title: PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene.
abstract: Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha gene. Here, we demonstrate that the human GM-CSF receptor alpha promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor alpha promoter contains an important functional site between positions -53 and -41 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU.1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU.1 site between positions -70 and -54 is involved in positive-negative regulation of the GM-CSF receptor alpha promoter activity. C/EBP alpha is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU.1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor alpha promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU.1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor alpha promoter PU.1 site. This is the first demonstration of a specific interaction with PU.1 on a myeloid PU.1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU.1 to extracts from certain nonmyeloid cell types which do not express PU.1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU-SF complex binds to PU.1 sites found on a number of myeloid promoters, and its formation requires an intact PU.1 site adjacent to a single-stranded region. Expression of PU.1 in nonmyeloid cells can activate the GM-CSF receptor alpha promoter. Deletion of the amino-terminal region of PU.1 results in a failure to form the PU-SF complex and in a concomitant loss of transactivation, suggesting that formation of the PU-SF complex is of functional importance for the activity of the GM-CSF receptor alpha promoter. Finally, we demonstrate that C/EBP alpha can also active the GM-CSF receptor alpha promoter in nonmyeloid cells. These results suggest that PU.1 and C/EBP alpha direct the cell-type-specific expression of GM-CSF receptor alpha, further establish the role of PU.1 as a key regulator of hematopoiesis, and point to C/EBP alpha as an additional important factor in this process.
authors.affiliation: Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02115, USA.
authors.forename: S
authors.initials: S
authors.lastname: Hohaus
authors.forename: M S
authors.initials: MS
authors.lastname: Petrovick
authors.forename: M T
authors.initials: MT
authors.lastname: Voso
authors.forename: Z
authors.initials: Z
authors.lastname: Sun
authors.forename: D E
authors.initials: DE
authors.lastname: Zhang
authors.forename: D G
authors.initials: DG
authors.lastname: Tenen
mesh_terms: D000595:Amino Acid Sequence; D001402:B-Lymphocytes; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D006410:Hematopoiesis / Q000235:genetics; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009000:Monocytes; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D015682:Retroviridae Proteins, Oncogenic; D013601:T-Lymphocytes; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics*
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; C062022:v-Spi-1 protein, Friend spleen focus-forming virus; D004247:DNA