NRC-interacting factor 1 is a novel cotransducer that interacts with and regulates the activity of the nuclear hormone receptor coactivator NRC

Abstract

We previously reported the cloning and characterization of a novel nuclear hormone receptor transcriptional coactivator, which we refer to as NRC. NRC is a 2,063-amino-acid nuclear protein which contains a potent N-terminal activation domain and several C-terminal modules which interact with CBP and ligand-bound nuclear hormone receptors as well as c-Fos and c-Jun. In this study we sought to clone and identify novel factors that interact with NRC to modulate its transcriptional activity. Here we describe the cloning and characterization of a novel protein we refer to as NIF-1 (NRC-interacting factor 1). NIF-1 was cloned from rat pituitary and human cell lines and was found to interact in vivo and in vitro with NRC. NIF-1 is a 1,342-amino-acid nuclear protein containing a number of conserved domains, including six Cys-2/His-2 zinc fingers, an N-terminal stretch of acidic amino acids, and a C-terminal leucine zipper-like motif. Zinc fingers 1 to 3 are potential DNA-binding BED finger domains recently proposed to play a role in altering local chromatin architecture. We mapped the interaction domains of NRC and NIF-1. Although NIF-1 does not directly interact with nuclear receptors, it markedly enhances ligand-dependent transcriptional activation by nuclear hormone receptors in vivo as well as activation by c-Fos and c-Jun. These results, and the finding that NIF-1 interacts with NRC in vivo, suggest that NIF-1 functions to regulate transcriptional activation through NRC. We suggest that NIF-1, and factors which associate with coactivators but not receptors, be referred to as cotransducers, which act in vivo either as part of a coactivator complex or downstream of a coactivator complex to modulate transcriptional activity. Our findings suggest that NIF-1 may be a functional component of an NRC complex and acts as a regulator or cotransducer of NRC function.

FIG. 1.

Sequence and predicted domain structure of NIF proteins. (A) Comparison of human and rat NIFs. Schematic representations of the functional domains identified in human NIF-1, NIF-2, and the partial rat NIF clone are shown. D/E represents an Asp- and Glu-rich acidic amino acid stretch of ∼35 amino acids. The LXXLL motif corresponds to the amino acids LDLLL. Zinc fingers of the Cys-2/His-2 type are dispersed and represented by numbers 1 through 6. LZ indicates the leucine zipper-like motif localized at the C terminus. NIF-2 was identified by sequencing an EST clone (BE297231) and appears to be an alternatively spliced isoform of human NIF-1. Rat NIF is a partial clone isolated from the GH4C1 pJG4-5 cDNA library deposited in GenBank under accession no. AF309071 and AY079168. (B) Similarity of the zinc fingers, LXXLL, and leucine zipper-like domains in human, rat, and chicken NIFs. The region of comparison includes amino acids 592 to 1172 and contains zinc fingers 5 and 6 and the LXXLL and leucine zipper regions. (C) Amino acid sequence and functional domains of human NIF-1. NIF-1 mRNA contains an open reading frame of 1,342 amino acids. The initiator Met indicated by the arrowhead is preceded by a short open reading frame and an in-frame stop codon. DE, an acidic region rich in Asp and Glu, is underlined. Zinc fingers 1 through 6 are boxed. The leucine zipper-like motif is indicated in bold and boxed. The LXXLL motif is boxed and lightly shaded. The amino acid sequence within the arrows (which includes the DE stretch and zinc fingers 1 through 4) is absent in NIF-2, an isoform of NIF-1. The nucleotide and amino acid sequences of NIF-1 and NIF-2 have been deposited in GenBank under accession number AF395833.

FIG. 2.

NIF-1 is a nuclear protein. GFP-NIF-1 was transfected into Cos1 cells, and GFP fluorescence was detected in the nucleus (green). The nucleus was also stained with Hoechst stain (blue). GFP-NIF-1 fluorescence was also overlapped with the nuclear Hoechst stain as shown.

FIG. 3.

Northern blot of NIF-1 mRNAs in different tissues. NIF-1 mRNAs were detected with an MTN blot (Stratagene) containing poly(A)⁺ RNAs from the various tissues indicated. A NIF-1 mRNA of ∼5 kb was detected by probing the blot with ³²P-labeled human NIF-1 cDNA. Lanes 1 through 12 contain RNAs from the brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and blood, respectively. A shorter mRNA of ∼2.5 kb, designated NIF-2, was detected upon longer exposure of the blot (not shown) and is described in the text.

FIG. 4.

NIF-1 associates with NRC in mammalian cells. The mammalian GST expression vectors pEBG (expressing GST) and pEBG-NRC (expressing a GST fusion of full-length NRC) were cotransfected with pEX-FlagNIF-1 in 293T cells. Whole-cell extracts were prepared 36 h later, and the proteins remaining bound to the expressed GST proteins were purified with glutathione-agarose beads and processed for SDS-gel electrophoresis followed by Western blotting as described earlier (34). The Western blot was probed with M2 anti-Flag antibody to detect Flag-NIF-1. Lane 1, pEBG control (CON.); lane 2, pEBG-NRC.

FIG. 5.

NIF-1 interacts with NRC in yeast through a region containing zinc finger 6. Various NIF constructs were generated as B42 fusions and tested against each of the LexA fusions of NRC shown in Fig. 6A, a to g, in two-hybrid interaction assays. Rat NIF is the original isolate from the GH4C1 library while NIF-2 is an isoform of human NIF-1 that lacks amino acids 184 to 743, which includes the DE region and zinc fingers 1 to 4. The numbers correspond to amino acids. All of the NIF fragments containing the NRC-ID interacted with NRC in two-hybrid assays. +, positive; −, negative.

FIG. 6.

Identification of the NIF-ID of NRC. (A) Interaction of NIF-1 with NRC in yeast. Each of the LexA-NRC fusions was tested for interaction with various constructs of NIF-1 (Fig. 5) expressed as B42 fusions. All of the fragments of NRC containing the NIF-ID interact with NIF-1 clones containing the NRC-ID. MT fragments depicted are NRC clones containing mutations in the LXXLL-1 receptor interaction motif in which LVNLL was changed to AVNAA. +, positive; −, negative. (B) Binding of NIF-1 with NRC in vitro. NIF-1 was labeled with [³⁵S]l-methionine by in vitro transcription-translation with reticulocyte lysates. Bacterially expressed and purified GST-NRC.1a (a 147-amino-acid region of NRC that contains the NIF-1-ID) bound to glutathione-agarose beads was incubated with ³⁵S-labeled NIF-1. The samples were then electrophoresed in SDS gels and the ³⁵S-NIF-1 bound to GST or GST-NRC.1a was visualized by autoradiography. One fifth of the amount of ³⁵S-labeled NIF-1 used in the incubation was also electrophoresed in the same gel.

FIG. 6.

Identification of the NIF-ID of NRC. (A) Interaction of NIF-1 with NRC in yeast. Each of the LexA-NRC fusions was tested for interaction with various constructs of NIF-1 (Fig. 5) expressed as B42 fusions. All of the fragments of NRC containing the NIF-ID interact with NIF-1 clones containing the NRC-ID. MT fragments depicted are NRC clones containing mutations in the LXXLL-1 receptor interaction motif in which LVNLL was changed to AVNAA. +, positive; −, negative. (B) Binding of NIF-1 with NRC in vitro. NIF-1 was labeled with [³⁵S]l-methionine by in vitro transcription-translation with reticulocyte lysates. Bacterially expressed and purified GST-NRC.1a (a 147-amino-acid region of NRC that contains the NIF-1-ID) bound to glutathione-agarose beads was incubated with ³⁵S-labeled NIF-1. The samples were then electrophoresed in SDS gels and the ³⁵S-NIF-1 bound to GST or GST-NRC.1a was visualized by autoradiography. One fifth of the amount of ³⁵S-labeled NIF-1 used in the incubation was also electrophoresed in the same gel.

FIG. 7.

NIF-1 does not directly interact with nuclear receptor LBDs in yeast. NIF-1 was expressed as a B42 fusion and tested against LexA fusions of the following receptor LBDs: cTRα, hERα, hRXRα, hGR, hRARα, hPPARα, and NRC. T3-dependent interaction of LexA-cTRα was also verified against B42-NRC in the same assay as a positive control. Details of the β-galactosidase assay in yeast extracts are described in Materials and Methods. Vs, versus.

FIG. 8.

NIF-1 enhances ligand-dependent activation by Gal4-ER-LBD in HeLa cells. The Gal4 reporter pBL-G5-CAT2 was cotransfected in HeLa cells with vectors expressing the Gal4 LBD or the Gal4 LBD fusion of the mER-LBD with or without NIF-1. Cells were incubated with or without ligand, E2 (100 nM), for 40 h, and duplicate samples were then assayed for CAT activity. The experiment was repeated at least two times with similar results (refer to Materials and Methods for details about plasmids). +, with; −, without.

FIG. 9.

NIF-1 activates TR, RAR, and GR in HeLa cells. (A) HeLa cells were transfected with the ΔMTV-IR-CAT reporter and expression vectors for cTRα or hRARα and NIF-1 as indicated. The cells were incubated with T3 at a concentration of 1 μM and the RAR-specific ligand TTNPB at a concentration of 200 nM. All samples were analyzed in duplicate, and the experiment was repeated at least two times. Panel B is the same as panel A except that the mouse mammary tumor virus (MMTV)-long terminal repeat (LTR)-CAT reporter and an hGR expression vector were cotransfected with (+) or without (−) 500 nM Dex.

FIG. 10.

Ligand-dependent activation of endogenous nuclear receptors by NIF-1 in GH4C1 cells. (A) Cells were cotransfected with the ΔMTV-IR-CAT reporter alone and with (+) or without (−) the NIF-1 or NRC expression plasmids at various concentrations. T3 ligand was used at a concentration of 1 μM. Each sample was analyzed in duplicate, and the experiment was repeated at least two times with similar results (refer to Materials and Methods for details about plasmids). Panel B is the same as panel A except that the RXR-specific ligand LG100153 and the RXR/RAR-specific ligand 9-cis RA were each used at concentrations of 200 nM.

FIG. 11.

NIF-1 and NRC activate AP1 activity in HeLa cells. (A) The −73 collagenase-CAT reporter plasmid driven by AP1 (c-Fos and/or c-Jun) was transfected with 1 and 3 μg of the expression plasmids for NRC or NIF-1. The samples were analyzed in duplicate, and the experiment was repeated at least two times with similar results. (B) The amount of expression vector for NRC used was 0.7 μg. The amount of NIF-1 expression plasmid used was 0.7 μg in lane 3 and 1.2 μg in lanes 5 to 7. The amount of vector control (Vec.) used was 0.7 μg. +, with.