The role of HDAC3 in Notch signal transduction

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The Notch signaling pathway plays a pivotal role in the regulation of many cellular processes such as proliferation, stem cell maintenance, differentiation and has been also implicated in carcinogenesis. Post-translational modifications (PTMs) of the cleaved Notch Intracellular Domain (NICD) regulate Notch signaling strength and duration. Our preliminary results revealed that loss-of-function (LoF) of HDAC3 downregulates Notch target genes and destabilizes the NICD-protein. So far, this positive role for HDAC3 in Notch signaling is contrary to expectations, since HDACs are generally known to be corepressors. In aim-1 we want to identify the key lysines within the NICD protein regulated by HDAC3 using an already established acetylation/deacetylation assay coupled to mass spectrometry. In addition, NICD KR-mutants will be generated and a specific anti-acetyl-NICD antibody will be raised. Using these tools, we will elucidate in aim-2 the molecular mechanisms in regard to other PTMs, in particular ubiquitination. Phosphorylation and ubiquitination take place in the C-terminal destabilizing PEST-domain of the NICD, whereas acetylation occurs in the N-terminal region of the NICD. We want to elucidate whether acetylation, phosphorylation and ubiquitination are interdependent and determine if the acetylation of the NICD protein is a prerequisite for its ubiquitination. One scenario is that acetylated NICD forms the platform for E3 ubiquitin ligase Fbw7. In aim-3 we want to focus on HDAC3 function within Notch signal transduction. We will test HDAC3 specificity by analyzing all other class-I HDACs in acetylation-assays and by shRNA-mediated knockdown experiments followed by RT-qPCR analysis of Notch target genes. Since HDAC3 was described to be a histone deacetylase, we will investigate the HDAC3 function in the regulation of histone acetylation at Notch target genes using chromatin-immunoprecipitation experiments. The functional significance of NICD acetylation will be tested in vivo in an already established Notch-dependent neurogenesis assay in zebrafish embryos (collaboration with Drs. F. Oswald and S. Just, Ulm). The proposed project will elucidate the role of HDAC3 in Notch signal transduction. The major implications are that potentially a low dose (in the nanomolar range) of HDAC-inhibitor could have a major impact on Notch1-mediated diseases such as leukemia.