C/EBPalphap30, a myeloid leukemia oncoprotein, limits G-CSF receptor expression but not terminal granulopoiesis via site-selective inhibition of C/EBP DNA binding.
Heterozygous mutations of the CEBPA gene are present in 5% of acute myeloid leukemia (AML) cases and often lead to the expression of an N-terminally truncated, 30 kDa isoform, C/EBPalphap30, from an internal translation start site. We have assessed the effect of C/EBPalphap30 on granulopoiesis utilizing C/EBPalphap30-ER, containing the estradiol receptor ligand-binding domain. In contrast to C/EBPalpha-ER, C/EBPalphap30-ER did not induce 32Dcl3 myeloid cell differentiation in IL-3. However, both isoforms, when expressed at high levels, were capable of inhibiting E2F activity in 32Dcl3 cells and of slowing their G1 to S progression. C/EBPalphap30 repressed expression of the endogenous G-CSF receptor several-fold. To facilitate investigation of the effect of C/EBPalphap30-ER on granulopoiesis downstream of G-CSF signalling, we coexpressed exogenous G-CSF receptor. C/EBPalphap30-ER/GR cells expressed several granulocytic markers in G-CSF and demonstrated nuclear maturation. Rat C/EBPalpha-ER and C/EBPalphap30-ER, expressed in 293T cells, bound the C/EBP site from the NE gene with similar affinity, as did human C/EBPalpha and C/EBPalphap30. In contrast, C/EBPalphap30 bound the C/EBP sites in the PU.1 or GR gene with 3-6-fold reduced affinity. Thus, the selective inhibition of GR expression by C/EBPalphap30-ER is due in part to its variable affinity for C/EBP sites. Variation in affinity for selected cis elements among isoforms may affect the biology of basic region-leucine zipper (bZIP) proteins.
Cleaves
Rebecca
R
Division of Pediatric Oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA.
Wang
Qian-fei
QF
Friedman
Alan D
AD
D000818:Animals; D001483:Base Sequence; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016193:G1 Phase; D006098:Granulocytes / Q000166:cytology*; D006801:Humans; D011485:Protein Binding; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D022763:CCAAT-Enhancer-Binding Protein-alpha; D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D014157:Transcription Factors; D004247:DNA