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 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 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 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 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 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. A search of the protein (150mM NaCl/20 mM sodium phosphate, pH 7.3/0.1% Nonidet databases (ref. 35; BLAST, January 1995) demonstrated that P-40/5% glycerol/5 mM dithiothreitol/2 mM MgCl2/2 mM NAB1 was a novel protein, with homology only to an otherwise CaCl2/2 mM phenylmethanesulfonyl fluoride/0.25% bovine se- uncharacterized expressed sequence tag from a human mela- rum albumin) at 4°C for 1 hr. Beads were washed three times with noma cell line (EMBL no. S31927). Further analysis of the buffer BB and once with buffer BB lacking albumin. To confirm peptide sequence did not reveal any recognizable motifs that equivalent amounts ofprotein were bound to the glutathione (DNA-binding domain, LIM domain, etc.). beads, an aliquot of each set of beads was heated in Laemmli RNA blot analysis of NAB1 expression in adult mouse sample buffer at 95°C for 5 min to remove bound proteins, and tissues detected a 3.7-kb transcript that was present in all the proteins were analyzed by electrophoresis followed by blot tissues analyzed (Fig. 2B). A 1.1-kb transcript was also detected analysis with the anti-Myc monoclonal antibody 9E10 (38) (data in some tissues and was most prominent in brain. This smaller not shown). For the binding assay, beads coated with wild-type or transcript was also detected by a probe from the 5' end of the mutant (1293F) GST fusion proteins were resuspended in 500 j,l cDNA that encompasses the interactive domain (underlined in of buffer BB and incubated for 1 hr at 25°C with 10 ,ul of Fig. 2A). NAB1 was found to be widely expressed in day 16 35S-labeled NAB1 synthesized in vitro (Promega TNT kit) by mouse embryo as well, with the highest levels of mRNA using as template HA-tagged NAB1 cDNA inserted into pCITE1 detected in the forebrain, thymus, salivary gland, and cartilage at the Nco I and Xba I sites. Proteins were removed from the (T. Araki and J.M., unpublished observations). The wide- beads by heating at 95°C for 5 min in Laemmli buffer, electro- spread expression of NAB1 is similar to that observed for other phoresed in an SDS/7.5% polyacrylamide gel, and visualized by negative regulators, such as p107 and pRb (40-42), and is autoradiography. consistent with our previous functional experiments which showed that the NGFI-A-inhibitory factor was expressed in mammalian cell lines derived from a wide variety of tissues RESULTS AND DISCUSSION (28). Identification of NABI by Interactive Cloning. To identify NAB1 Directly Interacts with NGFI-A via the Rl Domain. the putative NGFI-A-inhibitory factor, the yeast two-hybrid NAB1 was isolated on the basis of its interaction with the system was employed (30). Strains containing wild-type NGFI- NGFI-A Rl domain in vivo in yeast, where the specificity of the A Rl domain (aa 269-304) linked to the GAL4 DNA-binding interaction resembled that seen before in mammalian cells as domain were used to screen a mouse embryo cDNA/VP16 indicated by its failure to interact with the 1293F mutant Rl activation domain fusion library (34). A single clone (E27.3) domain of NGFI-A (28). To confirm a direct protein-protein was identified that interacted with the wild-type but not the interaction between NAB1 and NGFI-A, radiolabeled full- 1293F mutant Rl domain (Fig. 1). The 378-nt cDNA fragment length NAB1 was synthesized in vitro and incubated with from this clone was used to probe a rat brain cDNA library, and bacterially expressed GST/NGFI-A fusion proteins. NAB1 Downloaded by guest on September 30, 2021 Biochemistry: Russo et al. Proc. Natl. Acad. Sci. USA 92 (1995) 6875 A MAFCQRLPRD SALRYI ISTO RPGHLNLFKK ILIFRESILN ARPPRPCPPR 50 B IN WARGRPGRRS RRRSPAEPLS RIAPQPRAAL SPSTVMATAL PRTLGELQLY 100 Izt:.S~ RILQKANLLS YFDAFI0QG!G DDVQQLCEAG EEEFLEIMAL VGdAK PLFV 150 r RRLQKALRDW VTNPGLFNQP LTSLPVSSIP IYKLPES5PT WLGISCNSY E 200 RSSSAREPHL KVPKCAATTC VQSLGOGKSE VGSLALQSVS ESRLWQGHHT 250 - 28S TESEHSLSPA DLGSPASPKE SSEALDAAAA LSVAECVERM APTLPKSDLN 300 EVKELLKNNK KLAKMIGHIF EMSDEDPHKE EEIRKYSAIY GRFDSKRKDG 350 KHLTLHELTV NEAAAQLCVK DNALLTRRDE LFALARQVSR EVTYKYTYRT 400 - 18S TRLKCGERDE LSPKRIKMED GFPDFQESVP TLFQQARAKS EELAGLSSQA 450 EKGMAKQMEL LCAQAGYERL QOERRLMAGL YRQSSGEQSP DGGLPSDGSD 500 GOGERPLNLR IPSVONROPH HFVVDGELSR LYSNEVKSHS SESLGILKDY 550 4, PHSAFTLEKK VIKTEPEDSR 5 7 0

FIG. 2. (A) Amino acid sequence encoded by rat NAB1 cDNA obtained from a brain cDNA library is shown. It encodes a 570-residue protein. The region of homology to the mouse probe isolated from the yeast interactive cloning system is underlined. (B) Survey of NAB1 expression in adult mouse tissues. RNA blot hybridization analysis was performed with total RNA (25 ,ug per lane) isolated from the indicated tissues. The hybridization probe was a 32P-labeled 1.8-kb NAB1 fragment isolated from a mouse thymus cDNA library (Stratagene) and confirmed by sequence analysis to be the murine NAB1 homolog. Inspection of the ethidium bromide-stained gel and analysis with a cyclophilin probe confirmed the integrity and equivalent loading of all RNA samples (data not shown). Positions of 28S and 18S rRNAs are indicated. bound to glutathione beads containing wild-type NGFI-A NAB1 could be rendered ineffective by the Ri competitor in fused to GST, but did not bind to beads bearing the I293F a similar fashion, NAB1, the NGFI-A/GAL4 chimera, and the mutant NGFI-A fused to GST or GST alone (Fig. 3), indicat- GAL4 luciferase reporter were transfected into CV-1 cells ing that NAB1 directly interacts with NGFI-A via the Ri along with increasing amounts of either wild-type or 1293F domain. mutant Ri-domain competitor. NABl-mediated inhibition of NAB1 Represses NGFI-A Transcriptional Activation. To NGFI-A was alleviated by increasing amounts ofwild-type, but examine the effects of NAB1 on NGFI-A transcriptional not mutant, Ri-domain competitor (Fig. 4B). Indeed, with activity, CV-1 cells were transfected with the following plas- high doses of competitor, activity actually increased over the mids: a NAB1 expression vector, a vector expressing NGFI-A level observed in the absence of NAB1, presumably because in which the DNA-binding domain has been replaced with the the endogenous inhibitory factor was also titrated at these GAL4 DNA-binding domain (NGFI-A/GAL4 chimera) [nec- levels. We conducted similar competition experiments at essary to reduce background expression (28)], and a luciferase different levels of NAB1, which demonstrated that higher reporter containing five GAL4 binding sites upstream of a levels of NAB1 required greater amounts of Ri-domain minimal promoter (28). Cotransfection of increasing amounts competitor to reverse the NABl-mediated suppression (Fig. of NAB1 cDNA resulted in a dose-dependent decrease in 4C). These results are consistent with the idea that the Ri transcriptional activity of the wild-type NGFI-A/GAL4 chi- competitor is directly countering NAB1 action and that NAB1 mera (Fig. 4A), indicating that NAB1 inhibits NGFI-A. The inhibits NGFI-A via its interaction with the Ri domain. specificity of the interaction was demonstrated by the lack of NABI Is a Nuclear Protein. It was important to determine NABl-mediated repression of a NGFI-A/GAL4 chimera the cellular location of NAB1, as one potential manner by lacking the Ri domain (data not shown) or of the mutant which it could inhibit NGFI-A-mediated transcription would NGFI-A(1293F)/GAL4 chimera (Fig. 4A4). The activity of the be to sequester NGFI-A in the cytoplasm. A HA-tagged nuclear receptor NGFI-B, a transcription factor unrelated to derivative of NAB1 was constructed and transfected into COS NGFI-A, was also unaffected by NAB1, indicating that NAB1 cells, and immunohistochemistry with the anti-HA monoclo- is not a general inhibitor of transcriptional activators (data not nal antibody 12CA5 (43) demonstrated that NAB1 resided in shown). the nucleus (Fig. 5). This result, along with the previous A key observation indicating that NGFI-A is modulated in demonstration that both wild-type and I293F mutant NGFI-A trans by a cellular inhibitory factor was that the inhibitory are located in the nucleus (28), indicates that NAB1 does not activity could be titrated out (i.e., NGFI-A activity was in- inhibit NGFI-A by sequestering it in the cytoplasm, as has been creased) in a dose-dependent fashion by coexpression of the observed for the IKB-mediated inhibition of NF-KB (2). Inhi- Ri domain (28). To determine whether exogenously added bition of transcription can also be the result of alterations in DNA binding. Previously, we had shown that the DNA-binding characteristics of wild-type NGFI-A and the highly active C 4*iC s90 t mutants were similar, indicating that the cellular inhibitory factor does not modulate NGFI-A activity via alterations in DNA binding (28). In addition, because NGFI-A binds DNA as a monomer (45), it is unlikely that NAB1 could operate in a manner analogous to the action of Id, which dimerizes with a number of myogenic transcriptional activators to form non-DNA-binding complexes (46). Transcriptional repression may also occur via interference with the function of a partic- FIG. 3. NABl interacts with NGFI-Ain vitro. Bacterially expressed ular activation domain. However, previous experiments dem- GST or GST fusion protein containing wild-type (WT) or mutant onstrated that repression through the Ri domain was not (1293F) NGFI-A Rl domain was incubated with in vitro translated, a 35S-labeled NAB1. Bound proteins were precipitated with glutathione- targeted to specific NGFI-A activation domain (28), sug- agarose beads and analyzed by electrophoresis in an SDS/7.5% gesting that this mechanism is unlikely to account for the polyacrylamide gel. Radiolabeled NAB1 was visualized by autoradiog- repressive effects of NAB1. It is possible that NAB1 represses raphy. For the lane labeled input, 20% of the radiolabeled NAB1 used the activation of transcription by NGFI-A via inhibition of the in the binding reaction was loaded directly onto the gel. general transcription apparatus itself, in a fashion similar to Downloaded by guest on September 30, 2021 6876 Biochemistry: Russo et al. Proc. Natl. Acad. Sci. USA 92 (1995)

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FIG. 4. (A) NAB1 represses the transcriptional activity of NGFI-A, but not the 1293F mutant. CV-1 cells were transfected with 100 ng of either NGFI-A/GAL4 (Left) or mutant NGFI-A(1293F)/GAL4 (Right) CMV expression construct (28), 4.9 jig of GAL4 luciferase reporter, and the indicated amount of CMV NAB1. Percent luciferase activity is defined as the activity relative to NGFI-A/GAL4 in the absence of NAB1, which is taken as 100%; activity of reporter alone is taken as 0%. Data shown are from two independent experiments with standard deviations of <5% in each case. Note the 10-fold difference in scale between the two panels. (B) NAB1 repression of NGFI-A is alleviated by competition with the Rl domain. CV-1 cells were transfected with 100 ng of NGFI-A/GAL4 expressor and 4.9 ,ig of GAL4 luciferase reporter, in the presence or absence of 2.5 jig of CMV NAB1, and the indicated amounts of expression vectors containing either the wild-type or mutant (1293F) Rl competitors (28). The activity of the expressor in the absence of CMV NAB1 is taken as 100% (denoted by dotted line), and that of reporter alone as 0%. Means and standard deviations of two independent experiments are shown. (C) Alleviation of NAB1 repression by Rl is dose-dependent. CV-1 cells were transfected with 4.9 jig of GAL4 luciferase reporter, 100 ng of NGFI-A/GAL4 expressor, and the indicated amounts of CMV NAB1 and the wild-type Rl competitor. The activity of the GAL4 luciferase reporter with NGFI-A/GAL4 is taken as 100% (denoted by dashed line), while that of reporter alone is 0%. Means and standard deviations of two independent experiments are shown.

the suppression of a number of yeast genes by the association nism by which these closely related proteins could be differ- of the Ssn6/Tupl complex with DNA-binding proteins (47- entially regulated. To test this idea, expression vectors for each 49). of these proteins were transfected into CV-1 cells in the Differential Regulation of NGFI-A Family Members by presence or absence of NAB1, along with a luciferase reporter NAB1. NGFI-A and its family members (Egr3, Krox2O, and containing two copies of the GCGGGGGCG response ele- NGFI-C) bind to very similar nucleotide sequences (A. H. ment. Although NAB1 repression is not as dramatic due to the Swirnoff and J.M., unpublished observations) and are coex- higher background activity of this reporter, it is apparent that pressed in some cell types in response to environmental stimuli NAB1 repressed the activity of NGFI-A and Krox2O to a (50, 51). It is unknown whether these proteins constitute a similar extent, whereas Egr3 and NGFI-C were unaffected redundant mechanism for the regulation of target genes, or (Table 1). The inability of NAB1 to suppress NGFI-C was whether regulatory mechanisms unique to individual family anticipated, since NGFI-C lacks the conserved region within members exist. We reasoned that NAB1 might interact with the Rl domain. However, the resistance of Egr3 to NAB1 some, but not all, of these proteins, thus providing a mecha- suppression was unexpected, as Egr3 is very similar to NGFI-A and Krox2O within this domain. An alignment of the sequences in this region shows a highly conserved core domain spanning NGFI-A residues 270-296. Within this domain, there are four positions where NGFI-A and Krox2O are identical, but where Egr3 differs (Fig. 6). One or more of these residues are presumably critical for the interaction with NAB1; alterna- Table 1. NAB1 differentially regulates members of the NGFI-A family Transcriptional Percent activity in the activator presence of NAB1 NGFI-A 19 ±14 NGFI-C 125 ± 12 Egr3 140 ±11 Krox2O 27 ± 6 CV-1 cells were transfected with 100 ng of a CMV expression vector for each of the indicated family members (NGFI-A, Krox20, NGFI-C, FIG. 5. NAB1 is localized to the nucleus. COS cells were tran- and Egr3) along with 4.9 ,ug of a luciferase reporter containing two siently transfected with a HA-tagged NAB1 expression vector and copies of the GCGGGGGCG response element (14) and 5 ,ug of either processed for immunohistochemistry (44). NAB1 was detected by use CMV NAB1 or nonrecombinant CMV vector. The percent activity of of the anti-HA monoclonal antibody 12CA5, followed by a fluorescein- each transcription factor was derived by dividing the activity in the conjugated goat anti-mouse secondary antibody. The nucleus stains presence of NAB1 by activity in the absence of NAB1 (defined as brightly, whereas the cytoplasm and several adjacent nontransfected 100% activity); activity of the reporter alone was taken as 0%. Means cells do not. Cells transfected with nonrecombinant pCMVneo dis- and standard deviations of relative luciferase activity in three inde- played only low, background staining (data not shown). pendent experiments are shown. Downloaded by guest on September 30, 2021 Biochemistry: Russo et aL Proc. Natl. Acad. Sci. USA 92 (1995) 6877

v NGFI-A 269 T[PDQ KPFQ-GLEN- NTQQPLTPLSTIKAFATQSGSQD 304 KROX20 193 G PDRKPF02L9SLR LTPLST FTLGGPGAG 229

EGR3EGR3 20O606 I IdEHKPQ-G!1EH -qUPPIRvPPPTPLnETIKAFKDKQIHP[gI NL | DQ Pi2242 FIG. 6. Alignment of Rl domain residues of the indicated proteins. The sequences shown are rat NGFI-A, murine Krox2O, and murine Egr3. Residues identical among these three proteins are boxed. Arrowhead marks the position of the disruptive Ile -- Phe mutation at position 293. tively, they may alter the inhibitory activity of NAB1 when it 17. Lemaire, P., Vesque, C., Schmitt, J., Stunnenberg, H., Frank, R. & interacts with Egr3. Charnay, P. (1990) Mol. Cell. Biol. 10, 3456-3467. To summarize, NAB1 is a widely expressed nuclear protein 18. Takimoto, Y., Wang, Z. Y., Kobler, K. & Deuel, T. F. (1991) Proc. that NGFI-A and Krox2O Natl. Acad. Sci. USA 88, 1686-1690. negatively regulates transcriptional 19. Drummond, I. A., Madden, S. L., Rohwer-Nutter, P., Bell, G. I., activity in a dose-dependent manner via an interaction with the Sukhatme, V. P. & Rauscher, F. J. (1992) Science 257, 674-678. Rl domain. The observation that NAB1 regulates only a 20. Kim, S. J., Park, K., Rudkin, B. B., Dey, B. R., Sporn, M. B. & subset of this family of immediate-early transcription factors Roberts, A. B. (1994) J. BioL Chem. 269, 3739-3744. suggests that it provides a potential mechanism for their 21. Colby, W. W., Chen, E. Y., Smith, D. H. & Levinson, A. D. (1983) differential regulation; however, its physiologic role remains to Nature (London) 301, 722-725. be defined. Although the mechanism of NABl-mediated 22. Molnar, G., Crozat, A. & Pardee, A. B. (1994) Mol. Cell. Biol. 14, remains to be its characteristics 5242-5248. repression elucidated, are 23. Motokura, T. & Arnold, A. (1993) Genes Cancer 7, reminiscent of the modulation of E2F and Myc activity by pRb 89-95. and p107, respectively (5, 7, 9). As NGFI-A is also induced 24. Nicolaides, N. C., Gualdi, R., Casadevall, C., Manzella, L. & Calla- upon entry into the cell cycle and regulates several genes bretta, B. (1991) Mol. Cell. Biol. 1, 6166-6176. associated with cell cycle progression, it is possible that NAB1, 25. Sham, M. H., Vesque, C., Nonchev, S., Marshall, H., Frain, M., Gupta, like pRb, may regulate cell growth. R. D., Whiting, J., Wilkinson, D., Charnay, P. & Krumlauf, R. (1993) Cell 72, 183-196. 26. Schneider-Maunoury, S., Topilko, P., Seitanidou, T., Levi, G., Cohen- We thank D. Geitz for advice on high-efficiency yeast transforma- tion, S. Hollenberg for the murine embryonic library, S. Elledge for Tannoudji, M., Pournin, S., Babinet, C. & Chamay, P. (1993) Cell 75, plasmid pAS1 and yeast strain Y187, D. Beach for yeast strain HF7c, 1199-1214. M. Johnston and K. Blumer for assistance with general yeast tech- 27. Nguyen, H. Q., Hoffman-Liebermann, B. & Liebermann, D. A. and D. (1993) Cell 72, 197-209. niques, Dean, J. Majors, S. Speck, and T. Wilson for their 28. Russo, M. W., Matheny, C. & Milbrandt, J. (1993) Mol. Cell. Biol. 13, comments on the manuscript. 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