Abstract
CCAAT enhancer binding protein alpha (C/EBPα) has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and binds to multiple myeloid-specific gene promoters at different stages of maturation. Mice nullizygous for C/EBPα display a selective early block in granulocytic differentiation. Furthermore, C/EBPα mutations leading to loss of C/EBPα function have been demonstrated in a subset of patients with AML. A complete understanding of the regulation of this key factor during myelopoiesis is therefore critical. Studies from our laboratory have demonstrated that lactoferrin (LF) gene expression in the developing neutrophil is dependent on a C/EBP binding site in the LF gene promoter. Using ChIP analysis of LF non-expressing and expressing cells, we demonstrated that C/EBPα binds to the LF promoter in uninduced myeloid cells, which do not express LF. Induction of differentiation, associated with LF expression, correlates with a loss of C/EBPα binding and a gain of C/EBPε binding coincident with increased C/EBPε expression. Hence, we reasoned that post-translational modification(s) of C/EBPα during neutrophil maturation alters its transcriptional activity, thus altering LF gene expression. C/EBPα was recently shown to be post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) within a region of the C/EBPα protein that can negatively affect transcriptional activity. Sumoylation at K159 is thought to hamper transactivation by preventing association of the SWI/SNF chromatin remodeling complex. We demonstrate that the levels of sumoylated C/EBPα decrease upon neutrophil maturation, and that transactivation of a LF promoter reporter is significantly enhanced by a sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, sumoylated C/EBPα binds to the C/EBP site in the LF promoter in uninduced myeloid cells while loss of sumoylation correlates with loss of C/EBPα binding and LF expression. We therefore conclude that sumoylated C/EBPα is associated with the negative regulation of LF in early myeloid cells. Several C/EBP family members, including shorter isoforms, have negative regulatory functions. C/EBP homologous protein (CHOP/C/EBPζ/gadd 153, CHOP) is one such factor. Transient co-transfection analysis of a LF promoter reporter with expression plasmids for C/EBPα and CHOP demonstrated a steep decline in C/EBPα mediated transactivation. This CHOP-mediated decline in transactivation was specific for C/EBPα, as CHOP induced no significant change in transactivation by C/EBPε. Additionally, EMSA analyses using extracts from 293T cells overexpressing C/EBPα, C/EBPε and CHOP demonstrated that increasing levels of CHOP could remove C/EBPα, but not C/EBPε, from the C/EBP site in the LF promoter. Since CHOP levels increase during neutrophil maturation and CHOP does not recognize C/EBP cis elements, we hypothesize that CHOP sequesters C/EBPα and prevents its binding. We propose the following model: Sumoylated C/EBPα binds the LF promoter in early myeloid cells and inhibits its expression. Upon induction of maturation, levels of CHOP increase, inducing heterodimerization with unsumoylated C/EBPα. Because CHOP-C/EBPα heterodimers will not bind canonical C/EBP binding sites, this decreases C/EBPα binding to the LF promoter. This change in binding dynamics allows C/EBPε to bind the LF promoter, resulting in high level LF expression.
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