INTRODUCTION

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Transcriptional repression is crucial in the regulation of developmental and differentiation processes from yeast to mammals (reviewed in references 21, 26, 30 and 46). Most transcriptional repressors and activators have an identifiable domain that regulates transcription and that is transferable to a heterologous DNA-binding domain (active repression or activation). The molecular mechanism of action of some of these transcriptional regulatory sequences is being uncovered, and these results may provide insight into relationships between regulatory sequences and how they selectively control tissue-specific processes. In most cases, there is a single regulatory domain. However, some activators and repressors contain more than one activator-repressor domain. For example, the T-cell factor NFAT-1 and the T-cell proto-oncogene RBTN-2 contain two independent transactivator domains (36, 39). Among repressors, Drosophila Krüppel contains two repressor domains that map to the N-terminal and the C-terminal regions of the protein (53). While the biological significance of multiple transcriptional regulatory domains remains unclear, it is likely that these domains are directed at different target genes or at different sets of transcription factors in a single promoter. Accordingly, there is some evidence that the repressor domains in Krüppel target a distinct but overlapping set of transcription factors in transfection assays (27).

We present evidence here that the zinc finger-homeodomain protein ZEB contains two independent repressor domains that target different sets of transcription factors and regulate distinct biologic processes. ZEB has been identified from Drosophila (where it was termed zfh-1) to vertebrates (where it received different names according to the species in which it was identified) (8, 16-20, 57). ZEB/zfh-1 is an active transcriptional repressor that binds to a subset of E boxes (with higher affinity for the CACCTG sequence) and E-box-like sequences in muscle and hematopoietic genes (20, 49-51, 56).

ZEB/zfh-1 regulates myogenic differentiation in both mammalian cells and Drosophila (49, 51, 56). In mammals, muscle differentiation is regulated by two families of positive factors: (i) the muscle regulatory factors (MRF; myoD, myf-5, myogenin, and MRF-4) which are basic helix-loop-helix proteins that induce muscle differentiation by binding E-box sequences in the promoter regions of muscle genes and activating their transcription (66); and (ii) the MEF-2 proteins, which synergize with MRF proteins to regulate muscle differentiation and activate transcription either by binding to specific DNA sequences or by interacting with the MRF proteins (44). Muscle differentiation is also under negative regulation by a number of factors such as ZEB, Id proteins, twist, and I-mfa (4, 11, 49, 57, 61); hence, myogenesis is the result of a fine balance between positive and negative factors. Binding sites for ZEB are present in the promoter regions of a number of muscle genes such as the MRF themselves (5, 15, 65), muscle creatine kinase (1), acethylcholine receptor  (58), etc. ZEB/zfh-1 is an active transcriptional repressor, and regulation of muscle differentiation by ZEB/zfh-1 requires this repressor domainthe DNA binding domain alone does not block myogenesis (49, 51).

We also found that the Drosophila homologue of ZEB, zfh-1 (16, 32, 33), is also an active transcriptional repressor that negatively regulates the onset of somatic and cardiac muscle differentiation in Drosophila embryos (51). zfh-1 expression is downregulated before muscle differentiation proceeds, but maintenance of its expression (using a heat shock-zfh-1 construct) blocks somatic and cardiac myogenesis (reference 51 and unpublished results). We also found that vertebrate and Drosophila homologues are interchangeable, since zfh-1 also represses mammalian muscle differentiation and ZEB represses in Drosophila cells (51).

ZEB is also critical for T-cell differentiation and function in vertebrates, since mice with targeted deletion of the ZEB/zfh-1 gene show a drastic decrease in thymocyte numbers and defective T-cell differentiation at several stages (29). In fact, ZEB was originally discovered as a repressor of the IL2 gene and further studies have demonstrated that negative regulation of the IL2 gene correlates with ZEB activity (68, 73). ZEB also regulates the activity of the immunoglobulin (Ig) heavy-chain enhancer (20), GATA-3 (24), and the ₄-integrin gene (29, 50). ₄-integrin (as part of the heterodimer ₄₁-integrin) is expressed on hematopoietic precursors and interacts with vascular cell adhesion molecule type 1 (VCAM-1) on stromal cells, and this interaction is crucial for hematopoietic differentiation (reviewed in reference 35). Binding to ₄₁ can also transmit a costimulatory signal in T-cell activation (reviewed in reference 35). Additionally, ₄₁ is critical in leukocyte trafficking to sites of inflammation (35). The ₄ gene is dependent upon the combination of c-myb and the ets family of transcription factors (as are a number of other hematopoietic genes) (50). ZEB blocks the activity of c-myb and ets proteins individually, but together the proteins synergize to prevent repression by ZEB (50). This imposes the requirement for both c-myb and ets for expression of these hematopoietic genes. Both c-myb and ets are required for normal hematopoietic differentiation (2, 45, 55, 63), suggesting that alterations in ZEB expression may adversely affect hematopoietic differentiation. Indeed, as outlined above, mice lacking ZEB show defects in T-cell differentiation (29). Additionally, in these mice, two genes that are dependent upon the combination of c-myb, ets, and ZEB (₄-integrin and CD4) are upregulated (29).

Here we show that ZEB contains two independent repressor domains. One domain regulates muscle differentiation and specifically blocks the activity of the myogenic transcription factor MEF2C. The other domain functions in lymphocytes and regulates the activity of hematopoietic factors such as c-myb and ets family members. ZEB is thus, to the best of our knowledge, the first example of a transcriptional repressor that contains independent domains with distinct transcription factor specificities to regulate different biological processes.

	MATERIALS AND METHODS

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Cell culture. The HT1080 fibrosarcoma (American Type Culture Collection [ATCC], Rockville, Md.) and C33A cervical carcinoma cell lines (ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) containing 5% fetal calf serum and 5% calf serum (Life Technologies). C3H10T1/2 fibroblasts (ATCC) were grown in DMEM containing 13% fetal calf serum. The T-cell line Jurkat (ATCC) was grown in RPMI 1640 supplemented with 10% FCS. 293T cells were obtained from S. J. Korsmeyer (Dana Farber Cancer Institute, Boston, Mass.). CV1-MLP-CAT cells were obtained by stably cotransfecting CV1 cells (ATCC) with a chloramphenicol acetyltransferase (CAT) reporter driven by the major late promoter (MLP) and a neomycin-resistant vector.

Plasmid construction. Gal4 and LexA fusion proteins of different ZEB fragments were obtained by PCR of the corresponding regions and in-frame cloning of the product in the BamHI-XbaI site of PM1 and PBXL3, respectively. RD (repressor domain)-ZEB refers to the region between the two zinc finger clusters (from nucleotides 906 to 2706). Region 1 comprises the cDNA between nucleotides 906 and 1626; region 2 comprises the cDNA between nucleotides 1626 and 2280; and region 3 comprises the cDNA between nucleotides 2280 and 2706. Out-of-frame GAL4 and LexA constructs for RD-ZEB were obtained by out-of-frame insertion of RD-ZEB in PM2 and PBXL3 and used as controls (data not shown). Most Gal4 activators were either previously described (67) (Gal4-CTF, Gal4-VP16, Gal-Sp1, Gal4-MEF2C [amino acids 1 to 465], Gal4-NFB-p65, Gal4-PU.1, Gal4-E2F-1 [amino acids 285 to 437], Gal4-c-fos [amino acids 21 to 380], Gal4-ITF-1 [amino acids 1 to 427], and Gal4-TFE-III [amino acids 2 to 216]) or obtained from the following investigators: Gal4-c-myb-CD (J. Lipsick, Stanford University, Stanford, Calif.), Gal4-myoD (amino acids 1 to 318) (G. Tomaselli, Johns Hopkins University, Baltimore, Md.), and Gal4-c-jun (C. Caelles, University of Barcelona, Barcelona, Spain).

Transient transfections and CAT assays. HT1080 cells were transfected by the calcium phosphate method, and Jurkat cells were transfected by electroporation as previously described (49, 50). After 48 h, lysates were collected and the transfection efficiency was corrected as previously described by cotransfection of a thymidine kinase-driven luciferase vector (49, 50). CAT assays were performed as described previously (49). DNA was brought to a total of 6 µg for 60-mm dishes (or to 20 µg for 100-mm dishes) with pBluescript (Stratagene, La Jolla, Calif.).

Myogenic conversion assays and immunostaining. For 10T1/2 cell myogenic conversion assays, 10T1/2 cells were transfected by the Lipofectamine method in Optimem medium (Life Technologies). After 12 h, medium was removed and replaced with differentiation medium (2% horse serum-DMEM). After 4 to 5 days, the cells were fixed in methanol and stained with anti-myosin heavy chain MF-20 (R. Kopan, Washington University, St. Louis, Mo.) monoclonal antibody as previously described (49). After being washed to remove unbound antibody, the cultures were incubated sequentially with horseradish peroxidase-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, Pa.) and with diaminobenzidine substrate (Vector Laboratories, Burlingame, Calif.). Nuclei were counterstained with hematoxylin (Zymed, South San Francisco, Calif.).

Western blot analysis. At 48 h after transfection with the appropriate plasmids, C33a cells were lysed in ELB (150 mM NaCl, 50 mM HEPES [pH 7.0], 5 mM EDTA, 0.1% Nonidet P-40), sonicated briefly, and centrifuged to remove nuclear debris as described previously (51). The lysates were then loaded onto a sodium dodecyl sulfate-4 to 15% polyacrylamide gradient gel (Bio-Rad, Hercules, Calif.) and transferred overnight to a polyvinylidene difluoride membrane (Immobilon P; Millipore Corp., Bedford, Mass.) in 10 mM CAPS [3(cyclohexylamino) 1-propanesulfonic acid] buffer (pH 11). The membrane was serially incubated with anti-Flag polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) and anti-rabbit IgG-horseradish peroxidase secondary antibody. After several washes, the membrane was then developed by the chemiluminescence method (Renaissance; NEN Life Sciences Products, Boston, Mass.) as specified by the manufacturer.

	RESULTS

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ZEB is a selective transcriptional repressor. In previous studies, we found that the central region of ZEB, between the N-terminal and C-terminal zinc finger DNA binding domains, contains the repressor domain (RD) (schematized below in Fig. 2A) (49). We found that this RD could function independently to repress transcription when fused to the DNA binding domain from the yeast transcription factor Gal4 (49). In this study, we asked which transcription factors are repressed by ZEB. We found that ZEB blocked the activity of some transcription factors but not others (Fig. 1). It is of note that the set of transcription factors blocked by ZEB includes factors that are important for both myogenesis (MEF2C) and lymphoid differentiation (i.e., c-myb, ets family members, TFE-III, and the NF-B protein p65).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   ZEB is a selective transcriptional repressor that blocks the activity of some transcription factors but not others. The repressor domain (RD-ZEB) was fused to the DNA binding domain of the bacterial protein LexA (L-ZEB) and tested for its activity to repress several transcriptional activators fused to the DNA binding domain of yeast Gal4 (G-activators). Eight-tenths microgram of the pGL construct containing LexA sites 30 bp upstream of Gal4 sites was cotransfected into HT1080 cells with 2 µg of L-ZEB and 0.1 to 0.5 µg of different G-activators. After 36 to 48 h, cells were harvested and the CAT activity was determined as described in Materials and Methods. Equal molar amounts of the control Lex A expression vector did not affect the activity of any of the different G-activators (data not shown). CAT results are the mean of duplicate assays and are all representative of at least five separate experiments with standard deviations below 15%.

ZEB contains two independent repressor domains. To further localize sequences important for repressor activity, we divided the RD into three parts (Fig. 2A). Region 1 includes sequences N-terminal of the central homeodomain; region 2 contains the homeodomain, and region 3 contains the C-terminal region of the RD. Region 2 did not show any repressor activity in these assays (Fig. 2B). We found that region 1 was sufficient to repress all of the transcription factors repressed by RD, as shown in Fig. 1, with the exception of the myogenic factor, MEF2C (Fig. 2). Factors repressed by region 1 include hematopoietic factors (e.g., c-myb, ets family members, and TFE-III) as well as several more general and ubiquitous factors (Fig. 2B and data not shown). Interestingly, region 3 blocked the activity of MEF2C but had no effect on the activity of any other transcription factor we tested (Fig. 2B and data not shown). These results indicate that ZEB has two independent repressor domains, which target different subsets of transcription factors.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   ZEB contains two independent repressor domains that target distinct sets of transcription factors. (A) Scheme of ZEB. The RD of ZEB lies between the zinc finger regions (the DNA binding domains [DB]) (49). Numbers indicate amino acids. (B) RD-ZEB contains two independent repressor regions. Regions 1, 2, and 3 of RD-ZEB (indicated by 1, 2, and 3) were fused to the DNA binding domain of LexA and tested for their ability to repress different transcriptional activators fused to the DNA binding domain of yeast GAL4 (G-activators as in Fig. 1). Eight-tenths microgram of pGL (as in Fig. 1) was cotransfected in HT1080 cells with 2 µg of L-ZEB constructs and 0.1 to 0.5 µg of different G-activators. After 36 to 48 h, cells were harvested and the CAT activity was determined. Equal molar amounts of the control LexA expression vector did not affect the activity of any of the different Gal4 activators (data not shown). CAT results are the mean of duplicate assays and are all representative of at least five separate experiments with standard deviations below 15%.

View larger version (58K): [in this window] [in a new window]   FIG. 3.   Effect of promoter position on repression by regions 1 and 3 of ZEB and regulation of c-myb and ets by domain 1. (A) Region 3 is a long-range repressor, whereas region 1 is a short-range repressor. Eight-tenths microgram of a reporter construct containing LexA sites either 30 or 1.7 kb upstream of Gal4 sites was cotransfected with 2 µg of expression vectors for fusion proteins between LexA and regions 1 and 3 along with 0.1 to 0.3 µg of G-c-myb, G-PU.1, and G-MEF2 expression vectors. LexA alone did not have any effect on the activity of the different Gal4 activators (data not shown). (B) ets and c-myb synergize in the 4-integrin promoter to overcome repression by ZEB. Three micrograms of a reporter containing 2.0 kb, 400 bp, or 76 bp of the 4 promoter region (52) was cotransfected in c-myb-negative HT1080 cells along with 3 µg of vector alone or with a vector expressing c-myb. (C) Region 1 regulates the activity of ets and c-myb sites in the 4-integrin promoter. Sixty micrograms of each of the reporters pETS, pETS-ZEB-MYB, pETS-ZEB plus pETSmut-ZEB-MYB, pETSmut-MYB, and pETS-MYB (50) was cotransfected into Jurkat cells by electroporation along with 30 µg of expression vectors encoding DB-ZEB or equal molar amounts of full-length ZEB (FL) or DB-ZEB fused to regions 1, 2, and 3. Note that domain 1 represses transcriptional activity mediated by c-myb and ets independently but not when the two factors are combined. CAT results are the mean of duplicate assays and are all representative of at least five separate experiments with standard deviations below 15%.

Region 1 of the ZEB RD regulates the activity of the hematopoietic transcription factors ets and c-myb. c-myb is essential for normal hematopoietic differentiation (45). The ets family contains a number of members, some of which are hematopoiesis specific and some of which are more widely acting (2). Mutations in hematopoietic ets genes have demonstrated that these proteins, as with c-myb, are essential for hematopoietic differentiation (55, 63). A number of hematopoietic genes contain binding sites for both c-myb and ets, and it has been demonstrated that the two factors synergize to activate transcription (40). In fact, the E26 virus encodes a v-myb-v-ets fusion protein, and it has been shown that the synergy between these two factors is essential in the generation of erythroleukemias in animal models (41, 42).

Region 1 of ZEB represses transcription by a mechanism involving histone deacetylase activity. p300/CREB binding protein (CBP) interacts with a number of transcription factors and activates transcription by acetylation of histones and disruption of the nucleosome structure (reviewed in reference 38). While p300/CBP itself has histone acetyltransferase (HAT) activity on its own, the C-terminal region of the protein interacts with PCAF, which contains HAT activity essential for p300/CBP function (47). p300/CBP was initially discovered by its interaction with the adenovirus protein E1A (62). The amino-terminal region of E1A (amino acids 2 to 36) also binds to the C-terminal region of p300/CBP and displaces PCAF (62, 72). In addition, E1A can directly block p300/CBP HAT activity (9).

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Region 1 of ZEB blocks transcriptional activity of c-myb and ets by targeting the coactivator p300/CBP. (A) Repression of ets and c-myb activity by ZEB is overcome by overexpression of p300. Thirty micrograms of each of the reporter constructs containing ets sites (764CAT [40]) or c-myb sites (as described in the legend to Fig. 3C and reference 50) with or without ZEB sites upstream (50) was cotransfected by electroporation with 60 µg of CMV-p300. The empty cytomegalovirus vector did not have any effect on transcriptional activity (data not shown). After 36 to 48 h, cells were harvested and the CAT activity was determined as described in Materials and Methods. (B) Repression of MEF2C by region 3 is rescued by overexpression of p300. Eight-tenths microgram of the pGL reporter construct containing LexA sites 30 bp upstream of Gal4 sites was cotransfected in 293T cells with 0.2 µg of Gal4-p65 or 0.5 µg of Gal4-MEF2C and with or without 2 µg of LexA-region 1-ZEB or LexA-region 3-ZEB, respectively, and with or without 2.5 µg of CMV-p300. (C) Region 1 of ZEB represses transcriptional activity mediated by p300. Eight-tenths microgram of the reporter pGL construct containing LexA sites 30 bp upstream of Gal4 sites was cotransfected into C33a cells with 0.5 µg of Gal4-p300 (amino acids 1737 to 2414) (74). After 36 to 48 h, the cells were harvested and the CAT activity was determined as described in Materials and Methods. (D) Region 1 of ZEB represses the activity of the histone acetylase-dependent MLP. CV1-MLP-CAT cells were transfected as described in Materials and Methods. After 4 days in selection medium, 300 nM trichostatin A (TSA) was added to the medium and the cells were cultured for an additional 48 h. Six days after transfection, the cells were collected and CAT activity was assayed as described in Materials and Methods. The CAT results are the mean of duplicate assays and are all representative of at least five separate experiments with standard deviations below 15%.

Region 3 of RD-ZEB blocks myogenesis. Expression of myoD is sufficient to trigger the myogenic pathway in a number of cell types (66). MyoD activates a cascade of transcription factors, including members of the MEF2 family, which collaborate with myoD to amplify the muscle differentiation program (44). A dominant negative form of MEF2 blocks vertebrate myogenesis (48), and somatic and cardiac myogenic differentiation is disrupted in Drosophila embryos with mef2 null mutations (6, 34), indicating that MEF2 function is essential in myogenesis from Drosophila to mammals. Negative regulation of muscle by ZEB is not due to ZEB binding E boxes and displacing myogenic factors (49). ZEB binds only a small subset of E boxes (and it also binds a subset of non-E-box sequences), and thus it cannot efficiently displace MRF proteins. Indeed, DB-ZEB has no effect on myogenesis (49). Instead, the entire molecule was necessary for inhibition of muscle differentiation (49); thus, ZEB is functioning as an active repressor when bound to promoters.

View larger version (70K): [in this window] [in a new window]   FIG. 5.   Region 3 of RD-ZEB selectively inhibits myogenic differentiation. (A) CH3-10T1/2 cells were transfected with 0.5 µg of a myoD expression vector and equal molar amounts of expression vectors for the DNA binding domain of ZEB (DB-ZEB) and DB-ZEB fused to RD-ZEB and regions 1, 2, and 3. The cells were switched to differentiation medium, and the formation of myotubes was assayed by immunostaining with myosin heavy chain as previously described (49). (B) Quantification of the results in panel A. Values represent the number of nuclei in myosin heavy-chain (MHC)-positive myotubes with the corresponding standard deviation. One hundred is an arbitrary value for MyoD + Vector. (C) Western blot analysis of the transfected proteins. Proteins were Flag tagged and detected with an anti-Flag antibody.

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Repression of MEF2C activity by region 3 of ZEB is rescued by MAP p38 kinase. A 0.8-µg portion of the reporter pGL construct containing LexA sites 30 bp upstream of Gal4 sites was cotransfected into C33a cells with 0.5 µg of Gal4-MEF2C or 0.2 µg of Gal4-c-myb and 2 µg of either LexA-region 1 or LexA-region 3 of ZEB in the presence or absence of 1 µg of a plasmid encoding MAP p38 kinase (pSR2-p38). Expression of the empty vector corresponding to p38 did not have any effect (data not shown). After 36 to 48 h, the cells were harvested and the CAT activity was determined as described in Materials and Methods. CAT results are the mean of results of duplicate assays and are all representative of at least five separate experiments with standard deviations below 15%.

	DISCUSSION

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ZEB/zfh-1 is a transcriptional repressor that functions in both muscle and T-cell differentiation (29, 49-51, 56). In this report we present evidence that distinct and independent repressor domains are responsible for these two biological activities (Fig. 7). To our knowledge, ZEB is the first example of a protein with independent repressor domains that target distinct sets of transcription factors.

View larger version (11K): [in this window] [in a new window]   FIG. 7.   ZEB contains independent repressor regions that target distinct sets of transcription factors and regulate different tissues. Region 1 of ZEB represses a number of hematopoietically restricted transcription factors including c-myb, ets family members and TFE-III, but it also represses other, more general transcription factors such as NFB p65 protein, E2F-1, CTF, c-fos, and c-jun. These results suggest that in addition to its role in regulating T lymphocyte differentiation, region 1 may be involved in other reported ZEB/zfh-1 functions such as bone morphogenesis and central nervous system (CNS) differentiation (29, 64). Region 3 is very specific in its activityit inhibits MEF2C activity to regulate muscle differentiation.

We have found that p300/CBP is able to overcome repression by regions 1 and 3 of ZEB. Most factors repressed by ZEB are known to bind p300 and are dependent on p300 for their activity. However, recent studies indicate that the requirements for the different components of the HAT coactivator complex vary among different transcription factors, with some factors requiring the HAT activity of p300, others requiring the HAT activity of pCAF, etc., (31). Therefore, it is likely that repression of different factors involves offsetting of different component of the HAT coactivator complex.

Although repression by both regions 1 and 3 is overcome by overexpression of p300/CBP, only region 1 represses p300 transcriptional activity. One mechanism by which region 1 is able to repress p300 activity could be by direct interaction with p300 and blocking of its ability to recruit other components of the histone acetylase complex. Another mechanism would be offsetting of p300 activity by recruitment of a deacetylase activity. Accordingly, we found that region 1 but not region 3 was able to repress the activity of the MLP and that the histone deacetylase inhibitor TSA is able to reverse that effect. Repression of this reporter by other repressors such as Mad or the retinoblastoma protein is fully dependent on the recruitment of histone deacetylase complexes (28, 37, 38). However, the precise mechanism of repression by region 1 remains to be determined, since we have been unable to detect interaction of RD-ZEB with either p300/CBP or histone deacetylase-1 (reference 37 and data not shown).

A number of hematopoietic gene promoters depend on the synergy between c-myb and ets for their activity. This is the case for p56^lck and CD13, where mutation of either the c-myb or ets sites abolished their promoter activity (40). In other genes, such as the ₄ gene, ets sites are active even in the absence of c-myb (the case in ₄-negative cells), but both factors are necessary to resist repression by ZEB (as in ₄-positive hematopoietic cells) (50). While the molecular basis for the transcriptional cooperation between ets and c-myb is not understood yet, it is clear that the factors together are still dependent on p300/CBP, since overexpression of E1A-12S (but not a 2-36 deletion mutant of E1A-12S) abolishes c-myb and ets activity (data not shown). Therefore, it is possible that the binding of both factors to p300/CBP renders the complex inaccessible to repression by ZEB. A similar mechanisms has been proposed for the cooperation between c-myb and C/EBP in the regulation of the myelomonocitic genes (43).

Results of experiments with mice where the ZEB gene has been knocked out indicate that this protein is essential for lymphoid differentiation, which correlates with the fact that ZEB is highly expressed during hematopoietic differentiation but it is downregulated as T cells differentiate and move into circulation (reference 29 and unpublished results). We have previously found that ZEB inhibits the activity of the factors c-myb and ets, which regulate the activity of several hematopoietic genes (including ₄ integrin, CD4, and p56^lck) (40, 49, 54, 59, 60). ZEB is able to block the activity of each of these factors separately, but together they synergize to resist repression (50). Although the mechanism of this synergy is not understood, the activity of both factors is essential for the expression of all of the above genes. Moreover, the E26 virus, which carries a fusion protein between v-myb and ets-1, is able to induce erythroleukemias where v-myb alone does not (41). This also points to a critical role for the synergy between c-myb and ets in the regulation of hematopoietic genes and indicates that the activity of these genes must be under strict control. Therefore, regulation of the activity of c-myb and ets is critical and to our knowledge ZEB is the only factor playing this regulatory role.

We found that region 3 of RD-ZEB regulates myogenic differentiation by specifically blocking the activity of the transcriptional factor MEF2C. MEF2C is the only target that we have found to date for this region. Although repression by region 3 is overcome by overexpression by p300, this region of ZEB failed to alter p300 transcriptional activity. Activation of MEF2C by p300 seems to be more complex than in the case of other factors. Transcriptional activity of MEF2C is completely dependent on its phosphorylation by MAP kinase p38, and mutations of the target residues for this kinase in MEF2C (or use of a dominant negative p38 kinase) render MEF2C inactive (25). It is tempting to speculate that phosphorylation by p38 may facilitate the formation of an active MEF2C-p300 complex. A similar mechanism regulates the activity of CREB, since phosphorylation by the calcium calmodulin-dependent CAMIV-kinase regulates its ability to interact with CBP/p300 (10, 66a). Calcium-dependent mechanisms have been recently linked to regulation of gene expression in muscle and to muscle differentiation and hypertrophy (12). We have found that overexpression of CAMIV kinase is also able to rescue repression of MEF2C by region 3 of ZEB (without affecting the repression of c-myb by region 1 of ZEB) (unpublished results), further suggesting a model where phosphorylation of MEF2C is critical for its transcriptional activity and where ZEB may work to offset the effects of this posttranslational regulation.

MEF2 activity is essential for muscle differentiation, since its mutation results in a block of embryonic muscle differentiation in Drosophila (6, 34) and a dominant negative form of MEF2 blocks myogenesis in mammalian muscle differentiation assays (48). Thus, blocking of MEF2 activity is sufficient to inhibit myogenic differentiation from Drosophila to mammals. The Drosophila homologue of ZEB, zfh-1, blocks somatic and cardiac muscle differentiation in embryos and targets MEF2 in vivo and in transfection assays (reference 51 and data not shown). These results provide compelling evidence that regulation of myogenesis by inhibiting MEF2 is a property of these orthologues that has been conserved during evolution. It is also important to note that ZEB/zfh-1 is the only active repressor of muscle differentiation that targets MEF2 activity. Whereas MRF proteins are essential myogenic triggers in mammals, this role in Drosophila is played by twist (3), which directly activates MEF2 transcription (13). Nevertheless, MEF2 is essential for myogenesis through this evolutionary period, and thus it is a logical target for inhibition of myogenesis by an evolutionarily conserved protein like ZEB/zfh-1.

In addition to muscle, Drosophila zfh-1 is important for differentiation of the central nervous system, heart, gonadal cells, and fat body (7, 32, 33). Thus, region 1 could be important in regulation of one or more of these other tissues. It is then of note that in addition to transcription factors specific for hematopoiesis, region 1 of the ZEB targets transcription factors that are expressed more generally. Moreover, ZEB also has functions beyond lymphoid and muscle differentiation in mammals, since mice lacking ZEB also have skeletal defects (64).

All members of the zfh family (as ZEB/zfh-1) contain zinc fingers and homeodomains. However, the family members differ dramatically in the number and location of these domains. The general structure of these proteins suggests that the zfh family is very modular and is the result of extensive genetic recombination events. Interestingly, the ZEB gene maps to chromosome 10p11 in a region of frequent translocations in leukemias (69), making it tempting to speculate that the independent repressor domains in ZEB arose from such a recombination event involving two repressor genes. Given the similarity in zinc finger and homeodomain modules in zfh family members, it would not be surprising if at least some other family members have functions analogous to ZEB/zfh-1.