Suppressor Of Fused Chaperones Gli Proteins To Generate Transcriptional Responses To Sonic Hedgehog Signaling

The proteins in Hedgehog(Hh)family regulate a variety of developmental processes and tumorigenesis,and as such dysregulation in the Hh pathway have been implicated in many developmental disorders and cancer.For instance,it is responsible for guiding the development from limb to central nervous system,and also has profound implications in basal cell carcinoma(BCC)and medulloblastoma(MB).The importance of Hh in cancer is underscored by the US-FDA’s approval of a Hh blocker in fighting metastasized BCC,and many clinical trials are still ongoing for other cancer indications as well.Hedgehog is a morphogen and it works by forming a concentration gradient along a developmental axis.In case of neural tube,it is expressed from notochord and floor plate,and projected dorsally.Hh proteins are ligands for the Patched(Ptc),which is a 12-transmembrane receptor and negatively regulates the 7-transmembrane protein Smoothened(Smo).Hh ligand bind to Ptch,resulting in activation of Smo.This allows Smo to translocate into primary cilia and Hh signaling to transmit.Downstream of Smo is the Gli family,which is a subset of zinc finger transcription factors.There are three of them in mammals,Gli1,Gli2 and Gli3.Sufu,a partner of Gli proteins and interact with all Gli proteins in a complex.The Gli proteins are repressed by Sufu through tethering Gli in the cytoplasm,Sufu can bind to a amino-terminal region of Gli with a highly conservered SYGH motif or masking Gli nuclear localization sequence(NLS).However,it is not clear how Sufu mediates Gli cytoplasm retention.As Hh stimulation,Gli proteins locate into nucleus,suggesting that the ability of Sufu to anchor Gli in the cytoplasm is somehow relived.Nevertheless,both biochemical and genetic studies have suggested that Sufu is capable of regulating Gli activity by another mechanism independent cytoplasm tethering.This indicates that Sufu is able to remain bound to Gli protein in the nucleus,In adult cerebellum,the Hh pathway is pretty much silent,but at one week after birth when cerebellar development is in full force,Sufu level is really high,as is Gli1,in granule neuron precursors in external germinal layer.The growth of these cells are highly dependent on Hh signal input.In medulloblastoma,which is a cerebellar tumor derived from granule neuron precursors,Hh pathway is reactivated,and as such,Sufu level is also highly up-regulated,as Gli1.Besides,Sufu is in both nuclear and cytoplasm.To look more closely of the nuclear and cytoplasmic distribution of Sufu,we examined MEFs.In normal MEFs,Sufu is both nuclear and cytoplasmic,with or without Shh treatment.What surprised us is that when we looked in Gli2/3 null cells,Sufu is mostly cytoplasmic,without Shh.With Shh,Sufu is induced nuclear.In Gli2/3 null cells,there are no repressors.This implys that in normal MEFs and without Shh treatment,Sufu is most likely brought into the nucleus by the repressors.In Gli2/3 null cells,the Shh induced localization is mediated by Gli1.To prove that,we generated a stable Gli2/3 null cells carrying shGli1 vector,so essentially converting them into all three Gli null cells.In these cells,Sufu can no longer be induced by Shh to enter the nucleus.Sufu recruits Sin3 A and HDAC to Gli to repress its transcriptional activity.We revisited this model and asked if Sufu can be found on the chromosomes at the Gli binding sites.We looked at both Gli1 and Ptch1 promoters.At least two sites,increasing amount of Gli-elements was recovered by the ChIP assay following Shh treatment,indicating indeed Shh induces the recruitment of Sufu onto its target gene promoters.Very interestingly,we also found SAP18 on Hh target gene promoters and Hh signaling causes it to disassociate.These data indicate that Hh signaling induces Gli1-Sufu complex latching onto its target gene promoters,and the binding of Sufu to chromatin is dependent on Gli1 and Gli3 R.To identify the sequence element on Sufu that determines its nuclear and cytoplasmic distribution,we did the deletion mapping,and identified a putative NES signal located between 308 to 318 residue in human Sufu.We mutated these two leucine residues to alanine and tested them in MEFs.The NES mutant Sufu is more nuclear than its wt counterpart.Moreover,the receptor of NES is CRM1 and we found that the mutant Sufu no longer binds CRM1 efficiently as the wt protein.These data indicate that we probably identified a bona fide NES of Sufu.Our lab previously identified a PKA and GSK3 dual phosphorylation site that controls the stability and ciliary trafficking of Sufu.This dual phosphorylation site sits next to the NES.We wondered if phosphorylation regulates NES recognition.In this CoIP experiment,we found that mutant Sufu that can not be phosphorylated at 342 or 346 position bind CRM1 very efficiently,but the Sufu mutant mimicking phosphorylation does not bind CRM1 at all.Finally,when we co-expressed wt Sufu-GFP with GSK3β or PKA alone or in combination,we found more Sufu in the nucleus,but these kinases have no effect on the non-phosphorylable double “A” mutant.These results illuminate that Sufu may not only participate in the Gli-mediated transcriptional control and inhibits Hh signaling pathway,but also promotes Gli1 protein stability and nuclear translocation in order to up-regulate Hh signaling pathway,which is required for generating maximal signaling output of Sonic Hedgehog.We have a new insight for the Sufu as a Gli proteins molecular chaperones.