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

Juliette Rambaud,Julien Desroches,Aurélio Balsalobre,Jacques Drouin

doi:10.1074/jbc.m809023200

Juliette Rambaud, Julien Desroches + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m809023200

Copy DOI

Abstract

In efforts to define mechanisms of transcriptional activation by the orphan nuclear receptor NGFI-B (Nur77), we identified TIF1beta by mass spectrometry within a nuclear protein complex containing NGFI-B. TIF1beta, 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. TIF1beta 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 TIF1beta 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 TIF1beta 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 TIF1beta 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 TIF1beta is recruited to the POMC promoter following CRH stimulation and that TIF1beta potentiates CRH and protein kinase A signaling through the NurRE; it acts synergistically with the SRC2 coactivator. However, the actions of TIF1beta and SRC2 were mapped to different NGFI-B AF-1 subdomains. Taken together, these results indicate that TIF1beta is an important coactivator of NGFI-B-dependent transcription.

Highlights

Glucocorticoids exert a negative feedback on POMC gene transcription
TIF1␤/KAP-1 Is a Coactivator of NGFI-B/Nur77 be involved in corticotropin-releasing hormone (CRH) activation and glucocorticoid repression of POMC transcription through the Nur response element (NurRE), we investigated CRH-dependent NGFI-B-associated proteins that may serve a transcription co-regulatory function in POMC-expressing cells
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]

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The reporter plasmids were constructed in pXP1luc [41] containing the minimal POMC promoter (Ϫ34 to ϩ63 bp). The extracts were diluted with 1 volume of IP 2ϫ buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 10% glycerol, 1.5 mM MgCl2, 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). The cells were washed twice with cold phosphatebuffered saline and 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.

Several Transcriptional Regulatory Proteins Interact with

NurRE and NBRE reporters in

DISCUSSION