Independent Repressor Domains in ZEB Regulate Muscle and T-Cell Differentiation

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 Kruppel 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 Kruppel 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 δ (58), etc. ZEB/zfh-1 is an active transcriptional repressor, and regulation of muscle differentiation by ZEB/zfh-1 requires this repressor domain—the 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 α4-integrin gene (29, 50). α4-integrin (as part of the heterodimer α4β1-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 α4β1 can also transmit a costimulatory signal in T-cell activation (reviewed in reference 35). Additionally, α4β1 is critical in leukocyte trafficking to sites of inflammation (35). The α4 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 (α4-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.