Cooperation between C/EBPalpha TBP/TFIIB and SWI/SNF recruiting domains is required for adipocyte differentiation.

Chromatin remodeling is an important step in promoter activation during cellular lineage commitment and differentiation. We show that the ability of the C/EBPalpha transcription factor to direct adipocyte differentiation of uncommitted fibroblast precursors and to activate SWI/SNF-dependent myeloid-specific genes depends on a domain, C/EBPalpha transactivation element III (TE-III), that binds the SWI/SNF chromatin remodeling complex. TE-III collaborates with C/EBPalpha TBP/TFIIB interaction motifs during induction of adipogenesis and adipocyte-specific gene expression. These results indicate that C/EBPalpha acts as a lineage-instructive transcription factor through SWI/SNF-dependent modification of the chromatin structure of lineage-specific genes, followed by direct promoter activation via recruitment of the basal transcription-initiation complex, and provide a mechanism by which C/EBPalpha can mediate differentiation along multiple cellular lineages.

Tumorigenic N-terminal deletions of c-Myb modulate DNA binding, transactivation, and cooperativity with C/EBP.

Oncogenic activation of c-myb by retroviral insertion has been implicated in tumor formation in chicken and mice. These genetic alterations result in deregulated expression of the c-myb gene and frequently in N-terminal truncation of the c-Myb protein. We demonstrate that truncation of the c-Myb N-terminus affects DNA binding and reporter activation. However, all three mutants, Myb Delta N20, Myb Delta N47 and Myb Delta N71 cooperated with C/EBP beta in reporter assays. In contrast to Myb Delta N20 and Myb Delta N47, however, the Myb Delta N71 mutant failed to activate the chromatin embedded endogenous mim-1 gene together with C/EBP beta. This suggests that an N-terminal region (amino acids 47-71) within repeat 1 (R1) of the murine c-Myb DNA binding domain affects activation of chromosomal target genes in collaboration with C/EBP beta.

Separation of C/EBPalpha-mediated proliferation arrest and differentiation pathways.

Cell proliferation and terminal differentiation are mutually exclusive in most cell lineages. The b-zip transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) induces proliferation arrest and differentiation in many cell types, suggesting that both activities are linked. Here we show that C/EBPalpha-mediated proliferation arrest and differentiation pathways can be separated by the E7 oncoprotein of the "high-risk" human papilloma virus 16. The E7 oncoprotein overrides C/EBPalpha-mediated cell cycle withdrawal without compromising the transactivation activity of C/EBPalpha or its ability to participate in differentiation. Uncoupling of both pathways depends on the casein kinase II site of the oncoprotein but not on its ability to neutralize pocket proteins or the cyclin-dependent kinase inhibitor protein p21. Our results suggest a bifurcation of C/EBPalpha-mediated proliferation arrest and differentiation pathways.

A C/EBP beta isoform recruits the SWI/SNF complex to activate myeloid genes.

The activation of many genes requires the concerted effort of two or more transcription factors. Although C/EBP beta is known to cooperate with Myb to induce transcription of the granulocyte-specific mim-1 gene, the molecular mechanism of this cooperativity is undefined. We show that the N terminus of the full-length C/EBP beta isoform, which is essential for induction of the mim-1 gene in chromatin, interacts specifically with the SWI/SNF complex. Grafting this domain onto Myb generates a chimeric activator that recruits SWI/SNF and induces mim-1 transcription in the absence of C/EBP beta. Interaction between C/EBP beta and SWI/SNF is essential for activating a subgroup of resident target genes in chromatin and may represent a major determinant of combinatorial gene regulation in eukaryotes.

Distinct C/EBP functions are required for eosinophil lineage commitment and maturation.

Hematopoietic differentiation involves the commitment of multipotent progenitors to a given lineage, followed by the maturation of the committed cells. To study the transcriptional events controlling these processes, we have investigated the role of C/EBP proteins in lineage choice of multipotent hematopoietic progenitors (MEPs) transformed by the E26 virus. We found that forced expression of either the alpha or beta isoforms of C/EBP in MEPs induced eosinophil differentiation and that in addition, C/EBPbeta could induce myeloid differentiation. Conversely, dominant-negative versions of C/EBPbeta inhibited myeloid differentiation. C/EBP-induced eosinophil differentiation could be separated into two distinct events, lineage commitment and maturation. Thus, eosinophils induced by transactivation-deficient C/EBPbeta alleles were found to be blocked in their maturation, whereas those expressing wild-type C/EBP proteins were not. Likewise, a 1-day activation of a conditional C/EBPbeta allele in multipotent progenitors led to the formation of immature eosinophils, whereas sustained activation produced mature eosinophils. These results show that C/EBP can induce both myeloid and eosinophil lineage commitment and that transactivation independent and dependent C/EBP functions are required during eosinophil lineage commitment and maturation, respectively.

The homeobox gene GBX2, a target of the myb oncogene, mediates autocrine growth and monocyte differentiation.

The homeobox gene GBX2 was identified as a target gene of the v-Myb oncoprotein encoded by the avian myeloblastosis virus (AMV). GBX2 activation by c-Myb requires signal transduction emanating from the cell surface while the leukemogenic AMV v-Myb constitutively induces the GBX2 gene. Mutations in the DNA binding domain of AMV-Myb render it independent of signaling events and concomitantly abrogate the collaboration between Myb and CCAAT Enhancer Binding Proteins (C/EBP), which are involved in granulocyte differentiation. Ectopic expression of GBX2 in growth factor-dependent myeloblasts induces monocytic features and independence from exogenous cytokines, reflecting distinct features of AMV-transformed cells. Our results suggest that Myb or factors it interacts with contribute to hematopoietic lineage choice and differentiation in a signal transduction-dependent fashion.

B-Myb, a repressed trans-activating protein.

B-Myb belongs to a family of related transcription factors which share a unique DNA binding domain. B-Myb plays an important role in regulation of the cell cycle. Its expression is upregulated by the human papilloma virus HPV16 E7 oncoprotein. Overexpression of B-Myb can bypass p53-mediated cell cycle arrest. The founding member of the myb gene family, c-Myb, and A-Myb are involved in hematopoiesis and neurogenesis, respectively, and are both activators of gene transcription. Whether B-Myb is a transactivator or a repressor, however, has remained a matter of discussion. We reviewed the transactivation potential of B-Myb in yeast, taking advantage of the fact that inducible gene activation is an evolutionarily conserved process. By mutational analysis we localized a conserved activation domain in B-Myb. In vertebrate cells the transactivation potential of B-Myb is concealed by the C-terminal part of the protein. We show that the cell cycle regulators cyclin A and cyclin E activate B-Myb by eradicating the inhibition mediated by its carboxy-terminus. Our data suggest that in vertebrates the trans-activating function of B-Myb is regulated during the cell cycle and link Myb functions to cell cycle progression.

Regulation of C/EBP beta/NF-M activity by kinase oncogenes.

CAAT Enhancer Binding proteins (C/EBP) belong to a family of transcription factors which are implicated in a number of developmental and growth regulatory processes. One member of this family known as C/EBP beta (called NF-M in the chicken system) is particularly important in myelomonocytic cells because it is targeted by kinases and collaborates with the Myb oncoprotein to induce the expression of myeloid specific genes. Experiments dissecting the structure of NF-M suggest that it is a repressed transcription factor. Using the yeast two-hybrid system we showed that a negative regulatory domain masks the transactivation domain. Examination of NF-M mutants suggests that kinase (proto-) oncogenes uncover the concealed transcriptional activity by phosphorylation of the negative regulatory domain.

NF-M (chicken C/EBP beta) induces eosinophilic differentiation and apoptosis in a hematopoietic progenitor cell line.

CAAT/enhancer binding proteins (C/EBPs) are transcriptional activators implicated in the differentiation processes of various cell lineages. We have shown earlier that NF-M, the chicken homolog of C/EBP beta, is specifically expressed in myelomonocytic and eosinophilic cells of the hematopoietic system. To investigate the role of NF-M in hematopoietic cell lineage commitment, we constructed a conditional form of the protein by fusing it to the hormone binding domain of the human estrogen receptor. This construct was stably expressed in a multipotent progenitor cell line transformed by the Myb-Ets oncoprotein. We report here that both NF-M-dependent promoter constructs and resident genes could be activated by addition of beta-estradiol to the NF-M-estrogen receptor expressing progenitors. At the same time, we observed a down-regulation of progenitor-specific surface markers and the up-regulation of differentiation markers restricted to the eosinophil and myeloid lineages. In addition to the onset of differentiation, cell death was induced with typical apoptotic features. Our results suggest that NF-M plays an important role in commitment along the eosinophil lineage and in the induction of apoptosis.

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.

Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types.

The c-Myb transcription factor regulates the differentiation of immature erythroid, lymphoid, and myeloid cells, although only the latter cells become transformed by the v-myb oncogene. These are also the only cells that express the Myb-regulated gene mim-1, suggesting that Myb requires tissue-specific, cooperating factors to activate such genes. Here, we investigated the tissue-specific regulation of the mim-1 promoter and found that it not only contains binding sites for Myb but also for NF-M, a myeloid-specific transcription factor that probably corresponds to mammalian C/EBP beta. Both types of binding sites were found to be required for full activity of the promoter. Remarkably, ectopic coexpression of Myb and NF-M proteins in erythroid cells or fibroblasts was sufficient to induce endogenous markers of myeloid differentiation, like the mim-1 and lysozyme genes. Our results indicate that c-Myb and NF-M proteins act as a bipartite, combinatorial signal that regulates the expression of myeloid-specific genes, even in heterologous cell types.

The NF-M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells.

Retroviral oncogenes encode nuclear regulators of gene expression or signal transduction molecules, such as protein kinases, which stimulate the activity of cellular transcription factors. Here we describe the cloning of NF-M, a myeloid-specific transcription factor related to C/EBP beta, which is a target of activated protein kinases. NF-M stimulates the expression of the gene encoding cMGF, a myeloid cell-specific growth factor, creating an autocrine growth loop crucial to oncogene transformation of myeloid cells. The NF-M protein bound directly to the cMGF gene promoter and activated its transcription, even in erythroid cells where the promoter is usually inactive. In addition, a truncated, dominant-negative form of NF-M inhibited cMGF expression in macrophages, indicating that NF-M is required for the normal activation of the gene. When multipotent hematopoietic progenitor cells were stimulated to differentiate, NF-M expression was induced at a very early stage, suggesting that the transcription factor plays a role in lineage commitment. The stimulation of transformed myelomonocytic cells or of normal peripheral blood macrophages with kinases or LPS or TPA respectively, led to the rapid redistribution of NF-M protein from the cell bodies to the nucleus, consistent with the notion that NF-M was directly affected by such treatments. Our data indicate that NF-M plays a key role in myelomonocytic differentiation, in signal transduction during macrophage activation and in the development of myelogenous leukemia.

Characterization of the nuclear proteins binding the CACCC element of a glucocorticoid-responsive enhancer in the tyrosine aminotransferase gene.

The nuclear proteins which act synergistically with the glucocorticoid receptor to induce transcription of the tyrosine aminotransferase gene include factors recognizing the CACCC element. We have purified and characterized the proteins from rat liver nuclei which bind to the CACCC motif in the glucocorticoid-inducible enhancer of the gene. Three protein-DNA complexes (C1, C2, and C3) were detected in band-shift assays. The protein component of complex C1 also binds a GC motif (a Sp1 binding site) and is recognized by anti-Sp1 antiserum. The proteins forming complexes C2 and C3 have been purified by DNA-affinity chromatography and their molecular masses (75-80 kDa and 35-40 kDa, respectively) have been determined by ultraviolet cross-linking to radio-labelled DNA and SDS/PAGE. The DNA-affinity-purified C2 and C3 activities do not bind significantly to the GC motif and are not recognized by anti-Sp1 antiserum. Methylation interference analysis indicates that the nucleotides of the CACCC element bound by the C2 and C3 proteins correspond to those of the glucocorticoid-responsive enhancer which are contacted in vivo following glucocorticoid administration. Our data suggest that these proteins contribute to glucocorticoid-induced transcription of the tyrosine aminotransferase gene.

Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation.