The Effect Of Sumo Modification On The Regulation Of Gli2Transcriptional Activity In Hedgehog Signaling Pathway

The family of secreted Hedgehog (HH) molecules plays an important role in embryonic development of both vertebrates and invertebrates. Loss of function in HH signaling in vertebrates is the cause of many birth defects, including holoprosencephaly,hyperdactyly,craniofacial defect and skeletogeny defect,whereas an inappropriate activation of the HH pathway is also associated with several types of human malignant tumor.such as glioma, medulloblastoma, esophageal cancer, rectal cancer, small cell lung cancer, breast cancer, prostatic cancer and pancreatic cancer. Thus, a thorough understanding of the molecular mechanism of HH signal transduction is essential not only for the elucidation of the molecular mechanism of the developmental process of embryos, but also for the prevention and treatment of HH-related cancers.In vertebrates, HH ligands bind to their cell surface12-transmembrane receptor. Patched (PTCH). This binding prevents PTCH from inhibiting a seventransmembrane protein known as Smoothened (SMO), allowing the HH pathway to be activated. There are three downstream GLI transcription factors:GLI1,GLI2and GLI3.Hh stimulation activate the full length proteins (GH2FL and Gli3FL) into Gli2A/Gli3A, and GLI2and GLI3act as transcriptional repressors (Gli2R/Gli3R) in the absence of HH signaling because the C-terminal activation domains of Gli2FL and Gli3FL are proteolytically processed. GLI1is one of downstream transcriptional targets activated by Gli2A/Gli3A,which only act as transcriptional activalor.So far. How the activities of Gli transcription factors are regulated by upstream Hh signaling events and protein modifications are not fully understood.The small ubiquitin-like modifier (SUMO) is a100-amino acid polypeptide that is covalently attached to target proteins in a way similar to the ubiquitination process. SUMO modification has various effects on target protein function. For example, sumoylation of transcription factors mostly results in repression of the transcription, although it is occasionally found to activate the transcription.In this study,through experiments in cell culture and transgenic mice in vivo,we try to find out the correlation between Sumo and Gli2,the effect of Sumo modification on the regulation of Gli2transcriptional activity,the factors that affect GH2sumoylation and the mechanisms of Gli2sumoylation.Chapter1Correlation research on Sumo and Gli2Section1Correlation research on Sumo and Gli2in vivoObjective:To identify the proteins that interact with the GH2protein in vivo,and to determine whether GH2is sumoylated in vivo.Methods:A mouse limb bud cDNA library for the yeast two-hybrid screen was constructed by isolating mRNAs from limb buds of E10.5mouse embryos and reverse transcribing them into cDNAs, which were then inserted into EcoRI and Xhol sites of pJG4-5vector. Use a Gli2C-terminal fragment (584-1024amino acids)as a bait to screen the library to identify the proteins that interact with the Gli2protein.Results:We use the bait to screen the cDNA library and two of the interactors identified were Sumo2and Piasl,one of the Sumo E3ligases.Through this experiment,we show that Gli2is sumoylated in vivo.Conclusion:The interaction between Gli2and Sumo2or Piasl could not be verified in the mammalian cells using coimmunoprecipitation assay, suggesting that the interaction might be transient. Similar results have also recently been reported.Section2Correlation research on Sumo and Gli2in HEK293cellObjective:To determine whether Gli2is sumoylated in the cell and whether two sumoylation sites in PDD(processing determinant domain-a domain in GH2C-terminal) are sumoylated.Methods:1. GIi2was coexpressed with HA-Sumo2in HEK293cells. After cells were lysed, the lysates were subject to either immunoblotting to determine Gli2expression or immunoprecipitation with Gli2antibody followed by immunoblotting with anti-HA antibody to detect sumoylated Gli2.2. Gli2、HA-Sumo2were coexpressed with Flag-UBC9(E2Sumo conjugated enzyme) or Flag-UBC9DN(Negative form of UBC9) in HEK293cells. After cells were lysed, the lysates were subject to either immunoblotting to determine Gli2expression or immunoprecipitation with Gli2antibody followed by immunoblotting with anti-HA antibody to detect sumoylated Gli2.3. Wild type or mutant GST-Gli2PDD (Lys→Arg substitutions in one or both sumoylation sites)was expressed alone or together with HA-Sumo2in HEK293cells. After cells were lysed, expression levels of GST-GH2PDD fusion proteins were examined by immunoblotting. The Sumo modification was detected by the GST pull-down, followed by immunoblotting with a HA antibody.Results:1. The result showed that the immunoreactive signals of sumoylated Gli2protein were delected only when Sumo2and Gli2proteins were expressed together but not separately.2. the coexpression of wild type UBC9with Gli2and Sumo2dramatically increased the level of Gli2sumoylation, whereas coexpression of UBC9DN completely inhibited Gli2sumoylation.3. The results showed that Sumo modification was detected in the wild type PDD, but significantly reduced or abolished in the single or double Sumo site mutants.Conclusion: Because the immunoprecipitation was performed alter proteins were denatured, the detected signals in the1st experiment should be Gli2that was sumoylated in the cell but not proteins that might be associated with Gli2.;lhe2’ul experiment show that Gli2sumoylation is specific;lhe3rd experiment indicates that Lys-630and Lys-716in PDD are sumoylated in the cell. Chapter2The effect of Sumo modification on the regulation of Gli2transcriptional activity in vivo and in vitroSection1The effect of Sumo modification on the regulation of Gli2transcriptional activity in vivoObjective:To determine the role of Sumo modification in Hh signaling and the effect on Gli2transcriptional activity in vivo.Methods:We employed the targeted gene knock-in approach and replaced Lys-630and Lys-716with an Arg residue at the Gli2locus to create a Gli2Sumo site mutant mouse line named Gli2k2R. we first determined whether the Gli2K2R mutant protein processing was affected in vivo. Following an enrichment of Gli2protein using a double-stranded oligonucleotide that contains specific GLI binding sites, immunoblotting analysis using Gli2antibody. Then we examined the neuronal cell specification and patterning in the developing neural tube using immunostaining to frozen sections made from E10.5embryos.Results:1. The extent of Gli2K2R protein processing is comparable with that of wild type Gli2protein in mouse embryos, indicating that Gli2processing is not compromised.2. In wild type embryos, the floor plate, V3progenitor cells, and motoneurons are specified and patterned in the distinct locations of the ven tral neural tube. They can be marked by transcription factors FOXA2, NKX2.2. and HB9and IS11expression, respectively.NKX6.1expression covers from the floor plate to the V1progenitors.In contrast, PAX6and PAX7expression is found in the dorsal neural tube and is absent in the ventral-most area and in the entire ventral area, respectively, because their expression is suppressed by HH signaling. In the Gli2K2R homozygous neural tube, the expression and patterning of these transcription factors are indistinguishable from those in the wild type. Ptchl expression in the neural tube was examined using lacZ reporter gene inserted in the Ptchl locus. We found that Ptchl-lacZ expression in the mutant neural tube was slightly higher and dorsally expanded than that in wild type.Conclusion:Gli2K2R is slightly more active than wild type Gli2in vivo, suggesting that sumoylation inhibits Gli2activity in vivo.Sect i on2The effect of Sumo modification on the regulation of Gli2transcriptional activity in the cellObjcctiver To determine the effect of Sumo modification on Gli2transcriptional activity in vitro using chick limb bud micromass culture.Methods:Chick limb bud mesenchymal cells were transfectcd with a Gli-responsive SxGliBS-luciferase reporter, TK-renilla (control), and Gli2. Gli2K630R, Gli2K716R. or Gli2K630/716R mutants. Two days later, firefly and renilla luciferase activities were examined. After normalized against renilla luciferase activity, firefly luciferase activities were plotted.Results:Overexpression of wild type Gli2protein activated the reporter almost10-fold. Gli2K630R and Gli2K716R each slightly increased the luciferase activity, but the Gli2K630/716R double mutant induced the reporter nearly30-fold, three times as high as Gli2.Conclusion:These data indicate that Sumo modification leads to suppression of Gli2transcriptional activity and that the suppression from each Sumo site is synergislic in the cultured cells. Chapter3The factors that affect Gli2sumoylation and the molecular mechanism by which sumoylation inhibits Gli2transcriptional activitySection1The factors that affect Gli2sumoylationObjective:Phosphorylation of Gli2by PKA induces Gli2proteolytic processing to generate the Gli2repressor. It may also exert an inhibitory effect on Gli2FL activity. In contrast,Hh signaling inhibits Gli2processing and activates GH2FL protein.Thus, we wanted to know whether Gli2sumoylation is affected by PKA-mediated phosphorylation and Hh stimulation.Methods:Gli2or its mutants containing mutations at different PKA phosphorylation sites were expressed alone or together with HA-Sumo2in chick limb bud mesenchymal cells. The cells were incubated with medium with or without ShhN conditioned medium overnight. After cells were lysed, the lysates were subject to either immunoblotting to detennine GH2expression or immunopreeipitation with Gli2antibody followed by immunoblotting with anti-HA antibody to detect sumoylated GH2.Results:Mutations at sites5and6(Gli2P5-6) only slightly reduced Gli2sumoylation level; mutations at sites1-4(Gli2P1-4) significantly decreased Gli2sumoylation. whereas mutations of all six sites (GH2P1-6) reduced Gli2sumoylation even further.Similarly, Hh stimulation also reduced GH2sumoylation level.Conclusion:These results indicate that both mutations at the six PKA phosphorylation sites and Hh stimulation inhibit Gli2sumoylation, suggesting that Gli2phosphorylation by PKA may positively regulate Gli2sumoylation. Section2The molecular mechanism by which sumoylation inhibits GIi2transcriptional activityObjectivc:To elucidate the molecular mechanism by which sumoylation inhibits Gli2transcriptional activity and to determine whether Gli2interacts with HDACs.Methods: Gli2and Gli2K630/716R mutant were separately coexpressed with Flag-HDAC4or Flag-HDAC5in HEK293cells. Protein lysates from the transfected cells were subject to either immunoblotting to determine Gli2expression or HDAC expression with Gli2antibody or Flag antibody,or immunoprecipitation with Gli2antibody followed by immunoblotting with anti-Flag antibody to detect Gli2.Results: Western blot showed that Flag-HDAC4expression level is lower than that of Flag-I1DAC5. Coimmunoprecipitation analysis showed that the Gli2antibody precipitated residual HDAC5even when Gli2was not overexpressed. When Gli2and Flag-I1DAC5were coexpressed, the significantly higher amount of Flag-HDAC5was coimmunoprecipitated. However, when coexpressed with Gli2K630/716R, the amount of HDAC5coimmunoprecipitated was similar to that when HDAC5was expressed alone. In contrast, the amount of Flag-HDAC4coimmunoprecipitated was similar when it was coexpressed with either Gli2or Gli2K630/716R.Conclusion: Taken together, these results indicate that Gli2preferentially binds UDAC5over HDAC4and that only Gli2but not Gli2K630/716R can bind HDAC5. Therefore, the inhibition of Gli2activity by sumoylation is most likely through its recruitment of HDAC proteins...