Summary The re1 family of proteins can be defined as a group of proteins that share sequence homology over a 300 amino acid region termed the re1 domain. The re1 family comprises important regulatory proteins from a wide variety of species and includes the Drosophila morphogen dorsal, the mammalian transcription factor NF-KB, the avian oncogene v-rel, and the cellular protooncogene c-rel. Over the last two years it has become apparent that these proteins function as DNA-binding transcription factors, and that their activity is regulated at the level of subcellular localization. Introduction The original member of the re1 family, v-rel, is an oncogene found in a turkey retrovirus REV-T (reticuloendotheliosis virus strain T). REV-T was described as an a ent that induced rapidly fatal tumors in young bird$). The tumors were initially reported to be reticuloendothelial in origin but later defined as REV-T is still considered one of the most virulent retroviruses and much attention has been focused on v-re1 and its cellular counterpart, c-rel, over the past 10 years. Molecular and biochemical studies have documented a wealth of information on the nature of v-re1and c-re1(e.g. size, sequence, associated factors, etc), but the mechanism of action of v-re1and the role of c-rel in the development of lymphoid cells has remained obscure. The discovery in 1987 that the Drosophila gene, dorsal, shared sequence homology with v-rel@) was intriguing but not very revealing. It was known that dorsal had an important regulatory role in determining the fate of cells along the dorsal-ventral (DV) axis of the Drosophila embryo(5). Embryos laid by dorsal mutant mothers develop only dorsal structures along the entire DV axis and never hatch into larvae. However, although the dorsal and v-re1 proteins were both known to be important regulatory molecules, the shared amino acid sequence did not shed any light on their nature or cellular function. An important insight was provided when further studies implicated dorsal as a transcription factor('). The dorsal protein was found to be distributed in a nuclear concentration gradient established b\7 the

unusual mechanism of regulated nuclear transport("'). In the developing embryo. ventrally located nuclei receive high levels of dorsal protein while nuclei in more lateral and dorsal positions receive lower amounts of the protein. This suggests that nuclear dorsal controls the expression oC target genes in a concentrationdependent manner. More recently it has been unambiguously shown that dorsul is in fact a sequence dependent DNA-binding protein('). Simultaneous with the latter finding was the exciting discovery that the subunits of the mammalian transcription factor NF-KB share qequence homology with v-re1 and NF-KB was originally identified as a nuclear factor that bound to the kappa ( K ) light chain enhancer in B lymphoid cells and activated K gene transcription(")). It was already known that NF-KB regulation occurs at the level of nuclear transport. NFKB is present ubiquitously but is active only in cells where it resides in the nucleus. In the past year it has become obvious that v-rd, crel, dorsal, and NF-KB - the re1 proteins - all bind similar DNA sequences and show characteristic patterns of subcellular localization. and do indeed constitute a new family of transcription factors. Structure of the re1 Proteins The turkey oncogene v-re/ was the first of the re1 proteins to be cloned and sequenced(15I'). Since then. the cognate proto-oncogene c-re1has been identified in many s ecies including chickens, mice, frogs and h u r n a n ~ ~ ~ Figure - ' ~ ) . 1compares the structural organization of several re1 proteins. It appears that v-rel is a truncated version of c-refi2*).In the virus REV-T, v-re1 is an env-rel-env fusion protein containing viral envelope sequcncef at the amino- and carboxylterminal ends. Relative to c-rel, it lacks two amino acids at the amino (N) terminus and is deleted for 118 amino acid at the carboxyl (C) terminus. The remaining 474 amino acids include a region of about 300 amino acids that is conserved between the various members of the re2 family. The re1 domain is present in the aminoterminal regions of dorsal(4)and the NF-KB subunits, pS0 and p6S('"-13).The degree of homology among re1 proteins varies from 45%-65% and can be up to 80% if conservative amino acid substitutions are taken into account. The re1 domain may be made up of subdomains that mediate nuclear localization, DNA binding and protein-protein interactions. All of the rel proteins contain an NTS (nuclear targeting sequence) which has been shown to be important for localization of v-rel("). In addition, they contain a potential phosphorylation site (arg-arg-X-ser) recognized by cyclic-AMP-dependent kinase that lies about 25 residues from the NTS. It ha5 been proposed that the NTS targets a protein to the nucleus while phos horylation at this site controls the rate of transport(23. The carboxvl-terminal regions of re2 nroteins show

P

re1 domain v-re1

5 0 3 AA

avian c-re1

595

human c-re!

1 0 -

P50

7 1 , 450 1969)

P65

dorsal

I

I

p r o pro

1-

Dr o

61 9

gln

549 gln

qln

1

678

Fig. 1. Schematic rcpresentation of the re1 proteins. The re1 domain (black box) spans approximately 300 amino acid residues and lies in the N-terminal portion of each of the re1 proteins. A putative phosphorylation site (P) and a nuclear targeting sequence (NTS) are located near the carboxyl end of the re/ domain. pS0 differs from other re1 proteins in that it contains an -40 amino acid insert midway through the rel domain. The C-terminal regions of the re1 proteins show littlc scqueiice homology. There are however characteristic repeats that may serve as transcriptional activation domains(z3.25).For example, p65 has an unusually high content of proline (pro) residues (IS%) over a stretch of 200 amino acids. dorsal also contains a pro-rich region (23%) over 42 amino acids, and several glutamine (gln) repeats. Moreover, the C-terminal halves of mouse and chicken c-rel have a net negative charge'""). The cDNA sequence that encodes the p50 subunit of NF-KB predicts a polvpcptidc of -100 kD. the N-terminal half containing the re1 domain and the C-terminal portion about six ankyrin (ank) repeats(''.''). The 100 kD polypeptide is clcaved to yield two smaller products, one of which is the rel-containing p50 subunit. It has been speculated that the ankyrin repeats form binding sites for cvtoskeletal proteins or integral membrane proteins(62)and may play a role in regulating the subcellular distribution of p d ' 'I.

little sequence homology, but in most cases share features characteristic of transcriptional activation domains such as acidic regions or stretches of proline residues or glutamine residues(24,25)(see Fig. 1). Regulation by Selective Nuclear Localization The most striking featurc of the re1 family of proteins is that their activity is primarily controlled by alteration in subcellular localization rather than at the level of transcription or translation. This regulatory strategy is unusual but not unique (reviewed in 26). For example, the glucocorticoid receptor is a zinc finger containing transcription factor that is present in the cytoplasm when uninduced. Following hormone stimulation, the receptor migrates to the nucleus where it regulates expression of its target gene^(^''. In this type of regulation the movement of protein from cytoplasm to nucleus is a critically controlled step. This has been clearly shown for d o r ~ a I ( ~ - ~ ) . In early Drosophilu development, nuclei divide as a syncitium without forming cells. dorsal protein is initially distributed evenly throughout the egg cytoplasm. At nuclear division cycle 10, dorsal protein moves from the cyto lasm into the nuclei in a spatially regulated manner('.' . In ventral region4 dorsal protein moves into nuclei, whereas in dorsal regions the protein is predominantly cytoplasmic. In between, in lateral regions, nuclei contain intermediate levels of dorsal protein such that a nuclear concentration gradient of dorsal forms along the DV axis. The nuclear gradient a5 well as the cytoplasmic localization of dorsal protein in dorsal regions can be visualized in Figure 2. Tt i q thoupht that the d o r w l nuclear sradient forms

P

Fig. 2, The dorsal gradient. A young clcavage-cyclc 11 embryo stained with anti-dorsal antibodies (white color); dorsal is up, anterior to the left. dor.sal proteins are localized to the nuclei in ventral regions (white dots) and to cytoplasm in dorsal regions. Note the lack of staining in dorcal nuclei (black dots; black arrow). The nuclear gradient is most evident in the ventral-lateral region (in between white arrows).

due to the asymmetric transport of protein from cytoplasm to nucleus along the DV axis. At least 10 genes, members of the dorsal group. are res onsible for the localization of dorsal to the nucleusg-'). In the absence of any of these genes, dorsal protein remains cytoplasmic and the embryos appear identical to those that have no dorsal protein at all. Cells in all regions of the mutant embryo behave like cells from the dorsal regions and the embryo is said to be dorsalized. Thus, despite its name, the dorsal gene is required for the wecification of ventral and lateral structures. In

contrast, in the absence of thc cactus gene product, dorsal protein is localized to the nuclei of all cells and the embryos have a ventralized phenotype(738). Like dorsal, cytoplasmic NF-KB is non-functional. Upon induction of pre-B cells. or stimulation of non-B cells by phorbol esters, cytoplasmic NF-KB becomes localized to the nucleus, whcre it can bind DNA and activate the transcription of target genes(=). NF-KB isolated from cytosolic fractions is unable to bind to DNA because it is associated with an inhibitor protein I K B ( ~ ~I n) . vitro phosphorylation of IKB by protein kinase C or hcme-regulated eIF2K kinase causes a change that prevents it from associating with NF-KB(”). The data suggest that phosphorylation of IKB in an NFKB-IKB cytoplasmic complex can free NF-KB from inhibition and allow it to translocate to the nucleus and bind DNA. The re1 domain (of the p65 subunit) is believed to mediate the interaction between NF-KB and 1~~(1”31). The importance of cytoplasmic versus nuclear localization is more ambiguous with respect to v-re1and c-rel. c-re1is present predominantly in the cytoplasm oi chicken lym hocytes and its function is not well understood(’ ). However, human c-rel(”) was isolated from nuclei of phorbol ester-induced lymphocytes and shown to bind JINA (see below)(”). Thus, like doual and NF-KB. c-re1 may function as an inducible transcription factor. For v-rel there is no simple correlation between its subcellular localization and its ability to transform cells(21). Interestingly, coprecipitation experiments have shown that v-rel is associated with at least five cytosolic proteins, including c-rcl and a protein of 36-40 kD ( p ~ 4 0 ) ( ~which ~ ) , is homologous to IKB(~~ Perhaps ). v-rel interacts with pp40 or c-re1 and thus interferes with the normal c-rel activity(”).

IKB-0,but not IKB-a. inhibits the binding of c-re1 to the KB site(”). The sequence similarity between MAD-3 and pp40 includes consenws phosphorylation sites that could regulate their ability to form complexes with re1 proteins(”). MAD-3 and pp40 also contain 5 ankyrin repeats similar to those which are found in the precurqor of the NF-KB pS0 subunit, the p10S molecule(10311,3s) (see Fig. 1 legend). Since the Cterminal half of p1OS can inhibit binding of pS0 to DNA(37),it could be yet another member of the IKB family of proteins.

cactus and IKB May Have Similar Functions In Drosoplzila embryos mutant for the cactus gene, dorsal protein is nuclear in all celld7 ’). The cactus gene product thus appears to have a similar function to IKB, but it is not yet known whether it is structurally related. Genctic dosage studies sug est that cactus and dorsal interact at the protein levef3’). They also suggest that the cactus product is evenly distributed along the DV axis and that its inhibitory effect on dorsal is overruled in ventral regions by the dorsal-group genes. The dorsal-group gems could act by modifying cactus (perhaps by phosphorylation, as in the case of IKB) or the dorsal protein itself. When expressed in Drosoplzila tissue culture cells, C-terminal truncations of dorsal show increased nuclear localization compared to the full length protein(‘). It is possible that the dorsal protein is cleaved by one of the dorsal-group gcncs, thus facilitating its entry into the nucleus. However it is more likely that the C-terminal region of dorsal interacts with cactus, and a modification of either protein disrupts this interaction. The nuclear localization of dorsal occurs in response to a signal generated by the dorsal-group genes. This signal is delivered through the Toll rotein which A Family of Proteins have I d Activity resembles a transmembrane receptor(’). It has an Recently, several proteins have been shown to be IKB- extracellular domain with leucine-rich repeats, similar like molecules. Purification of IKB from pre-B cell to those in platelet glycoprotein 1b and the Drosophila extracts revealed two forms: the major form of 35-37 cell adhesion molecule chaoptin. Toll’s intracellular kD (IKB-a) and a minor form of 40-45 kD (TKB-/;J)(~’). domain is similar to the cytoplasmic domain of the In addition, the 40 kD phosphoprotein (pp40) that interleukin-1 receptor (IL-1R)(“’.32). Although Toll associates with avian c-re1 and v-re1(35.37),and MADactivity appears to be highest in vcntral regions, Toll 3(”), a mammalian protein induced upon monocyte protein is evenly distributed along the DV axis(”). A adhesion, are also IKB-like. pp40 and MAD-3 have solution to this apparent paradox is suggested by recent been cloned and sequenced; they arc similar but not experiments. Between the cell membrane and the identical, and can inhibit the DNA-binding activity of eggshell is a fluid-filled space, the perivitelline space. NF-KB as well as ~-rel(”~”).Comparison of the Stein et al.(44)have demonstrated that the perivitelline predicted protein sequences with peptide sequences fluid contains an asymetrically localized substance that from IKB indicates that I ~ B - a imost s similar to MADis likely to be a ligand for the Toll receptor. Though the 3(35),and IKB-0to p~40(~’). It appears that a family of intermediate steps are still being worked out, one inhibitor proteins exists to regulate the family of re1 model is that the release of the ligand in ventral regions transcription factors. Multiple inhibitory molecules of the embryo results in the local activation of Toll and may provide cell type specificity and therefore greater thus the translocation of dorsal into nuclei in ventral control in the regulation of the various re1 proteins. cells of the embryo. Support for this idea is seen in the activity differences In this context. thc rcsemblance between Toll and TLobserved for different inhibitor proteins - for example 1R is intriguing because IL-1R plays a central role in the

P

activation of cells involved in the inflammatory and immune responses and can promote the nuclear localization of NF-KB(?

of these target genes. It has been shown that v-re1binds to KB sites and su resses transcription in coBP ). Moreover, non-transforming transfection assays(3534 mutants of v-re1 are unable to bind DNA(45).Thus, the transforming activity of v-re1 may be linked to its The re1 Family Includes Transcriptional inhibitory effect on cellular genes under NF-KB control, Activators and Repressors in addition to its possible interactions with pp40 and cOnce they are localized to the nucleus, the re1 proteins re1 alluded to earlier. regulate the transcription of various genes. NF-KB, In comparison with the complexity of NF-KB stimulus dorsal, and c-re1 all bind cis-regulatory se uences and response, the role of dorsal appears simple. We ) , discussed the activation of dorsal by Toll as well as known to be essential for normal e x p r e ~ s i o n ( ' ~ ' ~ ' ~ ~ ' ~have and this will be discussed in detail below. The best the possibility of additional regulation by phosphorylevidence that re1 proteins function as transcription ation/protein modification. The initial pattern of factors is the demonstration that purified NF-KB expression of most of the zygotic DV genes seems to be increases transcription of a target HIV-1 promoter in directly regulated at the transcriptional level by the ~ i t r o ( ' ~ )The . evidence that dorsal and c-re1 activate accumulation of nuclear dorsal protein(54). The genes transcription is less direct. mainly because neither has twist and snail are activated by high levels of dorsal, yet been purified to homogeneity for biochemical while Zen and dpp are repressed. Binding sites for studies. Instead transient gene expression assays dorsal have thus far been found in the zen, twist and provide evidence that they can activate transcription of snail promoters (9,50, see below: J. Jiang, T. Ip and M. target genes. In these studies, tissue culture cells are Levine, unpublished results). How dorsal effects transfected with an expression plasmid and a reporter transcriptional activation and repression remains a plasmid containing target cis sequences. When tethered mystery. Variation in the quality, number and location to a heterologous DNA-binding domain such as lexA or of binding sites could determine how dorsal protein gal-4 (to ensure DNA binding). c-re1was reported to act interacts with enhancers, silencers and the general as a potent transcriptional activator("-49). Similar transcription machinery. Alternatively. other unidentexperiments using Drosophila tissue culture cells ified proteins may determine the activity of dorsal. showed that dorsal also dramatically activated the Pcrhaps other dorsallrel hornologs exist in Drosophila. expression of a reporter The activation These homologs could dimerize with dorsal to evoke domains of dorsal and c-re1have been localized to their different responses in discrete regions of the embryo. A C-terminal regions in these assays. Interestingly, v-re1 comparison of dorsal binding sites in target gene does not appear to be a strong a~ tiv ato r('~ 3~ ~ ) . promoters may help differentiate amongst the various The re1 transcription factors act by controlling the mechanistic possibilities. expression of downstream target genes. About two dozen genes have been identified which contain NF-KB re1 Proteins Bind to Specific DNA Sequences binding sites, and are probable targets for NF-KB or some combination of its constituent subunits with other NF-KB was initially identified as a protein complex that cellular proteins (e.g. c-reQ('*). The large number of interacted with an enhancer in the K light chain gene target genes is not surprising given the range of stimuli promoter(5). Methylation interference experiments that can activate NF-KB, including viruses, parasites, Tshowed that the three G and two C (G on other strand) cell initogens, cytokines, DNA-damaging agents and residues in the binding site GGGACTTTCC, the KB bacterial lipopolysaccharides, some of which may act motif, are contact points for the protein. The binding of via rcactive oxygcn in terrn ~ d iatcs('~ .~ Many ~ j. of the NF-KB to this site is essential for the stage-specific target genes encode products which are important transcription of the gene(^^). NF-KBbinding sites have components of organismal or cellular defense mechanbeen identified in a number of immunoregulatory genes and viral enhancers('") (see Fig. 3). The consensus isms such as /J-intcrferon, the K light chain gene, the T binding site is an imperfect palindrome. Moreover, cell receptor, TNF-a. and fi, etc. The presence of KB sites in adenovirus, SV40, cytomegalovirus and the most NF-KB sites are decameric, compatible with the idea that NF-KB is a heterodimer recognizing two HIV LTR suggests that viruses can subvert this key pentameric half sites. Some binding sites show patterns factor involved in cellular defense to activate their own of methylation interference that span 11 or 12 bases, transcription. suggesting that various decameric motifs, which all Most of the genes classified as targets of NF-KB have fulfill consensus requirements. are recognized within been placed in this category because they contain NFthe 11 or 12 base pairs("). KB binding sites("). However, since c-rel (HIVEN86A) lnitial biochemical studies suggested that the NF-KB can bind to the same sequences(33), perhaps in pS0 subunit bound DNA while the 65 subunit was association with NF-KB, it is not clear how many of required for transcriptional activationF1'). However, it these genes are solely regulated by NF-KB or c-re1 in is now believed that both subunits bind to DNA as a vivo. An explanation for the oncogenic properties of vheterodirner(l2>"). Thus pS0 and p65 each bind a half rel may lie in its ability to repress transcription of some

re1 family binding sites KB

A-ACTTTEG

H2-kb

TWGATTCEA

HIV LTR

AWACTTTE

dorsal

dorsal mutant

GTGAATTCTA

Fig. 3. Comparison of DNA binding sites of thc re2 family proteins. The DNA binding motifs found upstream of the kappa light chain gene (KB), MHC Class I (H2-kB) gene. the dorsal target gene zen, and the HIV-1 LTR are compared. The residues that are contacted by the proteins are underlined (both strands denoted). An example of a mutant dorsal binding sitc is also listed.

site of the consensus sequence. In addition, they are able to bind DNA as homodimers. The p50 subunits can homodimerize (pS0-pS0) and recognize a slightly more palindromic binding site. Kieran et al. demonstrated that the nuclear factor that regulatcs the class I MHC promoter. KBF1, is in fact a p50 homodimer(''). This factor lacks transcriptional activation domains and is, in vitro, a transcriptional re ressor (although its physiological role is unknown)( 3'). The KBFl binding site GGGAAAGTCCC is similar to, but more symmetric than, the KB motif GGGACTTTCC (see Fig. 3). Later it was shown that p65 subunits can also homodimerize and bind DNA directly in vitro, although it is not known if p65 dimers bind to a specific subset of NF-KB sites in

t

viv0(13>311)

The c-re1 and v-rel proteins also bind specific s e q u e n c e ~ ( ~ ~Binding . ~ ~ ~ j of ~ )c-re2 . to the NF-KB site in the HIV-LTR was demonstrated indirectly when it was discovered that the previously characterized DNAbinding protein HIVEN86A, is in fact human c-rel("). c-rel binds DNA and may form dimers with itself and vrel and p50(" 49). This suggests that c-re2 can oligomerize with the NF-KB subunits zn vivo,although a rigorous demonstration has yet to be reported. Thus, the combination of various re1 proteins may confer differential regulation of target genes in vivo. The mechanism by which the re1 proteins bind to DNA is unknown. The re1 domain does not contain an well Characterized DNA-binding motifs. Zabel et al.("; have shown that NF-kB requires thc presence of Zn2+ in order to bind to DNA. Addition of the zinc chelating agent, 1,lo-orthophenantroline to DNA-binding reactions greatly impaired the formation of DNA-NF-KB complexes. This inhibition was reversed by addition of Zn2+ but not other divalent cations. Although binding activity depends on zinc, there is no evidence that the Cys and His residues within the re1 domain form a classic zinc finger motif. The re1 domain mediates not only DNA binding but

also dimerization('l,"). Mobility shift assays using truncated and internally deleted pS0, p65 and dorsal proteins indicate that most of the re1 domain is necessary for DNA binding(""). One p50 deletion was shown to act as a transdominant negative mutation(j6). Although it could not bind to DNA, it could form heterodimers with c-re1and v-ref and prevent them from binding DNA. These results suggest that the re1 proteins may bind to DNA as obligate dimers. The palindromic nature of the binding sites and interactions between various dorsal alleles suggests that dorsal also binds to DNA as a dimer(729). The dorsal protein can activate or repress its target promoters(9). For example, high levels of dorsal in ventral regions of the embryo activates the genes twist and snail, and represses the genes Zen and dpp. The regulation of Zen by dorsal has been extensively studied. A 600 bp segment of the zen promoter is known to be important for ventral repression(a). dorsal protein produced in bacteria binds to four specific sites within this segment which are similar to the KB site(9) (see Fig. 3). Mutations that change the middle G and one of the C residues (i.e. GTGAATTCT, see Fig. 3) abolish dorsal binding in vztro. The effect of thcsc mutations has been analyzed in the context of the full-length 1.6 Kb zen promoter attached to the lac 2 gene (C. Rushlow, T. Ip and M. Levine, unpublished results). Constructs containing mutagenized binding sites were introduced into embryos by P-element-mediated transformation and the distribution of lac2 transcripts was determined by in situ hybridization. An example of one such experiment is shown in Figure 4. The embryo in (a) carries the wild-type zen promoter-lac2 gene and expresses lac2 in the normal pattern, i.e. in dorsal but not ventral regions. The embryo in (b) carries point mutations in all four binding sites and expresses lac2 in both dorsal and ventral regions. Repression of Zen in ventral cells has been abolished due to the mutant binding sites. This is similar to what is seen in an cmbryo which lacks dorsal protein((''). In such an embryo, zen is transcribed in both ventral and dorsal regions. The correlation between the abolition of binding in v i m and the altered expression pattern in vivo provides strong evidence that the dorsal protein mediates ventral repression by binding to these sites and repressing transcription.

Concluding Remarks A common property of the re1 proteins is that they are present in a pre-synthesized, inactive form. In response to an extracellular signal they are activated, with no intervening lag time for transcription or translation, and can rapidly exert their effect on downstream genes. Teleologically it is clear why this rapid response is important in the case of NF-irB - because of its role in cellular responses. In Drosophila embryogenesis the D V polarity of the embryo is established in the form of

Fig. 4. Mutations in the dorsal binding sites abolish ventral repression. Whole mount in situ hybridization of cleavage stage-14 embryos using lacZ antiscnsc RNA probes. Sagittal (side) views of digoxigenin-labelled embryos - anterior is up, dorsal is lcft. (A) Transformant embryo carrying thc wild-type Zen promoter-lacZ fusion gene. Transcripts are localized to the dorsal side of the embryo. (B) Transformant embryo carrying the quadruple mutant Zen promoterlacZ fusion gene. All four dorsal binding sites contain point mutations that disrupt the 3 G and 2 C residues. Transcripts are no longer rcstricted to dorsal regions - ventral repression has been abolished.

the dorsal gradient between nuclear cycles 9 and 11 - a total of about 20 minutes. This requirement for speed in establishing polarity may be a consequence of the developmental strategy of long germ band insects like Drosophila. Despite similarities in structure, function and mode of action, the vertebrate re1 proteins appear to have very different regulatory roles from dorsul. NFKB is required for the maturation of pre-B cells and other aspects of the immune response, a developmental role which appears much more restricted than that l has global effects played by dorsal. The d o r ~ ugradient on cell fate in the developing embryo. To date, vertebrate re1 proteins have not been shown to have a role in embryogenesis. In the absence of such data, it may be too much to hope that a dorsal homolog will also regulate DV polarity in higher vertebrates. However, comparisons of the vertebrate and invertebrate systems will at least continue to illuminate details of regulatory mechanisms.

Acknowledgements The authors would like to thank Karina Yazdanbakhsh, Paula Enrietto, Tom Gridley and Mary Dickinson for helpful discussions and critical reading of the manuscript. We also thank Jin Jiang, Tony Ip and Michael Levine for sharing unpublished results.

References 1 Sevoian, M., Larose, R. N. and Chamberlain, D. M. (1964). Avian lymphomatosis. VI. A virus of unusual potency and pathogenicity. Avian Dis. 8, 336-347. 2 Thdlen, G. H., Zeigel, R. F. and Twiebaus, M . J. (1966). Biological studies with RE virus (strain T) that induces reticuloendothcliosis i n turkey?, chickens and Japanese quails. J. Narl. Cancer Insr. 37. 731-738. 3 Shibuyd, J., Chen, I., Howatson, A. and Mak, T. W.(1982). Morphological, immunological and biochemical analyses of chickcu spleen cclls transformed in vitro by reticuloendotheliosis virus strain T. Cancer Re.7. 42, 2722.2728. 4 Steward, R. (1987). Dor.ra1. an crnbryonic polarity gene in Druwphilu. is homologous to the vcrtehratc protooncogene, c - r d Science 238, 692-694. 5 Anderson, K. V. (1987). Dorsal-ventral embryonic patrcrn genes of Drosophilu. Trends. Genet. 3, 91-97. 6 Rushlow, C. A., Han, K.. Manley, .I. L. and Levine, 51. (1989). 'The graded distribution of the ONX XI niorphogen is initiated hy selective nuclear transport in Dro~uphila.Cell 59, 1165-1177, 7 Roth. S., Stein, D. and Nusslein-Volhard, C. (1989). A gradient of nuclear localization of the dorsal protcin detcrmines dorsoventral pattern in the Drosophila embryo. Cell 59, 1189-1202. 8 Steward, R. (1989). Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function. Crll 59, 1179.1188. 9 Ip, Y. T., Kraut, R., Levine, M. and Rushlow, C. A. (1991). The dorsal morphogen is a sequence-specific DNA-binding protein that intcracts with a long-range repression element in Drosophila. Cell 64, 439-446. 10 Ghoah, S., Gifford, A. M., Riviere, L. R., Tempst, P., Nolan, G. P. and Baltimore, D. (1990). Cloning of the p50 DNA binding subunit of NF-KB: homology to re/ and dorsal. Cell 62, 1019-1029. 11 Kierau, M., Blank, V., Logeat, R.. Vandekerckhove. J., Lottspeich, F., Le Bail, O., Urban, M., Kourilsky, P., Raeuerle, P. A. and Israel, A. (1990). The DNA hinding subunit of NF-KR is identical to factor KBFl a i d homologous Lo the re2 oncogene procluct. Cdl 62, 1007-1018. 12 Nolan, G. P., Ghosh, S., Linu, S . , Tempst, P. and Baltimore, D. (1991). DNA binding and IKB inhibition of the cloned p65 wbunit of NF-KB, a relrelated polypeptide. Cell 64, 961-969. 13 Ruben. S. M., Dillon, P. J., Scbreck, R., Henkel, T., Chen, C., Maher, M., Baeuerle, P. A. and Rosen, C. (1991). Isolation of a rel-related human cDNA that potentially cncodes the 65-kD subunit of NF-KB. Scierice 251. 1490-1493.

14 Lenardo, M. J. ;md Baltimore, D. (1989). NF-KB: a plciotropic mcdiator oi inducible and tissue-specific gene control. Cell 58? 227-229. 15 Stephens, R. M., Rice, N. R., Hiebsch, R. R., Bose, € R. I. and Cilden. R. t'. (1983). Nucleotide sequence of v-rel: thc oncogene of reticuloendotheliosis virus. Proc. Nntl. A c i d Sci. LISA 80, 6225.6233. 16 Wilhelmsen, K. C., Eggleton, K. and Temin, H. M. (1984). Kucleic acid sequcllccs of the oncogene v-re/ in reticuloendotheliosis virus strain T and its cellular homolog. the proto-oncogene c-rel. J. W r d . 52. 172.182. 17 Capobianco. A. J., Simmons, D. L. md Gilmore, T. 1). (1550). Cloning and cxprcssioii of a chicken c-re1 c D N h : unlike pS9 v-rel. p68 c-rel is a cytoplasmic protein in chicken embryo fibroblasts. On