Acetylation of C/EBPε Is Functionally Important During Neutrophil Development

Abstract 215

Dysregulation of myeloid differentiation can result in the development of a variety of pathological conditions ranging from bone marrow failure to myelodysplastic syndromes and leukemia. Neutrophil development is tightly regulated by key transcription factors including CCAAT enhancer-binding protein-alpha (C/EBPα) and CCAAT enhancer-binding protein-epsilon (C/EBPε). In recent years it has become clear that the expression and function of such proteins is regulated by post-translational modifications. Here, we have investigated the regulation and functional role of C/EBPε acetylation. Our results demonstrate that C/EBPε is indeed acetylated and that this can be increased by the lysine acetyltransferases (KAT) TIP60 and p300, as well as the sirtuin 1(SIRT1) inhibitor nicotinamide (NAM). In agreement with this, acetylation was decreased upon co-transfection of SIRT1. Despite normal expression levels and the capacity to form homo- and heterodimers, the C/EBPε-lysine dead mutant (C/EBPε K15xR) was transcriptionally inactive in luciferase reporter assays, suggesting that acetylation of C/EBPε is functionally important. Moreover, co-transfection of SIRT1 inhibited C/EBPε transcriptional activation. In order to investigate the functional relevance of acetylaton of C/EBPε, we retrovirally transduced CD34+ hematopoietic progenitors with C/EBPε or C/EBPε K15xR and differentiated sorted progenitors towards mature neutrophils. We observed a significant decrease in the percentage of mature neutrophils after transduction with C/EBPε K15xR compared to C/EBPε or control cells. Cytospin analysis demonstrated an immature phenotype of the C/EBPε K15xR-transduced cells, suggesting a differentiation block at the promyelocytic stage.

In order to determine the functionally important acetylated lysine residues, we performed mass spectometry and identified four C/EBPε acetylation sites including two sites in the repression domain and one site in the basic region, which includes the DNA-binding domain. To investigate the functional role of acetylation of these lysine residues, we designed specific C/EBPε lysine mutants. Utilizing the previously described ectopic expression system, we observed reduced levels of total C/EBPε acetylation upon transfection of two specific lysine mutants (K121R and K198R). Acetylation of these specific residues was confirmed by performing add-back experiments in the C/EBPε K15xR background. Furthermore, upon transfection of C/EBPε K121R and C/EBPε K198R in a luciferase reporter assay, we observed reduced transcriptional activation by C/EBPε, suggesting that acetylation of lysine 121 and lysine 198 is important for C/EBPε function. Currently we are investigating the functional role of acetylation of these specific lysine residues during neutrophil differentiation.

There is increasing knowledge concerning the role of epigenetic modifications in the development of myeloid malignancies. However, the specific role of acetylation of non-histone proteins, including key-transcription factors involved in myelopoiesis, remains largely unknown. We demonstrate, for the first time, the functional importance of C/EBPε acetylation during neutrophil differentiation. Together our data provide new insights in the regulation of both normal and aberrant myeloid differentiation.