1␤ to enhance POMC transcription. In contrast, mutagenesis of the NurRE abolished enhancement by TIF1␤ (middle). The in vivo importance of endogenous TIF1␤ protein in POMC transcription was assessed directly using a pool of four siRNAs against TIF1␤ in AtT-20 cells. The knock-down of TIF1␤ was ascertained by Western blot (Fig. 3C) and correlated with a reduction of POMC mRNA levels assessed by reverse transcription-quantitative PCR (Fig. 3D). Further, each siRNA from the pool resulted in varying degrees of TIF1␤ mRNA knockdowns that are directly correlated with their effect on POMC mRNA levels (Fig. 3E). Thus, basal POMC expression is partly dependent on TIF1␤ acting through the NurRE regulatory element. TIF1␤ Potentiates Both CRH and PKA Signaling through the NurRE but Not the Tpit/PitxRE-The NurRE and the cognate Nurs factors participate in basal activity as well as in CRH responsiveness of the POMC promoter (16,17). Consequently, we tested the ability of TIF1␤ to enhance the stimulatory effect of CRH on Nur-dependent transcription in AtT-20 cells. As shown in Fig. 4A, the activity of the NurRE reporter was activated by CRH, and this activation was reversed by Dex, a synthetic glucocorticoid. The overexpression of TIF1␤ alone did not affect basal reporter activity but potentiated both CRH and Dex/CRH responses.
The CRH signaling pathway involves a rapid increase in cAMP levels (14), followed by activation of PKA. Previous work had identified NGFI-B and Tpit as end point effectors of PKA signaling, through the NurRE and Tpit/PitxRE, respectively (18,50). We tested whether TIF1␤ modulates PKA-dependent transcription using the NurRE reporter in AtT-20 cells. PKA stimulation alone had a strong effect on NGFI-B-dependent transcription, and this effect was markedly potentiated by TIF1␤ (Fig. 4B). Similar results were obtained in CV-1 and 293T cells (data not shown). In contrast, TIF1␤ did not change the activity of the Tpit/PitxRE reporter with or without PKA (Fig. 4C). Thus, TIF1␤ potentiates both CRH and PKA signaling through the NurRE but not the Tpit/PitxRE.
TIF1␤ Is Recruited to the POMC Promoter-We next assessed recruitment of TIF1␤ to the POMC promoter upon treatment of AtT-20 cells with CRH, Dex, and CRH ϩ Dex by ChIP technique. In basal conditions, TIF1␤ was present at the promoter but only slightly above background. The recruitment of TIF1␤ was enhanced on the POMC promoter upon CRH and Dex/CRH treatments (Fig. 4D, left). Treatment with Dex, which represses POMC transcription (10,51,52), did not affect TIF1␤ recruitment to the POMC promoter, in accordance with previous data showing that Dex does not affect NGFI-B recruitment either (23). These results support a model in which TIF1␤ is recruited to the POMC promoter following CRH stimulation to enhance POMC transcription; this model is also supported by the greater amount of TIF1␤ associated with NGFI-B in extracts from forskolin-treated cells (Fig. 2D).
The Nur Factors Are Targets of TIF1␤ and Directly Bind TIF1␤-To test whether all three members of the Nur subfamily are targets of TIF1␤, we investigated the effect of TIF1␤ on NGFI-B, Nurr1, and NOR-1. Putative potentiation by TIF1␤ was assessed using luciferase reporters containing three copies of either the POMC gene NurRE (NurRE POMC ) (Fig. 5A) or a consensus NurRE (NurRE CON ) (Fig. 5B). We previously showed that the NuRE POMC has a preference for NGFI-B-containing dimers, whereas the NurRE CON does not exhibit a pref-
We showed interactions between TIF1␤ and NGFI-B (Fig.  2E). We therefore investigated the direct protein-protein interaction by using an in vitro pull-down assay (Fig. 5C), in which a resin-bound GST-TIF1␤ fusion protein was tested for interaction with in vitro translated NGFI-B, Nurr1, NOR-1, or luciferase. The three Nur factors bound specifically the GST-TIF1␤ column but not the empty vector-GST. Thus, all three Nur factors interact in vitro with TIF1␤.
TIF1␤ Enhances NGFI-B-dependent Transcription through the N-terminal AF-1A Domain-We previously showed that SRC coactivators exert their effects through the AF-1 N-terminal domain of NGFI-B (18). The AF-1 was subdivided into two regions comprised between amino acids 20 and 36 (AF-1A) and between amino acids 74 and 174 (AF-1B); both subdomains were found to contribute to SRC coactivator potentiation, but AF-1B mediated the PKA stimulatory effect (18). Expression vectors for NGFI-B and several deletions of NGFI-B were cotransfected in AtT-20 with increasing amounts of TIF1␤ expression plasmid and the NurRE-Luc reporter (Fig. 6A). Enhancement of NGFI-Bdependent activity by TIF1␤ was partially affected by deletion of NGFI-B N-terminal sequences between amino acids 3 and 13 (deletion ⌬N1). Complete loss of activity was observed upon deletion of the AF-1A subdomain (⌬N3). To support the interpretation that the AF-1A subdomain is entirely responsible for the action of TIF1␤, we created an internal deletion that only removed the AF-1B subdomain (mutant ⌬N7). This ⌬N7 mutant is fully competent to respond to TIF1␤ (Fig. 6A). Similar results were obtained in HEK293T and CV1 cells (data not shown). These results indicate that the AF-1A domain of NGFI-B is necessary and sufficient for TIF1␤ potentiation.
CRH/PKA Signaling Is Mediated through the N-terminal AF-1 Domain-The CRH signaling pathway involves activation of PKA. In order to gain further insight into mechanisms of PKA-or coactivator-induced transcriptional activity of NGFI-B, we used the series of deletion mutants in transfection experiments that included PKA stimulation using an expression vector for the catalytic subunit of PKA (Fig. 6B). PKA stimulation on its own had a strong effect on NGFI-B-dependent transcription, and this effect was potentiated by TIF1␤ (Fig. 6B, WT  NGFI-B).
Deletion between amino acids 17 and 74 of the N-terminal domain of NGFI-B (deletion ⌬N3) resulted in loss of the TIF1␤dependent enhancement (Fig. 6A), but this deletion did not abolish the effect of PKA. Indeed, deletion of the two AF-1 subdomains (deletion ⌬N5) was needed to abolish the PKA effect; however, the PKA-enhanced activity of mutant ⌬N3 was not increased by TIF1␤, in agreement with the interpretation that TIF1␤ can only act on the AF-1A domain. This activity is thus probably mediated through SRC coactivators, as previously described (18). In agreement with this, the ⌬N7 mutant that is only deleted of subdomain AF-1B was as sensitive to PKA and TIF1␤ as wild-type NGFI-B (Fig. 6B). These results suggest that both basal and PKA-stimulated TIF1␤ activities are mediated through the AF-1A domain, whereas prior work had localized PKA potentiation of SRC2-dependent activity to the NGFI-B AF-1B subdomain (18).  MAY 22, 2009 • VOLUME 284 • NUMBER 21

TIF1␤ Synergizes with the Coactivator SRC2 for NGFI-B-dependent
Transcription-SRC2 was previously implicated in CRH/PKAdependent activation of NGFI-B (18). Hence, we tested a putative collaboration between SRC2 and TIF1␤ using the NurRE reporter in AtT-20 cells. As shown in Fig. 7A, a limiting amount of SRC2 and TIF1␤ synergistically enhanced NGFI-Bdependent activity on the NurRE reporter, suggesting that TIF1␤ and SRC2 may be part of the same transcriptional complex. We next assessed a putative interaction between TIF1␤ and SRC2 in vivo using coimmunoprecipitation in  HEK293T cells. Immunoprecipitation of FLAG-tagged SRC2 brought down HA-TIF1␤ (Fig. 7B).
We wanted to verify that SRC2 and TIF1␤ are present at the POMC and whether their recruitment is increased by CRH. Using ChIP, we assessed recruitment of SRC2 and TIF1␤ to the POMC promoter (Fig. 4B). In basal conditions as well as following CRH activation and Dex repression, both TIF1␤ and SRC2 are recruited in parallel to the POMC promoter, in agreement with previous data (19). Thus, TIF1␤ and SRC2 may be part of a common transcriptional complex and thus synergistically enhance NGFI-B-dependent transcription. It is noteworthy that the combined action of TIF1␤ and SRC2 surpasses their individual action (Fig. 7A).

DISCUSSION
The present work documented a coactivator function for TIF1␤ and implicated this coactivator in hormone responsiveness mediated through NGFI-B and related NRs. Multiple experiments support the role of TIF1␤ as a hormone-responsive coactivator of NGFI-B, starting with the identification of this protein by mass spectrometry within protein complexes associated with NGFI-B. Not only was TIF1␤ identified within affinity-purified complexes, but further, the amount of TIF1␤ was shown to be enhanced by cAMP signaling (Fig. 2). We showed that TIF1␤ enhances transcription dependent on the POMC promoter and its NurRE (Fig. 3) and that this action potentiates responsiveness to the hypothalamic hormone CRH and to PKA (Fig. 4). These actions were mapped to the N-terminal half of the NGFI-B AF-1 domain (AF-1A; Fig. 6). The in vivo relevance of these interactions was supported by ChIP and siRNA knockdown experiments.
In the general context of NR-dependent activation of transcription, it is also noteworthy that we report coactivation by both TIF1␤ and the SRC2 coactivator that was previously implicated in the action of many NRs. We have shown recruitment of both coactivators in response to CRH at the POMC promoter, and we showed that both can be present within the same protein complexes in cells, thus providing a molecular basis for their synergistic coactivation of transcription dependent on NGFI-B and the NurRE. Interestingly, the activity of each coactivator was mapped to a different subregion of the NGFI-B AF-1 domain. Whereas SRC2 was previously shown to mediate the activator effect of CRH-dependent signaling through both NGFI-B and Tpit, TIF1␤ appears to be only involved in NGFI-B-dependent transcription. The involvement of two different coactivators in enhancement of NGFI-B-dependent transcription may increase the possibilities of signaling inputs into the control of target genes; it will ultimately be interesting to assess whether other NGFI-B-dependent target genes also use these two coactivators.
TIF1␤ as Coactivator-TIF1␤ was initially identified as the universal transcriptional co-repressor for the large family of vertebrate-specific KRAB domain-containing zinc finger transcription factors (53). TIF1␤-mediated gene silencing involves the recruitment of histone deacetylases (37,54), and of histone methyltransferases (36). Only one study had previously described TIF1␤ as a coactivator. Indeed, TIF1␤ was found to act as coactivator of the bZIP transcription factor CCAAT/ enhancer-binding protein-␤, which mediates together with GR the induction of the ␣1-acid glycoprotein gene by inflammatory cytokines and glucocorticoids (40).
The present work supports the function of TIF1␤ as a transcriptional activator of NGFI-B. It was previously shown that the AF-1 domain of NGFI-B mediates coactivator recruitment through direct protein-protein interaction (55). SRC1, -2, and -3 as well as Rb were shown to exert coactivator effects through the AF-1 N-terminal domain of NGFI-B (18,19). Prior work (18) subdivided the AF-1 into two regions comprised between amino acids 20 and 36 (AF-1A) and between amino acids 74 and 174 (AF-1B) and indicated that SRC1, -2, and -3 were acting through both subdomains but that PKA signals were primarily targeting the AF-1B-dependent activity. In contrast, all TIF1␤dependent actions (basal and PKA-induced) on NGFI-B appeared to depend on the AF-1A subdomain (Fig. 6). Taken together, these data indicate that TIF1␤ acts on the AF-1A subdomain and SRCs act preferentially, but not exclusively, on the AF-1B subdomain.
It was suggested that sumoylation is important for the repressor activity of TIF1␤ (45). Sumoylation was found to stabilize the association of the bromodomain with the chromatin modifiers SETBD1 and the NurD complex, thereby promoting establishment of the silent gene expression state. When we tested whether the sumoylation sites of TIF1␤ have an impact on NurRE activation, we found the TIF1␤ sumoylation-deficient mutant to be unaffected in its ability to activate the NurRE reporter (data not shown). Transcriptional co-activation by TIF1␤ therefore appears independent of its sumoylation, but it may be modulated by other post-translational modification, such as phosphorylation (38).
Coactivator Synergism-We observed synergistic activation of NGFI-B-dependent transcription by TIF1␤ and SRC2 (Fig.  7A). When assessed separately for coactivation of NGFI-B dimers, TIF1␤ is of similar potency as SRC2. Both act on the NurRE target of NGFI-B dimers but not on the NBRE monomer target (Fig. 3A) (18). TIF1␤ may be recruited as part of a transcriptional complex together with SRC2 for NGFI-B-dependent transcription, as suggested by the fact that both can be coimmunoprecipitated (Fig. 7B) and that both are recruited to the POMC promoter following CRH activation (Fig. 4D). The data may be taken to suggest that TIF1␤ and SRC2 are co-recruited to NGFI-B dimers upon CRH stimulation. However, there are differences in the action of these two co-activators on NGFI-B and on different POMC promoter targets. Indeed, the two coactivators target different AF-1 subdomains. In addition, the CRH signaling pathway acting through PKA and mitogen-activated protein kinase targets SRC2 to both NGFI-B and Tpit through the NurRE and Tpit/PitxRE, respectively (50). In contrast, TIF1␤ potentiated PKA activation of NGFI-B-dependent transcription but not the activity of the Tpit/PitxRE with or without PKA (Fig. 4, B and C). SRC2 enhanced the intrinsic activity of Tpit on its cognate POMC target as well as its synergistic activity with Pitx1. These results underline functional differences in the mechanisms of action of these two co-activators and may reflect the involvement of multiple signals for CRH activation.