Multiple Functional Domains of AML1: PU.1 and C/EBPα Synergize with Different Regions of AML1

In order to understand the mechanisms that control monocytic commitment and differentiation, we have investigated the tissue-specific regulation of the human macrophage colony-stimulating factor (M-CSF) receptor. We have previously identified three factors required for M-CSF receptor transcription in monocytic cell lines, PU.1, C/EBPα (CCAAT/enhancer-binding protein alpha), and AML1, and demonstrated that mutations in any of the three DNA-binding sites decreases promoter activity significantly in transient transfection studies (77–79). In addition, PU.1 transactivates the M-CSF receptor promoter, and although C/EBPα has little transactivation potential alone, it synergizes with AML1B to increase the activity of the promoter an average of 90-fold (78, 79). All three of these transcription factors play important roles in hematopoiesis. AML1 (also known as CBFα2 and PEBP2αB) contains a domain that is highly similar to the DNA-binding domain of the Drosophila runt transcription factor, which mediates both DNA-binding and heterodimerization abilities (25). The heterodimerization partner of AML1, CBFβ, does not bind DNA directly but increases the affinity of AML1 for DNA (37, 51, 75). In addition to the M-CSF receptor, the target genes of the AML1-CBFβ heterodimer include granulocyte-macrophage colony-stimulating factor (GM-CSF), T-cell receptor (TCR) subunits, interleukin-3, osteocalcin, neutrophil elastase, and myeloperoxidase (2, 3, 6, 15, 18, 21, 50, 67, 70). In several cases AML1 functions in concert with neighboring factors. For example, AML1 binds cooperatively with another member of the ets family, Ets-1, to the TCRα, TCRβ, and Moloney murine leukemia virus enhancers (18, 67). AML1 exhibits functional synergy with c-Myb in the absence of cooperative binding in the context of the TCRδ and myeloperoxidase enhancers (6, 21). Both AML1 and CBFβ are frequently involved in genetic rearrangements identified in human leukemias (12, 19, 35, 40, 47–49). Furthermore, mice which have homozygous disruptions of either gene, or are heterozygotes containing either the AML/ETO or CBFB/MYH11 fusion genes, have strikingly similar phenotypes. All die in midgestation, exhibit multiple hemorrhages in the central nervous system, and have severely impaired hematopoiesis (7, 53, 61, 73, 74, 76). These data support the theory that AML1 function is critical for normal hematopoietic development. C/EBPα, a basic region leucine zipper (bZip) transcription factor (31, 32), regulates not only a variety of hepatocyte and adipocyte genes which are important for energy homeostasis but several myeloid-specific genes as well (9, 10, 16, 17, 22, 24, 50). For example, in addition to the M-CSF receptor promoter, C/EBPα has also been shown to regulate the G-CSF receptor and GM-CSF receptor α promoters (23, 66). Mice with a homozygous disruption of the C/EBPα gene die at birth from hypoglycemia (14, 72) and exhibit hematopoietic defects as well. Analysis of the fetal and newborn hematopoietic tissues revealed a profound absence of mature neutrophils. In addition, there were no neutrophils observed after transplantation of the fetal liver into an irradiated recipient, implying that the block in neutrophil development was intrinsic to the cell and not a defect in the environment (80). Therefore, it is clear that C/EBPα plays a critical role in normal granulocyte development. PU.1, the product of the spi-1 oncogene and a member of the ets family, is upregulated during hematopoietic development and is specifically expressed in myeloid and B cells (8, 29, 55, 59, 71). The pivotal role that PU.1 plays in hematopoietic differentiation is established by the following observations. There are a number of genes that are regulated by PU.1 in both myeloid and B-cell lineages, including those encoding CSF receptors and immunoglobulin subunits (45, 57, 64, 69). Overexpression of PU.1 early in erythroid development blocks erythroblast differentiation (62), and addition of PU.1-binding oligonucleotides to human CD34+ bone marrow cells decreases in vitro colony formation (71). In addition, mice with a disruption in both alleles of the PU.1 locus die in utero (63) or shortly after birth (36) and exhibit major defects in hematopoiesis, including a block in myeloid development. The DNA-binding ets domain shows sequence similarity with other members of the ets family and is contained within amino acids 171 to 267 of the C terminus (29). The activation domain of PU.1 is located within the N terminus and consists of several regions rich in either acidic amino acids or glutamines and a region from amino acids 118 to 160 which has a high number of prolines, glutamic acids, serines, and threonines (PEST domain) (28). PU.1 has been shown to interact with TATA-binding protein (TBP) and the retinoblastoma protein in vitro, requiring amino acids 1 to 75 (20). There are multiple examples where PU.1 functions in concert with other transcription factors, including NF-IL6β (C/EBPδ) (44) and NF-EM5/PIP (11, 56, 58), c-Myb and C/EBPα (50), c-Fos and c-Jun (5, 56), and Ets-1 (13). We are interested in determining the events that control myeloid differentiation so that we can better understand the aberrant differentiation that is exhibited in the leukemic state. For example, it is not clear how the fusion gene, AML/ETO, and other genomic abnormalities associated with myeloid leukemia contribute to the changes in differentiation and proliferation of the myeloid lineage. Alternative theories include inhibition of normal AML1 function (15, 27, 38) or increased activation by AML1 (60), or even direct activation by AML/ETO itself (39), resulting in the dysregulation of genes such as those encoding GM-CSF, the M-CSF receptor, or Bcl-2. Therefore, we have investigated the mechanism by which the transcription factors regulating the M-CSF receptor promoter interact in an effort to reveal the next layer of complexity in myeloid-specific transcriptional activation. Here we show that, in addition to C/EBPα, AML1B interacts with PU.1 to synergistically activate the M-CSF receptor promoter but requires different regions contained within the C terminus for each function.