C/EBPalpha binds and activates the PU.1 distal enhancer to induce monocyte lineage commitment.
The PU.1 gene contains a 237-base pair distal enhancer located 14 kilobases upstream of its promoter. We have identified 2 sites within the PU.1 enhancer that strongly bind C/EBPalpha in a gel shift assay, and interaction with endogenous C/EBPalpha was confirmed by chromatin immunoprecipitation. Mutation of these DNA elements reduced activity of a distal enhancer-promoter construct 2- or 5-fold in a myeloid cell line, while mutation of a weaker C/EBPalpha-binding site located in the promoter minimally reduced activity in this context. These findings strengthen the link between C/EBPalpha and PU.1 expression. Reduction of C/EBPalpha activity in cases of acute myeloid leukemia may therefore contribute to transformation by reducing PU.1 levels. In addition, induction of PU.1 by C/EBPalpha during normal hematopoiesis may contribute to stem cell commitment to the myeloid lineages and further commitment to monopoiesis. Consistent with a requirement for C/EBPalpha induction of PU.1 during myeloid development, we demonstrate that C/EBPalpha induces monocytic development when expressed in PU.1(+/+), PU.1(+/-), or PU.1(+/kd) marrow myeloid progenitors but induces granulocyte lineage commitment in PU.1(kd/kd) cells lacking the PU.1 distal enhancer and does not induce either lineage in PU.1(-/-) cells.
Division of Pediatric Oncology, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD 21231, USA.
Christine
C
Yeamans
Dehua
D
Wang
Ido
I
Paz-Priel
Bruce E
BE
Torbett
Daniel G
DG
Tenen
Alan D
AD
Friedman
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism* / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics; D002478:Cells, Cultured; D004742:Enhancer Elements, Genetic; D006410:Hematopoiesis / Q000235:genetics; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D009000:Monocytes / Q000166:cytology*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*; D020298:U937 Cells; D000099232:Proto-Oncogene Protein Spi-1
D022763:CCAAT-Enhancer-Binding Protein-alpha; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1