Charakterisierung der Rolle von ETO und AML1/ETO als SHARP interagierende Proteine bei der RBP-Jkappa/SHARP vermittelten Transkriptionskontrolle von Notch-Zielgenen

Abstract

The transmembrane receptor Notch plays an important role during many developmental and differentiation processes. Ligand binding induces a proteolytic cleavage of the intracellular domain (IC) of the Notch receptor. The released IC translocates to the nucleus and associates with the DNA-binding protein RBP-J?. This leads to the recruitment of further coactivators and to the transcriptional activation of notch target genes. In the absence of IC RBP-J? represses transcription through the recruitment of corepressor complexes. SHARP has been identified as a part of aa RBP-J? repressor complex. SHARP directly interacts with RBP-J? and recruits HDACs and HDAC associated proteins to RBP-J&. This study is about the characterisation of new RBP-J?/SHARP associated proteins. The corepressor ETO was found as a SHARP interacting protein in a yeast-two-hybrid-screen. Further experiments identified other members of the ETO-family, the murine ETO-2 and Xenopus laevis ETO (xl), as SHARP interacting proteins. The chromosomal translocation t(8;21) is typical for the human acute myeloid leukaemia (FAB M2). It leads to the generation of the aberrant fusion protein AML1/ETO. ETO and AML1/ETO directly interact with SHARP through NHR1 and NHR2 as shown by GST-pull-down assays and immunoprecipitation experiments. Purification of endogenous RBP-J? complexes by DNA affinity chromatography showed that ETO and AML1/ETO are part of these complexes. In contrast to AML1/ETO ETO enhances HDAC-dependently the SHARP dependent transcriptional repression of Notch target genes. Regarding the results of this work it can be suggested that (A) ETO is a new component of the RBP-J?/SHARP repressor complex and (B) AML1/ETO in contrast to ETO leads to the activation or derepression of Notch target genes. According to this it has been shown for the first time that AML1/ETO is directly involved in the deregulation of Notch target genes. This deregulating effect of AML1/ETO may contribute to the onset of AML.