CCAAT enhancer binding protein alpha (C/EBPα) is the founding member of a family of basic region/leucine zipper (bzip) transcription factors and has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and has been shown to bind to the promoters of multiple myeloid- specific genes at different stages of myeloid maturation. Profound hematopoietic abnormalities have been reported for mice nullizygous for C/EBPα, including a selective early block in the differentiation of granulocytes. Recently mutations in C/EBPα have been demonstrated in a subset of patients with AML presenting normal karyotypes. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform which contributes to loss of C/EBPα function. Since the molecular basis for this observation remains unknown, a complete understanding of the regulation of this key transcription factor during myelopoiesis is critical. C/EBPα was recently shown to be post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) which lies within a region of the C/EBPα protein that can negatively affect transcriptional activity. Sumoylation at K159 in the C/EBPα protein is thought to prevent association of the SWI/SNF chromatin remodeling complex with C/EBPα, thereby hampering transactivation. In order to demonstrate the role of sumoylation in mediating C/EBPα activity, we examined the effect of both the full length (p42) and the dominant negative (p30) isoforms of the C/EBPα protein on myeloid gene expression. We demonstrate that the levels of sumoylated p42C/EBPα decrease upon normal neutrophil maturation, and that transactivation of the myeloid-specific lactoferrin promoter reporter is significantly enhanced by a p42 sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, we show that sumoylated p42C/EBPα binds to the C/EBP site in the LF promoter in immature myeloid cells while loss of sumoylation correlates with loss of p42C/EBPα binding and LF expression in more mature cells. Based on these observations we conclude that sumoylated p42C/EBPα is associated with the negative regulation of LF in early myeloid cells. We show in addition, that p30 C/EBPα can also be sumoylated. In transactivation assays, however, sumoylated p42C/EBPα suppresses LF promoter activity more efficiently than p30C/EBPα in 293 cells. We are now in the process of confirming this finding in leukemic cell lines. This observation leads us to hypothesize that the differential transactivation potential of sumoylated p30 versus the p42 C/EBPα proteins may contribute, in part, to the aberrant activity of C/EBPα in acute leukemias.
Disclosure: No relevant conflicts of interest to declare.