Hematopoiesis is the process through which mature blood cells of distinct lineages are produced from pluripotent stem cells. Like many differentiation systems, transcription factors that activate lineage-specific genes are essential to the commitment and development of specific hematopoietic lineages (57
). One such transcription factor essential for commitment to and development of the granulocytic lineage is CCAAT/enhancer binding protein α (C/EBPα).
C/EBPα is a basic leucine zipper protein (bZIP) that forms homodimers or heterodimers with other C/EBP proteins to activate the transcription of target genes (reviewed in references 34
). In addition to granulocytes, C/EBPα is highly expressed in many differentiated cell types such as hepatocytes and adipocytes. A number of reports indicate that C/EBPα has a crucial role in regulating the balance between cell proliferation and differentiation, which is crucial for lineage commitment of any cell type. First, C/EBPα has been shown to cause growth arrest in adipocytes as well as in hepatocytes (18
). C/EBPα initiates growth arrest through its ability to stabilize the expression of the cyclin-activating kinase inhibitor (CAK), p21, as well as through disruption of E2F transcriptional complexes during the G1
phase of the cell cycle (64
). Additionally, expression of antisense C/EBPα RNA prevents both growth arrest and terminal differentiation of 3T3 L1 adipocytes (36
). Finally, C/EBPα−/−
mice exhibited improper development of lung and liver with increased hepatocyte proliferation, supporting the role of C/EBPα in the differentiation of these tissues (17
). A striking feature of the C/EBPα−/−
mice was the complete absence of any mature neutrophils (73
). This result demonstrates the indispensability of C/EBPα for the granulocytic differentiation pathway.
mice exhibit a block in granulocytic differentiation that is early in the developmental pathway. Fluorescence-activated cell sorter analysis of embryonic and newborn animals demonstrated no detectable expression of the granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) receptors, and mRNA levels for both were drastically reduced (73
). Consequently, C/EBPα−/−
mice exhibit a reduced response to those respective cytokines. These results suggested that much of the C/EBPα−/−
phenotype could be attributed to the decrease in the levels of both the G-CSF receptor and the IL-6 receptor and their respective signaling pathways. However, neither G-CSF receptor−/−
mice nor IL-6−/−
mice exhibit serious defects in granulocytic differentiation (39
). Therefore, it was hypothesized that a cross between G-CSF receptor−/−
mice and IL-6−/−
mice would mimic the phenotype observed with the C/EBPα−/−
mice alone. However, this cross did not result in a severe defect in granulocytic differentiation, which indicates that there must be additional C/EBPα target genes in myeloid progenitor cells necessary for mature neutrophil development.
c-Myc is a basic helix-loop-helix (HLH) leucine zipper protein that dimerizes with its partner Max to activate gene transcription through consensus E-box elements located on the promoters of certain genes (6
). Myc was discovered to be an oncogene causing leukemia in birds and inducing in vitro transformation of avian myeloid cells (56
). Dysregulated c-Myc expression has been implicated in the development of lymphoid malignancies and other tumors (13
), as well as in the induction of genomic instability (15
). This demonstrates the importance of appropriate c-Myc regulation and the role of c-Myc for proper maintenance of the cell cycle (46
). c-Myc is expressed in proliferating cells, and both c-Myc mRNA and protein levels are virtually undetectable in terminally differentiated cells (21
). These studies indicate that down-regulation of c-Myc is a critical event for a cell to commit to a differentiation pathway (12
). This is particularly true in differentiation of myeloid cells (25
), and treatment of myeloid cell lines with antisense oligonucleotides that inhibit c-Myc expression induces myeloid cell differentiation (26
). Failure to down-regulate c-Myc in transgenic mice can lead to myeloid leukemia, a condition characterized by a block in differentiation (16
As proliferation and differentiation are mutually exclusive, c-Myc, a proliferative factor, and C/EBPα, a differentiation factor, act in opposition to each other. First, c-Myc and C/EBPα act reciprocally during adipogenesis (18
). Overexpression of c-Myc blocks the ability of adipoblasts to terminally differentiate, while the introduction of C/EBPα overcomes this c-Myc-induced differentiation block (37
). Next, c-Myc can activate cyclin E complexes, which results in increased active E2F transcription complexes. This leads cells into the G1
/S transition of the cell cycle. Moreover, expression of c-Myc can overcome growth arrest imposed by the p21, p27, and p16 cyclin-dependent kinase (cdk) inhibitor proteins (61
). In contrast, C/EBPα achieves growth arrest through increased p21 CAK inhibitor protein, which ultimately results in decreased numbers of active E2F transcription complexes (65
). Most importantly for their opposing effects in cells, c-Myc and C/EBPα can reciprocally regulate the expression of their respective genes. c-Myc has previously been shown to negatively regulate C/EBPα expression and block C/EBPα transactivation function (2
). However, the effects of C/EBPα on c-Myc regulation have not been investigated.
In order to identify C/EBPα targets in myeloid cells, we performed both representational difference analysis (RDA) and oligonucleotide array screening. From both of these independent screens, we identified c-Myc as a target gene of C/EBPα. We show that C/EBPα can directly down-regulate human c-Myc promoter activity. Moreover, we have identified a consensus E2F site located between the P1 and P2 c-Myc promoter elements as being critical for C/EBPα negative regulation. This is the first investigation to show that C/EBPα directly affects c-Myc expression levels and thus further elucidates the mechanisms through which C/EBPα induces cellular differentiation.