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Novel mechanism of C/EBP beta (NF-M) transcriptional control: activation through derepression.

Phosphorylation of transcription factors is regarded as a major mechanism to control their activity in regulation of gene expression. C/EBP beta is a transcription factor that becomes activated after phosphorylation to induce genes involved in inflammation, acute-phase response, cytokine expression, cell growth, and differentiation. The chicken homolog NF-M collaborates with Myb and various kinase oncogenes in normal myeloid differentiation as well as in the leukemic transformation of myelomonocytic cells. Here, we examined the structure of NF-M and its mechanism of activation. We show that NF-M is a repressed transcription factor with concealed activation potential. Derepressed NF-M exhibits enhanced transcriptional efficacy in reporter assays. More importantly, NF-M activates resident chromatin-embedded, myelomonocyte-specific target genes, even in heterologous cell types such as fibroblasts or erythroblasts. We identified two regions within NF-M that act to repress trans-activation. Repression is abolished by deletion of these regions, activation of signal transduction kinases including v-erbB, polyoma middle T, ras and mil/raf, or point mutation of a critical phosphorylation site for MAP kinases. We provide evidence that phosphorylation plays a unique role to derepress rather than to enhance the trans-activation domain as a novel mechanism to regulate gene expression by NF-M/C/EBP beta.

CAAT/enhancer-binding proteins (C/EBPs) are a family of transcription factors with highly similar carboxy-terminal DNA-binding domains (DBDs) and basic leucine zipper dimerization (LZ) domains. In contrast, the amino-terminal effector domains are not conserved. Whereas C/EBPa constitutively activates genes in cell cycle-arrested terminally differentiated hepatocytes or adipocytes (Christy et al. 1989;Friedman et al. 1989), C/EBPp and C/EBP8 are implicated in signal transduc tion as nuclear targets for cytokine-induced gene expres sion in various dividing and nondividing cell types (Akira et al. 1990;Poll et al. 1990; Akira and Kishimoto 1992;Juan et al. 1993). The expression of C/EBPa, C/EBPp, and C/EBPS is regulated during cellular differ entiation of adipoblasts or myeloblasts as well as during Diosophila melanogaster embryo development (Cao et al. 1991;Rorth and Montell 1992;Scott et al. 1992). Their expression kinetics together with the fact that C/EBPa antisense RNA inhibits terminal differentiation into adipocytes suggest important roles for C/EBP pro teins during cellular differentiation (Samuelsson et al. 1991;Freytag and Geddes 1992;Lin and Lane 1992).
The cell type specificity of C/EBP-regulated gene ex pression is thought to result from combinatorial inter actions with other transcription factors such as NF-Y (Milos and Zaret 1992), glucocorticoid receptor (Williams et al. 1991a), or the Myb (proto-) oncogene . Additionally, C/EBPp in particular requires activation through kinases for efficient tiansactivation of various reporter constructs. Cyclic AMPdependent kinase (Metz and Ziff 1991), calcium-depen dent kinases (Wegner et al. 1992), retrovirally derived oncogenic kinases (Sterneck et al. 1992b;Katz et al. 1993), mitogen-activated protein (MAP) kinase (Nakajima et al. 1993), or protein kinase C (PKC) (Trautwein et al. 1993) have all been shown to mediate phosphoryla tion of C/EBPp at different sites and, consequently, en hance tr<32is-activation efficacy.
In the hematopoietic system of mammals and the chicken (Scott et al. 1992;Katz et al. 1993), C/EBPp/ NF-M is solely expressed in the myelomonocytic lin eage, the same cell type that also becomes transformed by retrovirally encoded v-myb genes. We have shown recently that in myelomonocytic cells of the chicken, the C/EBPp homolog NF-M collaborates with Myb as well as with kinase (proto-) oncogenes to activate my eloid specific genes (Sterneck et al. 1992b;Ness et al. 1993). Intriguingly, ectopic expression of NF-M together with cellular or viral Myb activates the resident granulocyte-specific mim-1 gene as well as the macrophagespecific lysozyme gene in cells such as erythroblasts and fibroblasts. Hence, NF-M and Myb act as a combinatorial switch to induce myeloid-specific gene expression in heterologous cell types .
Whereas the function of NF-M in v-myb-induced my eloblast transformation has still to be elucidated^ its role to mediate the activity of protein kinase-encoding onco genes in v-myb-or v-myc-transformed myeloid cells has recently been established. In these cell types, NF-M is the critical target of activated kinase oncogenes to in duce expression of the myelomonocytic growth factor (cMGF) gene (Adkins et al. 1984;Graf et al. 1986;Sterneck et al. 1992a,b). cMGF is required to sustain growth of v-myb-or v-myc-transformed leukemic cells in an au tocrine fashion (Leutz et al. 1990;. Sim ilar to the differentiation-specific mim-1 and lysozyme genes, cMGF is normally expressed in mature macro phages after activation with the bacterial cell wall prod uct lipopolysaccharide (LPS). It therefore appears that NF-M mediates important myelomonocytic functions involved in cellular differentiation as well as in leuke mic transformation.
Comparison of the NF-M amino acid sequence with its mammalian homologs [CEBPp, NF-IL6, IL6-DBP, LAP, CRP2, and AGP/EBP (Akira et al. 1990;Chang et al. 1990;Descombes et al. 1990;PoH et al. 1990;Cao et al. 1991;Williams et al. 1991b) showed that the DNA-binding and dimerization motifs are contiguous and highly conserved with other C/EBP family members including C/EBPa, C/EBPp, and C/EBP8 from various species. In contrast, comparison of the more amino-terminal se quence of NF-M with its mammalian homologs revealed seven small, discreetly conserved regions (CR1-CR7), in terrupted by Ala-, Gly-and Pro-rich segments of variable length (Katz et al. 1993). Because the murine and rat C/EBPp proteins display indistinguishable activity to NF-M in chicken cells (E. Kowenz- Leutz and A. Leutz, unpubl.), we hypothesized that these conserved regions could reflect functional domains of NF-M/C/EBPp. Such regions may form a flexible "beads on a string" type structure connected by variable "spacer" sequences. On the basis of such a hypothetical arrangement of fvmctional regions, we constructed a series of deletion mu tants and examined their activity in reporter activation assays and their ability to activate chromatin-embedded endogenous genes.
Here, we show that NF-M is a repressed transcription factor with concealed activation potential that becomes unmasked by kinase oncogenes. Deinhibition of NF-M results in enhanced transcriptional efficacy of reporter constructs and, more significantly, of resident, myeloidspecific target genes in heterologous cell types. Inhibi tory functions locate to two evolutionary conserved re gions. Both regions contribute to silence the tians-activating domain. The inhibitory function can be neutralized by deletion. We show that one of the regions, CR7, can be neutralized by activation of the signal trans duction cascade involving either a conditional v-erbB cell surface kinase, polyoma middle T (PMT), ras, or v-mil/iaf oncogenes or by a point mutation of a phos phorylation site for mitogen-activated kinases (MAPK). Taken together, our data suggest that C/EBPp is a target of kinase oncogenes and delineate a novel mechanism of C/EBPp activation.

Results
Comparison of the NF-M amino acid sequence with its mammalian counterparts revealed seven discreetly con served regions (CR1-CR7) in addition to the highly con served DBD and LZ dimerization domains as shown in Figure 1. The regions between CRl and CR7 are not con served and are particulary rich in the amino acids Ala, Gly, and Pro (60%) when compared with total amino acid content (25%). The arrangement of conserved and nonconserved regions has been published (Katz et al. 1993) and is schematically depicted in Figure 1. The ac tivation of reporter and resident genes by mammalian C/EBPp in chicken cells is indistinguishable from that of NF-M ; data not shown), suggesting a strong evolutionary conservation of those regions that determine the activity and/or specificity of the transactivator proteins.

Inhibitory functions conceal the ttSLns-activation potential of NF-M and its ability to activate chromatin-embedded genes
To test whether the conserved regions CR1-CR7 could reflect functional domains of NF-M/C/EBPp, deletion mutants were constructed in expression plasmids and examined for their ability to activate reporter constructs as shown in Figure 2. The deletion endpoints were lo cated in the divergent regions of the protein between Pro-Gly residues, thereby minimizing disruption of sec ondary structure. All resultant mutants were analyzed for RNA expression (data not shown), protein integrity, and ability to bind DNA (bandshift assay in Fig. 2A,B). By these means they were foimd to be highly similar (see also Fig. 4B,C,below,or relevant mutants). Using these constructs we asked which deletion mutants could activate reporter gene expression in fibroblasts. As seen in Figure 2A, sequential amino-terminal dele tions of the first 140 amino acids gradually abolished NF-M-induced reporter expression. This indicates that  Forty-eight hours after transfection, cell extracts were prepared, normalized, and analyzed for reporter and bandshift activity {lower] as described (Sterneck et al. 1992a,b). (5) A modified version of the cMGF promoter containing 4x Gal4-binding sites replacing the NF-M-binding sites as indicated was transfected and analyzed as described above. The data represent means of triplicates; standard deviations were within the 10% range. Nuclear extracts were examined as in A.
several amino-terminal CRs contribute to the trans-activating function of the protein (a comprehensive analy sis of the trflns-activation region of NF-M w^ill be pub lished elsewhere). In contrast, internal deletion of amino acids 116-229 (CRS, CR6, and CR7) enhanced the transactivation five-to sevenfold in comparison to full-length NF-M (FLNF-M). Thus, the region between amino acids 116 and 229 partially represses the activity of NF-M. Internal deletions within this region enabled a more de tailed analysis of this possibility ( Fig. 2A). Removal of CRS or CR7 alone or in combination enhanced reporter expression five-to sevenfold over FLNF-M. Likewise, ad dition of CRS or CR7 to a construct consisting of the trcms-activating domain (CR1-CR4; amino acids 1-113) diminished reporter activity to wild-type level. Hence, two motifs, CRS and CR7, repress the full trans-activa tion potential of NF-M. In contrast, CR6 appeared to partially antagonize the inhibitory effects of CRS or CR7 because its deletion (ACR6) strongly reduced reporter ex pression, whereas its presence masks the negative effect of either single inhibitory region CRS or CR7 alone (as in the constructs ACRS and ACR7). Among other possibil ities, it is tempting to speculate that CR6 is required to maintain a conformational state that is crucial for NF-M derepression.
Are the trans-activation and inhibitory domains inde pendent modules of NF-M or are they intrinsically de pendent on the carboxy-terminal portion of the protein? To address this question we exchanged the carboxy-ter minal 96 amino acids of NF-M for the DNA-binding dimerization domain of the yeast transcription factor Gal4 [Gal4( 1-147)] (Ptashne 1988;Sadowski and Ptashne 1989). Transient reporter expression (Fig. 2B) revealed that activatory and inhibitory domains functioned in a Cold Spring Harbor Laboratory Press on July 22, 2018 -Published by genesdev.cshlp.org Downloaded from similar way as in the original mutants. These results indicated that neither the activating nor the repressing hmctions of NF-M depended on the native DNA-binding or dimerization domains.
Reporter assays however do not prove conclusively that the inhibitory and activating domains of NF-M have similar functions on endogenous genes. Transient ex pression of NF-M alone is sufficient to activate a panel of myelomonocytic-specific genes in unrelated cell types (E. Kowenz- Leutz and A. Leutz, in prep.). This enabled us to examine whether the repression mediated by the in hibitory regions within amino acids 116-229 was also applicable to chromatin-embedded genes (see Fig. 3). Un der normal conditions, fibroblasts do not express RNA for gene 126 [encoding a potential Ca^"^-binding protein that is specifically expressed in myelomonocytic cells (Nakano and Graf 1992)]. Transfection of the FLNF-M expression vector into QT6 fibroblasts already caused a weak induction of the 126 message. Here, too, removal of the inhibitory regions strongly enhanced 126 expres sion. Taken together, our data suggest that inhibitory functions within NF-M conceal its potential to activate reporter and resident genes.

Inhibition of NF-M is negatively regulated by kinase oncogenes
The fact that in fibroblasts NF-M is partially repressed suggests that its normal mode of activation in cells may be through derepression. Trans-activation of NF-M/C/ EBPp is enhanced by a calcium ionophore, phorbol ester treatment, bacterial lipopolysaccharide (LPS), by ras, or by various kinase oncogenes as found by us and other workers (Akira et al. 1990;Stemeck et al. 1992b;Wegner et al. 1992;Nakajima et al. 1993;Trautwein et al. 1993). Furthermore, activation of mammalian C/EBP(i by phor bol esters or oncogenic ras coincides with increase of Figure 3. Inhibitory regions of NF-M conceal its ability to ac tivate chromatin-embedded genes. Expression plasmids as indi cated at the top were transfected in QT6 fibroblasts. After 36 hr, cells were lysed and their poly(A) "^ fraction examined by Northem analysis with #126 and GAPDH probes. phosphorylation at positions that are within inhibitory motifs CR5 and CR7 (Nakajima et al. 1993;Trautwein et al. 1993). To investigate whether an oncogenic receptor tyrosine kinase can enhance NF-M activity by neutral izing its inhibitory regions, we took advantage of an erythroid cell line (HD3) that harbors a temperature-sensi tive version [ts] of the v-erbB oncogene (Graf et al. 1978;Beug et al. 1982). In contrast to QT6 fibroblasts, ectopically expressed FLNF-M induces #126 expression to a similar level as ACR7 in HD3 cells (Fig. 4A). This indi cates that in HD3 cells, FLNF-M is already activated at the permissive temperature. That the activity of NF-M was dependent on the v-erbB kinase was confirmed by the finding that at the nonpermissive temperature where the conditional tsv-erbB kinase is inactivated (Beug and Hayman 1984), the expression of #126 by FLNF-M was completely abrogated. Importantly, both ACR5 or ACR7 induced #126 expression even in the absence of func tional tsv-erbB. These results imply that tsv-erbB over comes the inhibition of FLNF-M at the permissive tem perature and that removal of the inhibitory regions cir cumvents the requirement for the kinase at the nonpermissive temperature. The observed differences were not the result of the particular gene (#126) exam ined, because another myelomonocyte-specific gene, #325 [encoding the chicken homolog of the granulocytespecific goose-type lysozyme (Nakano and Graf 1992)], was activated in a similar fashion (Fig. 4A). Furthermore, protein levels were comparible between the wild-type and mutant proteins even at the nonpermissive temper ature (Fig. 4B). Therefore, the differences were not be cause of altered mutant protein expression, intracellular localization, or changes in the ability of mutant proteins to bind to DNA, respectively, as shown by the controls (Fig. 4B,C).
We then examined whether or not kinase-induced ac tivation of NF-M was dependent on protein synthesis. To do so, tsv-erbB kinase was reactivated by backshif ting to the permissive temperature in the presence of cycloheximide as indicated in Figure 5. Under these condi tions, tsv-erbB kinase induced #325 expression even when protein synthesis was blocked. These results sug gest that the inhibitory function is mitigated by tsv-erbB or downstream mediators through a post-translational mechanism.
Finally, we asked whether oncogenes that act down stream of or in parallel to v-erbB in the signaling cascade can rescue FLNF-M activity. We transfected HD3 cells with expression vectors encoding E]-ras, v-mil, or PMT oncogenes and incubated the cells at the nonpermissive temperature to inactivate the tsv-erbB kinase as shown in Figure 6a. In the presence of FLNF-M, each of the signaling oncogenes at least partially induced the resi dent #126 gene or #325 gene (data not shown) and thus complemented the inactive v-erbB kinase. In the absence of NF-M, none of the signaling oncogenes examined were capable of inducing #126 or #325 (data not shown; Fig. 6B). To rule out cell lineage-specific effects, we asked whether induction of the signaling cascade in fi broblasts could also activate NF-M. Figure 6B shows that HD3 cells harboring the conditional tsv-erbB oncogene (Beug and Hayman 1984) were transfected with expression constructs as indicated (cf. with Fig. 2a) and incubated for 24 hr at the permissive temperature (+, 36°C) or the nonpermissive temper ature {-, 42°C, the normal body temperature of chicken). Acti vation of resident NF-M target genes and GAPDH controls were monitored by Northern analysis.
[B] Total cellular protein ex tracts were prepared in parallel, equivalents of 5 x 10^ cells were subjected to SDS-gel electrophoresis, blotted, and probed with an NF-M antiserum. (C) Nuclear protein extracts were prepared in parallel and examined for DNA binding by bandshift analysis as described (Stemeck et al. 1992b). NF-M shifts were controlled by competition analysis with a 100-fold excess of unlabeled oligonucleotide (oligo comp.) and supershift (+ aS) using a 1:500 dilution of a specific NF-M antiserum (Katz et al. 1993).
coexpression of PMT together with NF-M induced #126 expression to a similar level as ACR7, whereas the ac tivity of ACR7 was not further increased by PMT. PMT on its own did not induce #126 at all, confirming that trans-activation occurs through NF-M.
If phosphorylation of the inhibitory motifs is respon sible for activation of NF-M, their removal should be accompanied by a reduction in phosphorylation of the mutant molecules. To examine this possibility, we com pared specific phosphorylation levels of transiently ex pressed NF-M derivatives following metabolic labeling of HD3 cells as shown in Figure 7. To do so, NF-M con structs were modified to encode the FLAG-tag epitope at their carboxyl teminus to ensure equal antigenicity of the constructs. These constructs were then transfected into HD3 cells that were subsequently metabolically la beled with radioactive inorganic phosphate, lysed, immunoprecipitated with a FLAG-specific monoclonal an tibody, and the ^^P label was quantified by Phosphorlmage analysis. To determine specific ^^P incorporation into wild-type and mutant proteins quantitatively, the same blot was incubated with anti-FLAG followed by an antimouse ^^^I Fab fragment and subsequently analyzed as above. The ratio of ^^P to ^^^I label in wild-type NF-M was set to 1.0 and plotted as shown in Figure 7. Deletion of CR5, CR7, or both together diminished specific phos phate incorporation to 50%, 10%, and 5%, respectively. Thus, the inhibitory regions CR5 and CR7 provide the majority of phosphorylation sites in NF-M. It also ap pears that phosphorylation of CR5 and CR7 may to some extent be interdependent on each other. Importantly, ^^P incorporation was lowest in mutants with highest gene activation potential. It therefore appears that phospho-

Con-tsv-erbB
Time ( , and the addition of cycloheximide by an arrow. Briefly, following transfection, cells were incubated at the nonpermissive temperature (with exception of one of the vector controls) for 16 hr or backshifted after 12 hr to the permissive temperature for 4 hr. Where indicated by an arrow, cycloheximide (at 50 p-g/ml) was added 20 min before back shift to block protein synthesis. Northern blots were eval uated quantitatively by Phosphorlmage analysis (BaslOOO, Fuji) and normalized to the GAPDH control, and #325 expression induced by ACR7 was set to 1.0 (arbitrary units). rylation of the inhibitory regions abolishes their ability to suppress trans-activation. It appeared unlikely that the v-erbB kinase directly phosphorylates NF-M, as we failed to detect phosphorylated tyrosine residues on NF-M foUow^ing metabolic la beling of HD3 cells (data not shown). On the other hand, we had pointed out previously that a consensus sequence for MAPK within CR7 (Pro-Gly-Thr-Pro) is conserved between avian and mammalian C/EBPp (Katz et al. 1993). Subsequently, a peptide from NF-IL6 (human C/EBPp) comprising this site was shovm to be phosphorylated by purified MAPK and to be a target for the las oncogene in P19 cells (Nakajima et al. 1993). Hence, we examined whether a point mutation at this site (thr218 to asp218) could also derepress NF-M. Figure 7B shows that mutation of the MAPK target in NF-M218D miti gated the requirement for v-erbB to activate the resident #126 and #325 genes. Thus, the inhibitory function could be overcome either by deletion of repressing re gions, activation of signaling oncogenes, or by a point mutation at a MAPK phosphorylation site within the inhibitory CR7.

Inhibitoiy and tians-activating regions of NF-M interact
We decided to probe the mechanism by which NF-M inhibition is accomplished using the yeast "two-hybrid" interaction trap system. This approach allowed us to as certain whether the inhibitory regions interact with the trans-activating region. To do so, we cloned various parts of NF-M into yeast expression vector pairs that express the peptides of interest as fusion proteins (Fields and Song 1989). Here, blue colonies appear on X-gal plates when two protein sequences interact to induce galactosidase expression. As shown in Table 1, we found that the trans-activating region CR1234 of NF-M (cloned into the activation hybrid vector, pGAD424) induced galactosidase activity when cotransf erred with either CR5 or CR7 (cloned into the DNA-binding hybrid pGBT9). Moreover, CR7 domains can be seen to interact with each other imder these conditions, an observation that is discussed later in this paper. The kinetics of color appearance in these experiments was similar to the color development observed in the positive control as shown in Table 1 (p53 protein interaction with SV40 large T antigen). Likewise, we examined the effect of the point mutation at amino acid 218 in the MAPK site. As shown in Table 1, CR7D218, which harbored the T218 to D218 mutation, was completely impaired in its interaction with CR1234. This was not because of the failure to express this mu tant, because it still retained the ability to interact with CR7, as shov^m in the table. These results suggest that the inhibitory regions interact with the trans-activating To obtain equal antigenicity, NF-M constructs were tagged with the FLAG peptide (tg) fused in-frame to their carboxyl termini. These constructs were trans fected into HD3 cells. After 16 hr, cells were labeled with [^^Pjorthophosphate for 4 hr, lysed, and immunoprecipitated (Trautwein et al. 1993) with monoclonal anti-FLAG antibody, separated by SDS-PAGE, blotted to PVDF membrane, exposed to reveal ^^P label, and quantitatively evaluated by Phosphorlmage analysis. To determine protein levels, the blot was subse quently incubated with anti-FLAG antibody followed by ^^^Ilabeled second antibody and quantitatively analyzed as above.
The ratio between ^^P label (phosphate incorporation) and ^^^I label (protein level) was calculated, normalized to 1.0 for FLNF-M, and plotted as shown.
[B] HD3 erythroblasts were trans fected with expression constructs encoding NF-M derivatives, incubated for 20 hr under permissive or nonpermissive condi tions as shown, and analyzed for RNA expression by Northern analysis. NF-M218D is a derivative of the full-length protein, where threonine 218 was replaced with aspartic acid by sitedirected mutagenesis.
Activation of C-EBPp by deieptession  region probably by masking its activation potential. The finding that interaction of CR7 with the tians-activating domain can be abrogated by mutation in the MAPK site lends support to the idea that deinhibition of NF-M oc curs by kinase-induced unmasking of its trans-activation domain. Such a hypothetical mechanism is schemati cally shown in the model presented in Figure 8.

Discussion
Our data strongly favor a novel mechanism to control the activity of C/EBP^. We suggest that activation of signaling pathways results in phosphorylation of the in hibitory domains of C/EBPp, thereby eradicating the in hibition by exposing its trans-activating functions. Therefore, inactivation of inhibitory functions (negative regulation) rather than enhancement of the trans-activa tion domain (positive regulation) appears to be a major driving force of C/EBPp-induced gene transcription. The results obtained with reporter assays on defined promoter constructs demonstrate that activation of gene expression by deinhibition maps to the cis-regulatory el ements known to bind NF-M or its derivatives. How ever, replacement of the carboxy-terminal one-third of the protein (containing the b-Zip motif and DNA-bind ing domain) with the DNA-binding domain of the yeast trans-activator Gal4 creates a fusion protein that could still be activated by the same deinhibition mechanism as the wild-type protein. This suggests that the highly con served DBD-LZ region of NF-M is not essential for the activation mechanism. It does, however, not exclude that the DBD-LZ region participates in the regulation of NF-M/C/EBPp or the activation of some genes, for ex ample, in a cell type-dependent fashion as observed by others (Wegner et al. 1992).
We provide evidence that two inhibitory domains, CR5 and CR7, interact with the trans-activation domain of NF-M and silence its function. One of the pathways to activate C/EBPp occurs through MAPKs that phosphorylate a highly conserved site in CR7 (Nakajima et al. 1993). We demonstrate that activation is achieved by deletion of the entire region that harbors the MAPK site or by a point mutation at the MAPK site that mimics the negative charge of a phosphate residue. Along these lines, a mutation that inhibits phosphorylation of the homologous site in NF-IL6 was shown by others to result in loss of activation by the ras oncogene (Nakajima et al. 1993). However, data presented in Figure 7A as well as peptide maps of in vivo-labeled NF-M mutants (data not shown) or human C/EBPp (Nakajima et al. 1993) indi cated several additional phosphorylation sites in the in hibitory regions with unknown function. At the mo ment we do not know the biological significance of these sites. However, the results obtained with the two-hybrid system indicates that the MAPK site is indeed a critical target of region CR7, because its mutation abrogates its interaction with the trans-activating domain of NF-M but not with CR7. The observation that the CR7 inhib itory regions interact with each other in the two-hybrid system also deserves attention. The major implication from this finding is that inhibition/activation may also be achieved by intermolecular interactions between NF-M molecules, as opposed to the intramolecular mechanism proposed for CR5 and CR7. Because NF-M/ C/EBPp readily forms heterodimers with other members of the C/EBP family, it will be interesting to investigate whether CR7 could also inhibit activation in trans. It would probably also help in understanding the function of the natural dominant-negative C/EBPp that is trans lated from an internal start codon at the beginning of CR7 by a ribosomal scanning mechanism (Descombes and Schibler 1991).
The mechanism that targets the other inhibitory re gion, CR5, is less clear. Interestingly, PKC was suggested to mediate enhanced transcriptional efficacy through phosphorylation of a serine residue (Ser-105) in the rat C/EBP(3 version (Trautwein et al. 1993). According to our amino acid alignment of C/EBP proteins (Katz et al. 1993), the equivalent position of rat C/EBPp SerlOS would be at the amino-terminal border of CR5. There fore, it appears that the apparent activation of rat C/EBPp may also be occurring through disruption of CR5. However, this particular phosphorylation site in the rat protein is conserved neither in human nor in chicken C/EBPp (Katz et al. 1993). It therefore appears possible that multiple ways exist to inactivate the inhib itory regions of C/EBPp. Like rat and human C/EBP(3, we also observe activation of NF-M by phorbol ester or LPS treatment. However, activation through both path ways is cell type-dependent. In myeloblasts that express large amounts of NF-M, neither LPS nor phorbol ester activate the NF-M-dependent cMGF promoter. On dif ferentiation of myeloblasts into macrophages, however, NF-M-dependent cMGF expression and cMGF promoter activity is mediated by phorbol ester or LPS through the NF-M-binding sites . In contrast, kinase oncogenes activate the cMGF promoter through its NF-M sites irrespective of the differentiation status of myelomonocytic cells (Adkins et al. 1984;Graf et al. 1986;Stemeck et al. 1992a,b). This suggests that at least some of the pathways that mediate NF-M activation in myelomonocytic cells are sensitive to a differentiation program. Taken together, our data suggest that two re gions within NF-M, CR5, and CR7 mask the trans-acti vation domain and prevent its interaction with the basic transcription machinery.

Inactive Active
Many of the above data were obtained by exploiting the ability of NF-M to activate endogenous, chromatinembedded target genes and by using the ability of a con ditional oncogene to regulate the activity of NF-M. These experiments confirm the relevance of the deinhibition mechanism in vivo. Modulation of NF-M activity and, in particular, its ability to regulate resident genes is somewhat reminiscent of the myogenic helix-loop-he lix proteins, where removing negative regulation of a sin gle transcription factor can override constraints in the activation of cell lineage-specific genes (Weintraub 1993). Why is the trans-activation function of NF-M reg ulated by deinhibition? One advantage of this type of activation is that it could be achieved easily by many different means. They only need to have in common the ability to inactivate a specific protein function rather than to install one as it is the case with positive control mechanisms. We therefore anticipate that a range of sig naling pathways, effector molecules, and protein-pro tein interactions might converge on the inhibitory re gions of NF-M/C/EBPp. Conceivably, a model approxi mating this mechanism is shown in Figure 8 and might involve neutralization of the inhibitory domain causing a structural change that in turn reveals the previously concealed activation domain. Of course, more complex mechanisms involving dimerization partners or ancil lary proteins may also be possible and will be examined.
The derepression mechanism described in this paper provides an explanation why the cMGF gene (which con tains binding sites for NF-M but not for Myb) becomes transcribed only after activation of oncogenic kinases in myelomonocytic cells that contain high amounts of NF-M (Stemeck et al. 1992a,b). In contrast, the mim-\ gene (which contains binding sites for both NF-M and Myb) becomes cooperatively activated by NF-M plus Myb in cells lacking an oncogenic kinase. How can this discrepancy be resolved? It is conceivable that on pro moters that contain adjacent cis-acting elements for both NF-M and Myb, derepression might occur through interaction with adjacently bound Myb, omitting the ki nase activation step. Activation of NF-M in this instance may occur through modulation of the same inhibitory regions by interaction with Myb. As mentioned earlier, it is possible that the local activation of NF-M by tran scription factors binding to neighboring sites determines the cell type-restricted transcriptional activity of NF-M and other C/EBPs. The requirement of accessory tran scription factor NF-Y in the case of C/EBPa-regulated albumin promoter activation has already been shown and supports this idea. Whether such a combinatorial site-restricted activation mechanism occurs between NF-M and Myb as well as how NF-M participates in v-myi>-induced transformation are currently under in vestigation.
Mutant constructions were done as follows: To 100 ng of linearized plasmid encoding wild-type NF-M, 100 pmoles of the respective primer pairs was added and cycled through eight rounds of PCR amplifications, cut with HindUl-Xbal, separated by agarose gel electrophoresis, and cloned into pCDM8. Internal deletions were constructed by choosing appropriate primer pairs between Pro-Gly residues in the intervening, nonconserved parts of NF-M as indicated in Figure 1. The internal deletion primers harbored Smal-Xmal sites encoding Pro-Gly at the de letion endpoints and were used to amplify the respective parts of NF-M as described above. The following primers were used to construct intemal deletions: ID881, ATCCCGGGGAGT-CCTCCAAGTCGGCC; ID747, TACCCGGGCCCCGCTCC-CCCTTCTTC; ID743, ATCCCGGGCCAGGACCGGGGGG-CATG; ID645, TACCCGGGCAGCACCCCCGGCTTGTG; ID633, ATCCCGGGGTGCTGCCGGGCTGCTTC; ID561, TACCCGGGGCCGCCGCTGCCTTTATA. After amphfication, products were cut with Hindlll-Xmal and Xmal-Xbal, respectively, purified by DNA-agarose gel electrophoresis and joined by three-factor ligation into pCDM8. By a similar strat egy, the translation initiation and multiple cloning site of Gal4 (1-147) in the plasmid pSG424 (Sadowski and Ptashne 1989) were modified to a Nail site (which was in-frame with the NF-M Narl site) at the 5' end and an Xbal-translation stop codon at the 3' end (5' primer sequence: GAL4DBSEH, GCAC-TAGTGAATTCAAGCTTACCATGGCGCTACTGTCTTCT; 3' primer sequence, GAL4DBBXC, GCATCGATCTAGAGTG-GATCCGATACAGTCAACTGTCT). Appropriate NF-M con structs were ligated as Hindlll-Nail fragments to the modified Gal4DBD. Point mutation of the NF-M MAPK site was achieved with the primer NF-MBX and NF-M218D, CTCCGACCCCCCCGGCACCCCGAACCCC-TCCGAGTCCTC. Amphfication from wtNF-M template was performed as described above. The PCR product was digested with Bgll and Xbal and ligated to the Hindlll-Bgll fragment of WtNF-M in pCDM8. This exchanged the threonine at position 218 to aspartic acid.

Two-hybiid system
The two-hybrid system, yeast strains, and hybrid plasmids pGBT9 and pGAD424 were purchased from Clonetech. Gal4-NFM chimeric proteins were constructed in two steps: NF-M fragments were first obtained by PCR and subsequently subcloned in-frame to Gal4 sequences as described above. The fol lowing primers were obtained as synthetic oligonucleotides and used to construct the fragments containing CR1234 (primers IF and 4R), CR5 (primers 5F and 5R), CR7 (primers 7F and 7R), CR89 (primers 8F and 9R): IF, GCGAATTCCCCGGGATGC-AACGCCTGGTGGCC; 4R, GCGGATCCGCCGCCGCCGC-TGCCTTTATAGTC; 5F, GCGAATTCCCCGGGGGCGGC-AAGAAGCCCGACTAC; 5R, GCGGATCCCAGCACCCCC-GGCTTGTGGCTCTG; 7F, GCGAATTCCCCGGGGGCAT-GTCCTCGCCCTAC; 7R, GCGGATCCTCACGGCCCCGA-GTAGCCCCCGGC; 8F, GCGAATTCCCCGGGGCCGCCG-GCGCCGGGGGCTAC; 9R, GCGGATCCTCAGCAGCGG-GGCGAGGAAGCGAGCAG. All primers contain £coRI or BamHl restriction sites in-frame with the Gal moiety and clon ing sites of the vectors. After PCR amplification, fragments were cut with £coRI and BamHl, separated by agarose gel elec trophoresis, and ligated into appropriate vectors. Maxi-plasmid preparations were sequenced. All the experiments were per formed as described in the Clontech kit, except that plasmids were transformed by the method developed by Gietz et al. (1992) using the yeast strain SFY526. Coloration of filter-transferred and lysed yeast colonies was analyzed in all cases after 6 hr at 30°C of incubation with lacZ buffer containing 0.35 mg/ml of X-gal. The ability of p53 to interact with large T was used as positive control, which was provided with the Clonetech kit. All experiments were repeated at least three times with identi cal results.

Cell culture, tiansfection, metabolic labeling, and imm unopiecipi tation
The quail fibroblast cell line QT6 and the chicken HD3 (erythroblast) line have been described (Beug et al. 1979). All cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 8% fetal calf serum, 2% heat-inactivated chicken serum, 10 mM HEPES-NaOH (pH 7.2) plus penicillin, and streptomycin in a humidified incubator in 5% CO2 at 36°C. Temperature shifts were done by incubating cells at 42.5°C to inactivate the tsv-ezbB kinase. Cells were harvested 14-24 hr after the temperature shift. Backshift experiments were per formed by incubating the cells for 12 hr at the nonpermissive temperature (42.5°C) followed by incubation for 4 hr at the per missive temperature (36°C). Where indicated, cycloheximide (50 |xg/ml) was added 20 min before back shift to block protein synthesis. The transfection procedures using DEAE-dextran have been published in detail (Katz et al. 1993;Stemeck et al. 1992a,b). Briefly, for reporter assays, 2x10*^ cells were transfected for 30-45 min at 36°C with 200 ng of expression vector and 1 | jLg of reporter plasmid in 0.3 mg/ml of DEAE-dextran (Sigma) in STBS solution (Ausubel et al. 1987). Cells were pel leted, seeded in 4 ml of tissue culture medium, and harvested after 48 hr. For Northern analysis, transfection protocols and cell number were scaled up 10-fold. COS-7 cells were transfected with lipofectin (GIBCO-BRL) according to the manufac turer's instructions at 3x10^ cells in 60-mm tissue culture dishes and harvested after 3 days. Twelve hours after transfec tion, metabolic labeling with [^^Pjorthophosphate was per formed for 4 hr. Cells were washed three times in phosphatefree DMEM (Sigma) and incubated with 0.5-1 mCi/ml of P^Pjorthophosphate (5000 Ci/mmole) in phosphate-free DMEM supplemented with 10% dialyzed fetal calf serum. Cells were washed twice in TBS (25 mM Tris-HCl at pH 7.5, 150 mM NaCl, 100 mM sodium orthovanadate, 1 mM DTTj and lysed in RIP A buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM DTT, 100 mM so dium orthovanadate, 10 mM aprotinin, 10 mM leupeptin). To equalize antigenicity of NF-M derivatives, the FLAG peptide (Kodak) tag was fused in-frame to the carboxyl terminus by the PCR method as described above. The sequence of the oligonu cleotide encoding the FLAG tag was DTKDDDDK. NF-M-FLAG chimeras were immune-precipitated with anti-FLAG an tibodies (Kodak) and washed three times with RIPA buffer. '^^P-Labeled immunoprecipitates were separated by SDS-PAGE, transferred to PVDF membranes (Millipore), and evaluated quantitatively by Phosphorlmage analysis (Fuji-Bas 1000). Fol lowing exposure to determine ^^P contents, protein blots were blocked with dry-milk solution (6% in 25 mM Tris at pH 8.0, 150 mM NaCl), and probed with anti-FLAG M2 monoclonal antibody (Kodak). Quantitative evaluation of NF-M constructs were performed with ^^^I-labeled second antibody and Phospho rlmage analysis. The ratio of ^^P to ^^^I-specific radioactivity was determined.

Reportei and resident gene activation assays
The cMGF reporter construct (M82) has been described (Sterneck et al. 1992b). The Gal4-responsive cMGF promoter was constructed by replacing the two NF-M-binding sites of the -82 cMGF promoter with four tandemerized binding sites for the Gal4 DBD. Transient expression assays, luciferase assays, and normalization were performed as described elsewhere (Stemeck et al. 1992a,b;Katz et al. 1993).
To monitor endogenous gene activation, total RNA was ex tracted from transfected cells using a guanidinium-isothiocyanate method (Chomczynski and Sacchi 1987). The polyadenylated RNA fraction was isolated using magnetic oligo(dT) beads (Dynal) as recommended by the manufacturer. RNA was sepa rated on 1.2% formaldehyde-agarose gel electrophoresis and transferred by vacuum blotting to nylon membranes (Hybond, Amersham). Blots were probed in QuickHyb hybridization so lution (Stratagene) at 65°C for 2 hr with appropriate random ^^P-labeled (Pharmacia, Amersham) DNA fragments as shown in Figures 3-7. Before exposure, filters were washed twice for 15 min with 2x SSC, 0.1% SDS at room temperature and twice for 15 min with 0.2 x SSC, 0.1% SDS at 60°C and exposed to Kodak XAR5 X-ray film or to Phosphorlmaging screens. Phosphorlm age and quantitation analysis was performed using the manu facturer's software packages (Fuji or Molecular Dynamics). For reprobing, filters were stripped in 0.04x SSC, 0.1% SDS at 95°C for 2 min.

Nuclear extracts and DNA-binding assays
Nuclear extracts from HD3 or COS-7 cells were prepared as described by a mininuclear extract procedure (Schreiber et al. 1989) in the presence of protease inhibitors. The doublestranded oligonucleotide C/EBP-binding site  or Gal4-binding site was labeled with [a-'^^P]dCTP by end-filling with Klenow polymerase. Mobility shifts were performed ex actly as described previously (Stemeck et al. 1992b;Katz et al. 1993). Where indicated, polyclonal rabbit antiserum raised against bacterially expressed NF-M was diluted with PBS (1:500 final concentration) and added to the binding reaction 10 min after mixing nuclear extracts and DNA. Samples were incu bated on ice for an additional 10 min before electrophoresis. Electrophoresis was performed on 5% polyacrylamide gels in 0.5 X TBE at 25 mA at room temperature.