C/EBPalphap30, a myeloid leukemia oncoprotein, limits G-CSF receptor expression but not terminal granulopoiesis via site-selective inhibition of C/EBP DNA binding.

Abstract

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.