Reduced Binding of C/EBPα to Myeloid Specific Promoters with Altered Gene Expression in the Presence of PML/RARα.

The hallmark of acute promyelocytic leukemia is the reciprocal translocation t(15;17) resulting in the fusion protein PML/RARα . According to the current concept of the pathogenesis of APL, PML/RARα interferes with RARα target gene expression. Until now, the crucial target genes have not been found. The transcription factor C/EBPα is essential for myeloid development and mutations have been found in 10% of AML patients. No mutations of C/EBPα in APL have been described. However, using the U937 cell line with a Zinc-inducible PML/RARα (U937PR9), a decrease of C/EBPα binding to the G-CSF-R promoter in the presence of PML/RARα was detected in EMSA as well as chromatin immunoprecipitation. This finding indicates, that the function of C/EBPα might be impaired in APL. To test whether the effect is limited to the G-CSF-R promoter, C/EBPα binding to other myeloid promoters was analyzed upon Zn-induction of PML/RARα . In those experiments decreased binding of C/EBPα to other promoters of myeloid specific genes, such as neutrophil elastase, was detected. In contrast, no effect of Zn-induction on the binding of Sp3 to the thymidine-kinase promotor was found. Next, the functional importance of the alteration in binding was demonstrated. In the presence of PML/RARα , decreased G-CSF-R RNA expression as well as G-CSF-R surface expression was found in U937PR9 cells. In addition, the RNA expression of elastase was reduced after Zn-induction. To investigate the effect of PML/RARα on C/EBPα function in t(15;17) positive cells, a knockdown of PML/RARα by lentivirally delivered shRNAs in NB4 cells was used. After lentiviral transduction, a marked decrease of PML/RARα RNA and protein was found. Consistent with the results in U937PR9 cells a 5–6 fold increase of elastase RNA expression and a 2 fold increase in G-CSF-R-RNA was observed after PML/RARα knockdown. Taken together, decreased binding of C/EBPα to promoters of myeloid specific genes was found in the presence of PML/RARα . This alteration in binding was associated with a reduction of RNA expression. Decreasing PML/RARα with siRNA had the opposite effects. Currently, the mechanism for this effect is being investigated.