br The GLI code The GLI code model considers the

The GLI code The GLI code model [21], [22] considers the total GLI function as a balance of positive activator (GLIA) and negative repressive (GLIR) activities with GLI1 being mostly a positive transcription factor and GLI3 mostly a transcriptional repressor. The GLIA:GLIR ratio is thus critical, being highly regulated, species- and context-specific, and highly dynamic (Fig. 1). GLI proteins belong to the superfamily of zinc finger transcription factors with five sequential zinc fingers of the C2H2 type constituting the sequence specific DNA binding domain. GLI1 (originally GLI) was first identified as an amplified gene in a human glioblastoma cell line [23], [24]. Later on and independently, what turned out to be its fly homolog, Cubitus interruptus (Ci) was identified and placed in the Hh pathway [7], [25], [26], [27]. GLI1 was not linked to the vertebrate Hh pathway until later [12], [28]. While the Drosophila C646 encodes only one GLI protein, the mouse and human genomes comprise three: GLI1, GLI2 and GLI3. One of the most remarkable features of GLI proteins is that in canonical HH signaling they can act as both transcriptional activators and repressors [29], [30], [31], [32], [33]. The situation is likely to be complex as all GLI proteins can act as activators or repressors in a stage-dependent and target gene-dependent manner [34]. However, the basic idea of the GLI code is useful as a framework and generally considers GLI1 as an activator and GLI3 mostly as a repressor. In the absence of HH pathway activity positive GLI function is off, GLI1 is not transcribed [12] and the GLI code is tipped toward a GLIR output, thus leading to pathway silencing. In this context, GLI2/3 proteins are proteolytically processed into C-terminally truncated repressors consisting of an N-terminal repressor domain and the DNA binding zinc fingers, but lacking the C-terminal transactivation domain. There is also evidence for GLI1 isoforms but how these are produced is not clear [35]. GLI processing in the absence of HH signaling is triggered by sequential phosphorylation of Ci or GLI2/3 by Protein Kinase A (PKA), Glycogen Synthase Kinase 3-beta (GSK3β) and Casein Kinase 1 (CK1) [36] followed by proteasomal degradation of the C-terminal region [31], [33]. Truncated Ci/GLI repressor binds to GLI sites in HH target promoters, thereby shutting off target gene expression (e.g. [37], [38], [39] (reviewed in [16], [40])). Activation of canonical HH signaling abrogates GLI processing allowing full-length and active GLI (GLIA) to enter the nucleus and turn on target gene expression. HH-GLI signaling also has feed-forward and feedback loops. In the latter case, GLI1 directly regulates PATCHED1 (PTCH1), genetically a SMOOTHENED (SMOH) inhibitor, but it also autoregulates itself. GLI2/3A activity leads to GLI1 expression, which further positively boosts GLI1 transcription. How his apparently close loop is broken is unclear, in order to allow precise and reversible control of the GLI code, which is of utmost importance for proper development and health. It is also unclear how the GLI proteins act since there is evidence that the GLI code will be highly refined and meticulously regulated given that GLI1, GLI2 and GLI3 can act in a combinatorial manner [30], [34], [41], [42], [43]. The importance of the critical and tight regulation of the GLI code is illustrated on the one hand by the fact that varying levels of HH-GLI will induce different numbers of neural stem cells in normal development and homeostasis [35], [44], [45], [46], [47], [48], and also induce different cell fates in the ventral neural tube in response to a morphogenetic gradient of HH ligands [8], [9], [11], [49], [50], [51]. On the other hand, genetic and/or epigenetic changes leading to irreversible activation of GLIA, and GLI1 [52], can drive a variety of malignant states ranging from cancers of the brain, skin, breast, prostate and digestive tract to malignancies of the hematopoietic system (e.g. [16], [52], [53], [54], [55], [56], [57], [58], [59], [60]).