Molecular mechanisms of Gli protein activation in vertebrate Hedgehog signaling and Hedgehog-driven cancers.

Gli家族的转录因子是Hedgehog(HH)信号通路的最终执行者，Hedgehog(HH)信号通路在肢体发育过程中协调手指的模式，并沿着发育中的神经管的背腹轴建立细胞识别。然而，不受限制的HH信号导致结构性活性的Gli蛋白，并导致儿童和成人各种恶性肿瘤的形成。正常情况下，Gli蛋白受到三个水平的调节。在没有HH信号的情况下，全长Gli(GliFL)与其负调控抑制因子(SuFU)形成复合体，从而隔离在细胞质中。这使得蛋白激酶A(PKA)可以有效地磷酸化GliFL，并导致其随后被蛋白酶体系统部分降解。剩下的完整片段进入细胞核，发挥转录抑制因子(GIR)的作用。当HH途径被激活时，GliFL-SuFu复合体逃逸到初级纤毛，在那里这两种蛋白质可能是分离的。然后，GliFL离开纤毛进入细胞核，现在起到转录激活因子(胶质细胞)的作用。值得注意的是，核GliFL被过度磷酸化，并被蛋白酶体迅速降解。尽管Gli蛋白对发育和癌症具有基本的重要性，但我们对Gli蛋白激活的了解还远远不够。这尤其是从SuFu释放GliFL的过程和控制细胞核中神经胶质细胞活动的过程。这一建议致力于破译细胞质、纤毛和细胞核中Gli转录因子调控的潜在机制。首先，我将测试细胞质中PKA对GliFL的磷酸化如何调节与SuFU和纤毛运输的复合体的形成。这将通过创建GliFL的磷酸缺陷和模拟突变版本并测试它们(I)亚细胞定位，(Ii)与SuFU的关联和(Iii)执行HH信号的能力来完成。其次，我将使用生化方法来阐明GliFL在细胞核中过度磷酸化和蛋白酶体介导亚稳定状态的作用，以及这些特征是如何将GliFL转化为转录激活因子(Glia)所必需的。最后，我将进行无偏见的遗传功能丧失和功能获得筛查，以确定参与Gli蛋白调控的新因素(使用最先进的CRISPR技术)。综上所述，我的工作不仅将促进我们对Gli蛋白调控的了解，还将促进我们对纤毛运输和蛋白酶体介导的转录反应微调过程的洞察。此外，它还将重点介绍新的治疗和诊断策略，以对抗HH驱动的肿瘤。

The transcription factors of the Gli family are the final executors of the Hedgehog (Hh) signaling pathway, which orchestrates the patterning of the digits during limb development and establishes cell identify along the dorso-ventral axis of the developing neural tube. Unrestrained Hh signaling however results in constitutively active Gli proteins and drives the formation of various malignant cancers in children and adults. Normally, Gli proteins are subject to three levels of regulation. In the absence of Hh signals, full-length Gli (GliFL) is in a complex with its negative regulator Suppressor of Fused (SuFu) and thereby sequestered in the cytoplasm. This allows the efficient phosphorylation of GliFL by Protein Kinase A (PKA) and causes its subsequent partial degradation by the proteasome system. The remaining intact fragment travels to the nucleus and acts as a transcriptional repressor (GliR). Upon activation of the Hh pathway, the GliFL-SuFu complex escapes to the primary cilium, where the two proteins are presumably dissociated. GliFL then leaves the cilium and enters the nucleus, now functioning as a transcriptional activator (GliA). Notably, nuclear GliFL is hyper-phosphorylated and rapidly degraded by the proteasome. Despite of its fundamental importance for development and cancer, our understanding of Gli protein activation is far from complete. This is in particular the case for the process of GliFL release from SuFu and the control of GliA activity in the nucleus. This proposal strives to decipher the mechanisms underlying Gli transcription factor regulation in the cytoplasm, cilium and nucleus. First, I will test how the phosphorylation of GliFL by PKA in the cytoplasm regulates complex formation with SuFu and ciliary transport. This will be done by creating phospho-defective and -mimetic mutant version of GliFL and testing their (i) subcellular localization, (ii) association with SuFu and (iii) ability to execute Hh signaling. Second, I will use a biochemical approach to clarify the role of hyper-phosphorylation and proteasome-mediated metastability of GliFL in the nucleus and how these features are required to convert GliFL into a transcriptional activator (GliA). Finally, I will perform unbiased genetic loss- and gain-of-function screens to identify novel factors involved in the regulation of Gli proteins (using state-of-the-art CRISPR technologies). Taken together, my work will not only advance our knowledge of Gli protein regulation, but also our insight into the processes of ciliary trafficking and proteasome-mediated fine-tuning of transcriptional responses. In addition, it will focus on introducing novel therapeutic and diagnostic strategies to combat Hh-driven tumors.