Cross-talk between regulators of myeloid development: C/EBPalpha binds and activates the promoter of the PU.1 gene.
CCAAT/enhancer-binding protein (C/EBP)alpha and PU.1 are required for myelopoiesis. Examination of the murine PU.1 promoter revealed several potential C/EBP-binding sites. Gel-shift assay demonstrated that C/EBPalpha expressed in 293T cells bound the site centered at -68 most potently. C/EBPalpha from 32D cl3 myeloid cell nuclear extracts also bound this site strongly, and endogenous C/EBPbeta did so to a lesser extent, whereas these C/EBP isoforms bound the neutrophil elastase promoter with equal affinity. The -68 site in the murine PU.1 promoter is conserved in the human PU.1 promoter. Mutation of the -68 C/EBP-binding site in a -85/+152 promoter segment linked to the luciferase cDNA reduced promoter activity fourfold in 293T cells in the presence of cotransfected C/EBPalpha and twofold in 32D cl3 myeloid cells. Induction of endogenous PU.1 RNA by C/EBPalpha-estradiol receptor (ER) in the presence of cycloheximide is obviated by mutation of the C/EBPalpha DNA-binding domain, and chromosomal immunoprecipitation demonstrated specific interaction of C/EBPalpha and C/EBPalpha-ER with the PU.1 promoter. Finally, PU.1 RNA is reduced several-fold in immortalized C/EBPalpha (-/-) compared with (+/-) cells. Together, these findings indicate that C/EBPalpha binds and activates the endogenous PU.1 gene in myeloid cells. Induction of PU.1 by C/EBPalpha may account for increased levels of PU.1 in myeloid as compared with B lymphoid cells and in this way, may contribute to the specification of myeloid progenitors.
Kummalue
Tanawan
T
Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland 21231, USA.
Friedman
Alan D
AD
D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000172:deficiency / Q000378:metabolism*; D002467:Cell Nucleus; D002478:Cells, Cultured; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D022423:Myeloid Cells / Q000166:cytology / Q000187:drug effects*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D000099232:Proto-Oncogene Protein Spi-1
D022763:CCAAT-Enhancer-Binding Protein-alpha; D008156:Luciferases; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1