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）信号传导通路的最终执行者，Hh信号传导通路在肢体发育期间协调手指的模式并沿着发育神经管的背腹轴沿着建立细胞识别。然而，不受限制的Hh信号传导导致组成型活性Gli蛋白，并驱动儿童和成人中各种恶性癌症的形成。通常，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）与其负调节因子Suppressor of Fused（SuFu）形成复合物，从而被隔离在细胞质中。这允许蛋白激酶A（PKA）有效磷酸化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，充当转录抑制因子（GliR）。在Hh途径激活后，GliFL-SuFu复合物逃逸到初级纤毛，在那里这两种蛋白质可能解离。GliFL然后离开纤毛并进入细胞核，现在作为转录激活因子（GliA）发挥作用。值得注意的是，细胞核GliFL被蛋白酶体过度磷酸化并迅速降解。尽管Gli蛋白对发育和癌症至关重要，但我们对Gli蛋白激活的理解还远未完成。这对于GliFL从SuFu释放的过程和细胞核中GliA活性的控制尤其如此。本研究试图阐明Gli转录因子在细胞质、纤毛和细胞核中的调控机制。首先，我将测试PKA在细胞质中磷酸化GliFL如何调节与SuFu和纤毛运输的复合物形成。这将通过产生GliFL的磷酸缺陷和模拟突变体版本并测试它们的（i）亚细胞定位、（ii）与SuFu的缔合和（iii）执行Hh信号传导的能力来完成。其次，我将使用生物化学的方法来阐明细胞核中GliFL的过度磷酸化和蛋白酶体介导的亚稳定性的作用，以及这些功能是如何将GliFL转化为转录激活因子（GliA）的。最后，我将进行无偏见的遗传功能丧失和获得筛选，以确定参与Gli蛋白调控的新因子（使用最先进的CRISPR技术）。综上所述，我的工作不仅将推进我们对Gli蛋白调控的认识，还将深入了解纤毛运输和蛋白酶体介导的转录反应微调的过程。此外，它将专注于引入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.