Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6873-6877, July 1995 Biochemistry Identification of NAB1, a repressor of NGFI-A- and Krox2O- mediated transcription MARK W. Russo, BRADLEY R. SEVETSON, AND JEFFREY MILBRANDT* Division of Laboratory Medicine, Departments of Pathology and Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110 Communicated by Stuart Kornfeld, Washington University, St. Louis, MO, March 6, 1995 ABSTRACT NGFI-A (also called Egrl, Zif268, or Ri domain was shown to be mediated by a titratable cellular Krox24) and the closely related proteins Krox2O, NGFI-C, and inhibitory factor in competition experiments in which in- Egr3 are zinc-finger transcription factors encoded by imme- creased NGFI-A activity was observed when the isolated Ri diate-early genes which are induced by a wide variety of domain was coexpressed with NGFI-A. These analyses indi- extracellular stimuli. NGFI-A has been implicated in cell cated that residues within the Ri domain mediated a func- proliferation, macrophage differentiation, synaptic activation, tionally significant interaction between NGFI-A and the in- and long-term potentiation, whereas Krox2O is critical for hibitory factor. Here, we describe the isolation of this inhib- proper hindbrain segmentation and peripheral nerve myeli- itory factor, NAB1,t and demonstrate that it represses the nation. In previous work, a structure/function analysis of transcriptional activity of NGFI-A and Krox20 through a NGFI-A revealed a 34-aa inhibitory domain that was hypoth- direct interaction. esized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid MATERIALS AND METHODS system, we have isolated a cDNA clone which encodes a protein that interacts with this inhibitory domain and inhibits the Cell Culture and Transfection. CV-1 and COS-7 cells were ability of NGFI-A to activate transcription. This NGFI-A- cultured as described (28). Transient transfection of CV-1 cells binding protein, NAB1, is a 570-aa nuclear protein that bears (2 x 106 cells per 10-cm plate) was performed by calcium no obvious sequence homology to known proteins. NAB1 also phosphate precipitation (28). Indicated amounts of plasmid represses Krox2O activity, but it does not influence Egr3 or DNAs were added along with nonrecombinant pCB6 [a cyto- NGFI-C, thus providing a mechanism for the differential megalovirus (CMV) promoter-driven expression plasmid; ref. regulation of this family of immediate-early transcription 52] as necessary for a total of 10 ,ug per plate. Cells were factors. harvested 48 hr after transfection and assayed for luciferase activity (28). The results shown are the means of the indicated Negative regulation of transcription from specific promoters is number of experiments, each consisting of duplicate plates. At an important mechanism for controlling gene expression (1). least two independent preparations of each plasmid were One means by which negative regulators can function is tested and found to give indistinguishable results. through direct interactions with transcriptional activators. Plasmids. The NGFI-A/GAL4 chimera, NGFI-A, NGFI-A Examples include the regulation of NF-KB by its sequestration I293F mutant, Krox2O, NGFI-C, and Egr3 expression vectors in the cytoplasm by lKB (2), the prevention of glucocorticoid and the GAL4 and GCGGGGGCG luciferase reporter plas- receptor DNA binding by calreticulin (3, 4), and the modula- mids have been reported (28). CMV NAB1 contains the full tion of E2F and Myc activity by their association with the coding sequence of the rat NABI cDNA cleaved withEco47III retinoblastoma gene product pRb (5-8) and the related p107 and HindIIl and inserted into the blunted Mlu I and HindlIl (9), respectively. sites of pCMVneo. The hemagglutinin (HA)-tagged version of Expression of NGFI-A (10-12), Krox20 (13), NGFI-C (14), NAB1 was generated by introducing the HA epitope at the and Egr3 (15) is induced by a variety of proliferative, differ- amino terminus of NAB1 by PCR. Sequences of the PCR- entiative, and apoptotic stimuli. These closely related imme- derived product and junctions were confirmed, and the frag- diate-early proteins have very similar DNA-binding domains ment was inserted into the CMV expression vector. HA-tagged and activate transcription from a consensus sequence, 5'- NAB1 is functional, as it repressed the transcriptional activity GCG(G/T)GGGCG-3' (16, 17), present in promoters of a of the NGFI-A/GAL4 chimera to a similar extent as wild-type number of genes encoding growth factors [platelet-derived NAB1 (data not shown). growth factor A chain, insulin-like growth factor II, transform- Yeast Methods. The yeast interactive cloning system (30) ing growth factor ( (18-20)] and proteins involved in cell cycle used the yeast strain HF7c (31) bearing integrated GAL4- control [c-Myc, thymidine kinase, cyclin D, and c-Myb (21- responsive promoters for the reporter genes HIS3 and lacZ. 24)]. Krox2O has also been shown to regulate Hoxb-2 in the The "bait" plasmid encoded either the wild-type NGFI-A RI developing hindbrain (25), a structure that is severely mal- domain (aa 269-304) or its I293F mutant fused to the carboxyl formed in Krox20-deficient mice (26). NGFI-A activity ap- terminus of the GAL4 DNA-binding domain (aa 1-147) in a pears to be critical for monocyte differentiation: its inhibition modified pAS1 (Trpl marker) (32) lacking the HA epitope results in granulocyte formation, whereas its overexpression and polylinker region. Yeast transformation was performed as promotes macrophage development (27). described (33). A mouse embryo cDNA library constructed in NGFI-A contains a number of activation domains as well as the vector pVP16 (Leu2 marker) was obtained from S. Hol- an inhibitory domain, called Ri. When this Rl domain is lenberg (34). Five million transformants were screened for deleted (28, 29) or when an Ile -- Phe mutation is introduced clones capable of interacting with the NGFI-A Ri-domain at residue 293 (1293F) (28), a 15-fold increase in NGFI-A bait, as judged by growth of the transformants in the absence transcriptional activity is observed. The negative effect of the Abbreviations: CMV, cytomegalovirus; GST, glutathione S-trans- ferase; HA, hemagglutinin. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in fThe sequence reported in this paper has been deposited in GenBank accordance with 18 U.S.C. §1734 solely to indicate this fact. (accession no. U17253). 6873 Downloaded by guest on September 30, 2021 6874 Biochemistry: Russo et al. Proc. Natl. Acad. Sci. USA 92 (1995) of histidine. Initially, 13 positive clones were obtained. False NGFI-A RI Domain positives were identified by curing the yeast cells of the Mutant (1293F) Trp-bearing pAS1 plasmid, while retaining the pVP16 library Wildtype plasmid, and then mating them with yeast strain Y187 (32) bearing the wild-type or 1293F mutant bait plasmids. The resulting diploid yeast colonies were assessed for growth in the absence of histidine and for ,3-galactosidase (lacZ gene prod- uct) production. A single clone (E27.3) reacted only with the wild-type bait. The plasmid was recovered from this yeast SC -Leu-Trp t ! JF. colony and the cDNA insert was sequenced. |D X A5 Z Isolation and Sequence Analysis of NAB1 cDNA Clones. The cDNA fragment from the yeast clone E27.3 was labeled with [a-32P]dCTP and used as a hybridization probe to screen a rat brain cDNA library constructed in AZAPII (Stratagene). Three clones were obtained and the phagemid was excised by use of VCSM13 helper phage (Stratagene). These clones were sequenced with an Applied Biosystems model 373 automated DNA sequencer. Sequence comparisons were performed with SC - Leu -Trp- His BLAST (35) using the default parameters to search the National Center for Biotechnology Information nonredundant protein and DNA databases. A search for protein motifs was per- formed with PROSITE (36). NAB1/NGFI-A in Vitro Binding Assay. A construct encod- DNA-binding domain) (28) was FIG. 1. Yeast two-hybrid interaction of NAB1 clone with wild-type ing NGFI-A (with GAL4 and I293F mutant NGFI-A Ri domains. The library plasmid recov- modified to introduce a HA epitope near the amino terminus, ered from positive clone E27.3 was transformed into HF7c along with at residue 29, and a Myc epitope near the carboxyl terminus, either the wild-type NGFI-A or the I293F mutant NGFI-A Ri-domain at residue 531, and inserted into pGEX (Pharmacia). The bait. Growth of individual transformants was assessed by streaking I293F mutant differs from the wild type only at codon 293, onto synthetic medium plates containing histidine (SC -Leu -Trp) or where Phe has been substituted for Ile. Bacteria (Escherichia lacking histidine (SC-Leu-Trp-His) to identify cells with a func- coli DH5a) harboring the indicated pGEX constructs were tional cDNA product/Ri domain interaction. Plates were incubated at induced with 0.1 mM isopropyl P3-D-thiogalactopyranoside for 30°C for 4 days. 3-4 hr and lysates were prepared essentially as described (37) in a modified resuspension buffer [150 mM NaCl/20 mM several clones were obtained. The sequence of the largest clone sodium phosphate, pH 7.3/1% (vol/vol) Triton X-100/1% (vol/ contained an open reading frame that predicted a protein of vol) Tween 20/5% (vol/vol) glycerol/i mM EDTA/1 mM di- 570 aa which we have named NAB1 (NGFI-A-binding protein) thiothreitol/2 mM phenylmethanesulfonyl fluoride with pepsta- (Fig. 24). The sequence at the proposed translation initiation tin A and leupeptin at 5 ,ug/ml]. Lysates containing glutathione site conforms to the Kozak consensus sequence (39), and S-transferase (GST) or GST fusion proteins were incubated with several in-frame stop codons are present immediately up- 25 ,ul of glutathione beads (Pharmacia) and 500 ,l of buffer BB stream of the initiator methionine.