| Přispěvatelé: |
Cornwall, Gail, Coué, Martine, Pfarr, Curt M., Schneider, Brandt, Williams, Simon |
| Popis: |
CCAAT/enhancer binding protein epsilon (C/EBPε) is a myeloid-specific transcription factor that is critical for terminal differentiation of neutrophils and eosinophils. C/EBPε null mice are immunodeficient and display morphologically and functionally abnormal neutrophils with hyposegmented nuclei, absent specific granules and reduced chemotaxis and bactericidal activity in response to inflammatory stimuli. C/EBPε null mice also display myeloproliferation and eventually develop myelodysplasia, conditions that are commonly seen in certain forms of leukemia. In humans, mutations in the C/EBPε gene are associated with neutrophil-specific granule deficiency, a rare disorder that is characterized by functionally defective neutrophils and consequent immunodeficiency. Furthermore, altered C/EBPε activity has been implicated in acute promyelocytic leukemia (APL). Therefore, a complete understanding of the molecular mechanisms that regulate C/EBPε activity during neutrophil differentiation is clinically relevant and can aid in the development of novel therapies for these blood disorders. The purpose of this study was to gain insight into the regulation of C/EBPε transcriptional activity by post-translational modification with Small ubiquitin-like modifier (SUMO) during neutrophil differentiation. We first examined whether C/EBPε is a target for SUMOylation using all trans-retinoic acid (ATRA) stimulated NB4 APL cells as a model for neutrophil differentiation. Initially, using various biochemical approaches that inhibit the enzymes of SUMOylation pathway, we provided the first evidence that C/EBPε is a target for SUMO-attachment during neutrophil differentiation. Next, using RNA-interference to silence the expression of SUMO isoforms, we show that C/EBPε is primarily targeted by SUMO2/3 isoforms. To further investigate the role of SUMOylation in regulating C/EBPε activity, we examined the consequences of mutating the SUMO-acceptor lysine121 to alanine in C/EBPε. Non-SUMOylatable forms of C/EBPε were less active than the parental protein in transcription assays. Furthermore, stable expression of SUMOylatable and non-SUMOylatable C/EBPε in U937 promyelocytes showed that the non-SUMOylatable protein was significantly less efficient in activating expression of the bactericidal/permeability-increasing protein (BPI) and CD11b genes, two important markers of neutrophil differentiation. These data show that SUMOylation modulated the ability of C/EBPε to activate target genes and provides the first evidence for a role of SUMOylation in neutrophil differentiation. |
