TNIP1 is a corepressor of agonist-bound PPARs

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Abstract

Nuclear receptor (NR) coregulators include coactivators, contributing to holoreceptor transcriptional activity, and corepressors, mediating NR target gene silencing in the absence of hormone. We identified an atypical NR coregulator, TNFα-induced protein 3-interacting protein 1 (TNIP1), from a peroxisome proliferator activated receptor (PPAR) α screen of a human keratinocyte cDNA library. TNIP1’s complex nomenclature parallels its additional function as an NF-κB inhibitor. Here we show TNIP1 is an atypical NR corepressor using two-hybrid systems, biochemical studies, and receptor activity assays. The requirements for TNIP1–PPAR interaction are characteristic for coactivators; however, TNIP1 partially decreases PPAR activity. TNIP1 has separable transcriptional activation and repression domains suggesting a modular nature to its overall effect. It may provide a means of lowering receptor activity in the presence of ligand without total loss of receptor function. TNIP1’s multiple roles and expression in several cell types suggest its regulatory effect depends on its expression level and the expression of other regulators in NR and/or NF-κB signaling pathways. As a NR coregulator, TNIP1 targeting agonist-bound PPAR and reducing transcriptional activity offers control of receptor signaling not available from typical corepressors and may contribute to combinatorial regulation of transcription.

Highlights

► We report TNIP1is a corepressor of agonist-bound PPARs. ► TNIP1 was previously known to associate with HIV proteins and repress NF-κB. ► TNIP1 binds PPARs in a manner typical of coactivators but represses their activity. ► TNIP1 has separable activation and repression domains. ► Corepressors such as TNIP1 may contribute to combinatorial transcription regulation.

Introduction

The majority of ∼300 nuclear receptor (NR)2 coregulators identified to date are coactivators; far fewer corepressors have been isolated. Coactivators such as the p160/steroid receptor coactivator (SRC) and thyroid hormone receptor-associated proteins (TRAP) [1] associate with agonist-bound receptors via the coactivator’s NR box, a short, amphipathic, leucine-rich motif (LXXLL, L = leucine, X = any amino acid). Physical association of unliganded NRs with corepressors e.g., nuclear receptor corepressor (NCoR) or silencing mediator for retinoid and thyroid hormone receptor (SMRT) is mediated by amphipathic leucine-rich helices (L/I-XX-I/V-I or L-XXX-I/L-XXX-L; I = isoleucine, V = valine, X = any amino acid) in the coregulator referred to as corepressor/nuclear receptor (CoRNR) boxes [2]. For most NR-coregulator interactions, the aporeceptor associates with corepressors and upon agonist binding undergoes a conformational change, shedding the corepressor and associating with a coactivator. However, the discoveries of ligand dependent corepressor (LCoR), receptor interaction protein (RIP) 140, and in this report TNIP1 have recognized a distinct group of coregulators. These proteins represent a novel, functionally allied group of coregulators using NR boxes, targeting the agonist-bound receptor’s activating function-(AF) 2 region but reducing receptor-mediated gene transcription. This distinguishes them from typical NR corepressors such as SMRT and NCoR ([3] for review) and provides additional means of reducing NR activity without the more-extreme loss of activity conferred in the absence of ligand by SMRT or NCoR.

Three PPAR subtypes, PPARα, PPARδ, and PPARγ have been identified (NR1C1, NR1C2 and NR1C3) and each has been implicated in multiple aspects of keratinocyte response to natural and synthetic PPAR ligands [4], [5], [6], [7], [8]. As PPAR coregulators ([9] for review) would participate in these responses, we report here on a novel PPAR coregulator isolated via a two-hybrid screen of keratinocyte cDNA. Intriguingly, this PPAR-interacting clone matched coding sequences previously isolated via protein–protein interaction with (i) the HIV protein Nef, (ii) the HIV protein matrix, and (iii) the human zinc finger protein A20 [10], [11], [12]. In each case, different names were given to the isolate (Naf, VAN and ABIN, respectively). As found in the National Center for Biotechnology Information database, the sequence is named TNIP1 for TNFAIP3-interacting protein 1, and we use that designation in this report. Beyond the roles described for TNIP1 under its aliases of Naf, VAN, and ABIN are its recent gene-disease associations in lung cancer and psoriasis [13], [14].

Given the diversity of previous TNIP1 investigations and having isolated TNIP1 as a PPARα-interacting protein the objectives of this study became (i) to examine the expression range of TNIP1, (ii) to determine the ligand requirements and amino acid motifs within NR and TNIP1 necessary for their interaction, and (iii) to establish the consequences to PPAR activity from TNIP1 expression. Here we provide evidence for TNIP1 as an atypical NR corepressor. Despite association with PPARs in the presence of their respective ligands, TNIP1 partially represses their activity. TNIP1 does not interact with the PPAR heterodimer partner retinoid X receptor (RXR) even in the presence of that receptor’s 9-cis retinoic acid (RA) ligand. TNIP1’s wide tissue distribution suggests it may play an important regulatory role in multiple cell types. Corepressors such as TNIP1 and LCoR [15], using NR boxes, targeting the agonist-bound receptor’s AF-2 region, and yet reducing receptor-mediated gene transcription, may provide finer levels of control by lessening rather than completely silencing NR activity.

Section snippets

Yeast and mammalian expression constructs, one- and two-hybrid assays

Selection stringency, ligand concentrations, vectors for yeast two-hybrid screen of a human keratinocyte cDNA library with human PPARα ligand-binding domain (LBD), mutagenesis methods, and receptor mutants were previously described in detail [16]. Briefly, the mutations are consensus amino acids (italicized) replaced with conservative substitutions (underlined) e.g., second TNIP1 NR box LKKLL to LKKAA and PPARα AF-2 LLQEIY to AAQEIY [16]. In this report, a quantitative β-galactosidase assay was

Identification of TNIP1 as a PPAR interacting protein

A partial clone of TNIP1, designated 43a, was isolated as the most frequently occurring interacting partner for the LBD of human PPARα in a yeast two-hybrid screen of a human keratinocyte cDNA library which also yielded known NR coactivators and several novel interacting proteins [16], [18]. The 43a cDNA encodes 377 amino acids (Fig. 1A). The second-most frequently occurring clone was also TNIP1-derived, overlapped the 43a cDNA (not shown); both contained consensus NR (LKKLL) and CoRNR

Discussion

TNIP1 is a NR box-containing, receptor AF-2-targeting corepressor of agonist-bound PPARs. TNIP1 homologs exist across mammalian, amphibian and fish species with sequence conservation suggesting critical residues throughout the protein including its NR box and CoRNR box motifs. Additionally, a region with no canonical LXXLL motifs contributes to ligand-dependent association with PPAR. The preferential interaction of TNIP1 with PPARδ and γ, versus PPARα, and its undetectable interaction with

Funding

This work was supported by National Institutes of Health (NIAMS Grant Nos. AR048860, BJA; AR048483, AMF). Pre-doctoral fellowships from Boehringer–Ingelheim Pharmaceuticals (AMF and IG) and the American Foundation for Pharmaceutical Education (IG) provided partial stipend support. The University of Connecticut Summer Undergraduate Research Fund (TRD), the Center for Regenerative Biology Excellence in Graduate Research Award (VPR), and the Edward A. Khairallah Graduate Fellowship (IG and VPR)

Acknowledgments

We thank N. Fusenig for HaCaT keratinocytes, S. Gao for pGal4UAS-tk-CAT, M. Barber at the UConn Microscopy Facility for imaging assistance, and Aneskievich laboratory member P. Encarnacao for helpful discussions.

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    These authors contributed equally to this work.

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