Repression mechanisms of the Notch-signalling pathway in Drosophila

在高等真核生物中，缺口信号支配着细胞间的通讯。由于人类的几种疾病都与Notch信号的缺陷有关，因此了解Notch信号的调节是至关重要的。命名受体Notch的激活会导致细胞内Notch结构域(ICN)的释放，ICN在细胞核中作为一个大的激活复合体中的辅助激活因子来驱动Notch靶基因的表达，并引导细胞反应。确定了激活剂络合物的晶体结构。根据这一点，核心成分是转录因子CSL，它招募了除了ICN之外的几个辅助激活因子。在没有Notch信号的情况下，CSL与几个辅阻遏子一起组装了一个抑制物复合体，使Notch靶基因沉默。本项目的目的是在模型系统果蝇中对Notch抑制物复合体的结构特征进行描述。在这里，CSL同系物SU(H)组装了一个阻遏复合体，包含无毛(H)蛋白和额外的普通辅阻遏子。通过其C末端结构域(CTD)，SU(H)仅与跨越H的NT-box的16个氨基酸结合。在H-NT中，我们确定了SU(H)结合所必需的单一氨基酸。H对SU(H)结合的亲和力与ICN的亲和力一致。然而，ICN在体外能够将H从阻遏物复合体中置换出来，可能会引起SU(H)的构象变化。H-SU(H)抑制物复合体的结构将与瑞德·科瓦尔(辛辛那提大学)合作进行分析。首先，原始数据表明H-NT结构域与SU(H)的CTD之间存在疏水接触。基于这些数据，SU(H)应通过体外诱变来影响H结合。首先，已经取得了成果，而且非常有希望。新的SU(H)突变体的结合特性将通过生化和体外检测进行测试，并通过体内方法研究其生物活性。该提案的重点是允许在体内研究分别在体外产生的H和SU(H)突变体的基因工程。在第一步中，用attP靶序列分别替换野生型H和SU(H)基因座。AttP位点允许任何序列的位置特异性整合--这里是突变基因拷贝--随后可以在正常情况下直接在生物体中进行研究。因此，可以避免由于给定基因的过度表达而导致的典型负面影响。通过同源重组，我们已经在H基因座创建了attP-Fly线，并建立了原理证明。现在我们可以开始产生和研究第一个工程H突变体。随后将在SU(H)轨道进行工程设计。我们的目标是最终揭开Notch信号抑制的细节。

Notch signalling governs intercellular communication in higher eukaryotes. Since several human diseases are linked to defects in Notch signalling, understanding of its regulation is of fundamental importance. Activation of the name giving receptor Notch results in the release of the intracellular Notch domain (ICN), which in the nucleus acts as a coactivator within a large activation complex to drive expression of Notch target genes and to guide the cellular response. The crystal structure of the activator complex has been resolved. According to this, the central component is the transcription factor CSL which recruits several coactivators apart from ICN. In the absence of Notch signals, CSL together with several corepressors assembles a repressor complex that silences Notch target genes. The aim of this project is a structural characterization of the Notch repressor complex in the model system Drosophila. Here, the CSL-homologue Su(H) assembles a repressor complex containing the protein Hairless (H) plus additional general corepressors. With its C-terminal domain (CTD), Su(H) binds the just 16 amino acids spanning NT-box of H. In H-NT we identified a single amino acid essential for Su(H) binding. The affinity of H for Su(H) binding matches that of ICN. Nevertheless, ICN is able to displace H from the repressor complex in vitro, presumably provoking a conformational change of Su(H). The structure of the H-Su(H) repressor complex shall be analysed in collaboration with Rhett Kovall (University of Cincinnati). First raw data indicate a hydrophobic contact between the H-NT domain and the CTD of Su(H). Based on these data, Su(H) shall be changed by in vitro mutagenesis to affect H binding. First results have been achieved and are very promising. The binding properties of the new Su(H) mutants will be tested biochemically and by in vitro assays, and their biological activity investigated by in vivo approaches. The focus of to the proposal is 'gene-engineering' allowing the in vivo studies of the H and Su(H) mutants, respectively, generated in vitro. In a first step, the wild type H and Su(H) locus is each replaced by an attP target sequence. The attP site permits position specific integration of any sequence - here the mutant gene copy -, which can be subsequently studied directly in the organism within the normal context. Hence the typical negative effects resulting from the overexpression of the given gene can be avoided. Via homologous recombination, we have already created the attP-fly line at the H locus and established the proof-of-principle. Now we can start to generate and study the first engineered H mutants. Engineering at the Su(H) locus shall follow. Our goal is to eventually unravel the details of Notch signal repression.