The role of HDAC3 in Notch signal transduction

Notch信号通路在许多细胞过程如增殖、干细胞维持、分化的调节中起着关键作用，并且还与癌发生有关。切割的Notch胞内结构域（NICD）的翻译后修饰（PTM）调节Notch信号传导强度和持续时间。我们的初步结果表明，HDAC 3的功能丧失（LoF）下调Notch靶基因并使NICD蛋白不稳定。到目前为止，HDAC 3在Notch信号传导中的这种积极作用与预期相反，因为HDAC通常被认为是辅阻遏物。在aim-1中，我们希望使用已经建立的乙酰化/脱乙酰化测定结合质谱法来鉴定由HDAC 3调节的NICD蛋白内的关键赖氨酸。此外，将产生NICD KR突变体，并产生特异性抗乙酰基-NICD抗体。使用这些工具，我们将阐明在目标2的分子机制，在其他PTM，特别是泛素化。磷酸化和泛素化发生在NICD的C-末端不稳定的PEST结构域，而乙酰化发生在NICD的N-末端区域。我们希望阐明乙酰化、磷酸化和泛素化是否相互依赖，并确定NICD蛋白的乙酰化是否是其泛素化的先决条件。一种情况是乙酰化的NICD形成E3泛素连接酶Fbw 7的平台。在aim-3中，我们希望关注Notch信号转导中的HDAC 3功能。我们将通过在乙酰化测定中分析所有其他I类HDAC和通过shRNA介导的敲低实验，然后通过Notch靶基因的RT-qPCR分析来测试HDAC 3特异性。由于HDAC 3被描述为组蛋白去乙酰化酶，我们将使用染色质免疫沉淀实验研究HDAC 3在Notch靶基因处的组蛋白乙酰化调节中的功能。NICD乙酰化的功能意义将在斑马鱼胚胎中已经建立的Notch依赖性神经发生测定中进行体内测试（与F。Oswald和S.只是，乌尔姆）。该项目将阐明HDAC 3在Notch信号转导中的作用。主要的影响是，低剂量（纳摩尔范围内）的HDAC抑制剂可能对Notch 1介导的疾病（如白血病）产生重大影响。

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.