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. 2009 Aug 15;23(16):1910-28.
doi: 10.1101/gad.1794109.

Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved

Miao-Hsueh Chen  1 , Christopher W Wilson, Ya-Jun Li, Kelvin King Lo Law, Chi-Sheng Lu, Rhodora Gacayan, Xiaoyun Zhang, Chi-chung Hui, Pao-Tien Chuang
Affiliations

Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved

Miao-Hsueh Chen et al. Genes Dev. .

Abstract

A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. We focus on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveal that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. We propose that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop, a conserved Gli-degrading factor. Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. We also uncover a novel positive role of Sufu in regulating Hh signaling, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and cilium-independent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species.

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Figures

Figure 1.
Figure 1.
Endogenous Hh pathway components display dynamic patterns of ciliary localization in response to Hh signaling, while overexpressed Gli proteins localize to the primary cilium in the absence of Sufu. (A) Immunofluorescence of wild-type (wt), Sufu−/−, and Ptch1−/− MEFs using antibodies against acetylated tubulin (AC) (labeling primary cilia, red) and various endogenous Hh pathway components including Smo, Ptch1, Gli2, Gli3 and Sufu (green). Smo translocates to the primary cilium upon Hh pathway activation, which is associated with concomitant loss of Ptch1 from the cilium. Low levels of Gli2 and Gli3 can be detected on the primary cilium by immunofluorescence without Hh ligand stimulation (data not shown), and their levels on the cilium significantly increase upon exposure to exogenous Shh ligand. In contrast, ciliary localization and intensity of Sufu are unchanged upon Hh pathway activation (data not shown). Gli2, Gli3, and Sufu immunofluorescence is detected primarily at the end of the primary cilium in some cells, while in others it decorates the entire cilium, perhaps due to dynamic ciliary trafficking of these proteins. Gli2 and Gli3 localize to the primary cilium in the absence of Hh stimulation in Ptch1−/− MEFs, in which the Hh pathway is maximally activated. Ciliary localization of Gli2 and Gli3 is completely abolished in Sufu−/− MEFs. (B) Immunofluorescence of Sufu−/− MEFs expressing mouse Flag-tagged Gli1, Gli2, or Gli3 using antibodies against acetylated tubulin (red) and Flag antibodies against Gli1, Gli2, or Gli3 (green). Overexpressed Gli proteins localize to the primary cilium in the absence of Sufu, and ciliary localization is unaffected by Hh stimulation. We speculate that the amount of overexpressed Gli proteins exceeds the capacity of Gli-degradation machinery in the absence of Sufu (see Fig. 2A).
Figure 2.
Figure 2.
Mouse Sufu regulates Gli protein levels independent of the primary cilium. (A) Western blots of lysates derived from wild-type (wt), Gli2−/−, Gli3−/−, Sufu−/−; Ptch1−/− and Kif3a−/− MEFs probed with anti-Gli2 and anti-Gli3 antibodies. Endogenous Gli2 and Gli3 protein levels (including full-length Gli3 and Gli3 repressor) are greatly reduced in the absence of Sufu. Both full-length Gli3 and Gli3 repressor can be detected in Ptch1−/− albeit the Gli3 repressor level is reduced. The ratio of full-length Gli3 to Gli3 repressor is altered in Kif3a−/− MEFs as reported previously (Liu et al. 2005). Gli2 processing is known to be extremely inefficient, and the Gli2 repressor form cannot be readily detected without additional enrichment steps using specific Gli-binding oligonucleotides (Pan et al. 2006). We also cannot accurately assess the full-length to repressor ratios for Gli2 and Gli3 in Sufu mutants. Tubulin was used as the loading control, and numbers on the right indicate locations of protein size standards. (FL) Full-length; (R) repressor. (B) Isotopic in situ hybridization using 33P-UTP-labeled ribo-probes (pink) on paraffin sections of wild-type (wt), Sufu−/−, Kif3a−/−, Sufu−/−; Kif3a−/−, and Sufu−/−; Fu−/− mouse embryos at 9.5 dpc. Loss of Sufu resulted in global up-regulation of Hh signaling and ventralization of the neural tube. Shh, whose expression is restricted to the notochord and floor plate in wild type, is extended dorsally in the absence of Sufu. Similarly, Hh target genes such as Patched 1 (Ptch1) are expanded dorsally, suggesting ventralization of the neural tube. The expression domains of neuronal progenitor markers (Class I genes, including Pax7 and Pax6, repressed by Hh signaling and Class II genes, including Nkx6.1, Nkx2.2, and Foxa2, activated by Hh signaling) are shifted. For instance, Pax7, the dorsal-most marker, is not expressed, and Pax6 expression is confined to the dorsal neural tube of Sufu−/− embryos. Dorsal expansion of Nkx6.1, Nkx2.2, and Foxa2 was also observed in the absence of Sufu (data not shown). Similarly, the expression domains of markers for differentiated interneurons and motoneurons are changed. For instance, Sufu−/− neural tube displayed dorsal expansion of Islet1 and Oligo2 (data not shown), which label motoneurons. By comparison, marker analysis revealed a partially dorsalized Kif3a−/− neural tube. The neural tube defects in Sufu−/−; Kif3a−/− or Sufu−/−; Fu−/− embryos resemble those in Sufu−/− mutants. (n) Notochord; (fp) floor plate; (nt) neural tube. (C) Western blots of lysates derived from wild-type, Sufu−/−, Kif3a−/− MEFs and Kif3a−/− MEFs expressing Sufu shRNA probed with anti-Gli2 and anti-Gli3 antibodies. Efficient Sufu knockdown in Kif3a−/− MEFs was verified by anti-Sufu antibodies. Gli2 and Gli3 protein levels are reduced in Kif3a−/− MEFs expressing Sufu shRNA to the same extent as in Sufu−/− MEFs. (D) Hh reporter assays using the 8xGliBS-luc reporter in wild-type and Kif3a−/− MEFs. Expression of Gli1 or Gli2 (but not Gli3) activates the Hh reporter, and Hh pathway activation is repressed when Sufu is coexpressed. Gli2 is known to activate Hh reporters less efficiently than Gli1 (Gerber et al. 2007). Loss of the primary cilium in Kif3a−/− MEFs does not impair Sufu's ability in repressing Gli-mediated Hh pathway activation. Error bars are standard deviation (s.d.).
Figure 3.
Figure 3.
Loss of the primary cilium impairs ligand-independent Hh pathway activation in Ptch1−/− MEFs but has no effect on Sufu−/− MEFs. (A) Hh reporter assays using the 8xGliBS-luc reporter in Sufu−/− and Ptch1−/− MEFs expressing increasing amounts of a dominant-negative (dn) Kif3b construct (shown in the left panel) or Kif3a shRNA, both of which inhibits the function of the primary cilium. While dnKif3b or Kif3a shRNA have no effect on Hh pathway activation in Sufu−/− MEFs, increasing quantities of dnKif3b or Kif3a shRNA reduce Hh pathway activation in Ptch1−/− MEFs. (B) Western blots of lysates derived from wild-type (wt), Sufu−/−, and Ptch1−/− MEFs and Sufu−/− and Ptch1−/− MEFs expressing dnKif3b probed with anti-Gli2 and anti-Gli3 antibodies. Inhibition of ciliary function by dnKif3b has no effect on endogenous Gli2 and Gli3 protein levels in Sufu−/− MEFs, which are greatly reduced in the absence of Sufu. In contrast, defective ciliary function in Ptch1−/− MEFs changed the ratio of full-length Gli3 to Gli3 repressor. Tubulin was used as the loading control, and numbers on the right indicate locations of protein size standards. (FL) Full-length; (R) repressor. (C, left panel) Hh reporter assays using the 8xGliBS-luc reporter in Sufu−/− MEFs and Sufu−/− MEFs expressing Gli shRNA in the presence or absence of exogenous Shh. Hh pathway activation is significantly compromised in Sufu−/− MEFs in which Gli1 is efficiently knocked down, consistent with the notion that Gli1 contributes to Hh pathway activation in the absence of Sufu. Similar results were obtained using three pairs of Gli1 shRNA directed against different regions of Gli1. In contrast, Sufu−/− MEFs and Sufu−/− MEFs expressing Gli1 shRNA display normal responsiveness to the canonical Wnt ligand Wnt3a assayed by the SuperTOPflash reporter (data not shown). (Right panel) Semiquantitative RT–PCR demonstrates that Gli1 is efficiently knocked down via Gli1 shRNA; β-actin serves as the control.
Figure 4.
Figure 4.
Zebrafish and fly Sufu restore Gli protein levels in mouse Sufu−/− MEFs, while Drosophila Smo fails to rescue Hh defects in mouse Smo−/− MEFs. (A) Immunofluorescence of Smo−/− MEFs expressing Smo from different species including mouse (m), zebrafish (z), and Drosophila (d) using antibodies against acetylated tubulin (AC) (labeling the primary cilium, red) and Smo (green). While mSmo and zSmo introduced into Smo−/− MEFs via transient transfection led to ciliary localization of Smo, dSmo mainly resides in the cytoplasm and is not found on the cilium. (B, left panel) Hh activity assays using the 8xGliBS-luc reporter in Smo−/− MEFs expressing Smo from different species via transient transfection. Both mouse (m) and zebrafish (z) Smo restored Hh responsiveness in Smo−/− MEFs, while expression of Drosophila Smo (dSmo) has no effect on Hh activation. (Right panel) Sufu−/− MEFs were transfected with mouse Sufu (mSufu), mouse Sufu with the D159A mutation (mSufuD159A), zebrafish Sufu (zSufu), or Drosophila Sufu (dSufu). Both mSufu and zSufu repressed basal 8xGliBS-luc activity in the absence of ShhN and promoted an increase in ShhN-mediated response. In contrast, the mSufuD159A and dSufu constructs had a less pronounced effect, which may partially be attributed to their weaker Gli-binding capacity (Supplemental Fig. S9). The numbers indicate the ratios of Hh responsiveness in the presence and absence of exogenous Shh (e.g., the ratio is 1.23 when no Sufu is added and is 16.99 when mSufu is added). Error bars are s.d. (C) Immunofluorescence of Sufu−/− MEFs expressing Sufu from different species via retroviral infection using antibodies against acetylated tubulin (red) and Gli2 or Gli3 (green). Mouse, zebrafish, or Drosophila Sufu was capable of restoring ciliary localization of endogenous Gli2 and Gli3 to the cilium when expressed in Sufu−/− MEFs, suggesting an evolutionarily conserved function of Sufu. The percentage of cilia that exhibit Gli2 and Gli3 immunoreactivity is lower in Sufu−/− MEFs expressing dSufu compared with mSufu or zSufu, consistent with a partial rescue of Hh defects in Sufu−/− MEFs by dSufu. (D) Western blots of lysates derived from wild-type (wt), Sufu−/− MEFs, and Sufu−/− MEFs expressing mouse, zebrafish, and Drosophila Sufu via retroviral infection probed with anti-Gli2 and anti-Gli3 antibodies. Endogenous Gli2 and Gli3 protein levels are restored when Sufu from different species is expressed, suggesting an evolutionarily conserved biochemical function of Sufu. Tubulin was used as the loading control, and numbers on the right indicate locations of protein size standards. (FL) Full-length; (R) repressor.
Figure 5.
Figure 5.
Mouse Spop colocalizes with Gli proteins and antagonizes Sufu in controlling Gli protein levels. (A) Double immunostaining of MEFs transfected singly with Flag-tagged Gli1, Gli2, or Gli3 or cotransfected with Myc-tagged Spop using Flag and Myc antibodies against Flag-tagged Gli1, Gli2, or Gli3 (green) and Myc-tagged Spop (red). Both cytoplasmic and nuclear staining of Gli1, Gli2, and Gli3 was detected. Punctate Spop immunoreactivity in the nucleus and cytoplasm was evident, consistent with previous reports (Hernandez-Munoz et al. 2005). Immunoreactivity of Gli2 and Gli3 (and not Gli1) was reduced when coexpressed with Spop, and Gli2/3 distribution extensively overlaps with Spop, particularly in the cytoplasm. Loss of the primary cilium in Kif3a−/− MEFs has no effect on the subcellular distributions and interactions of Gli1, Gli2, Gli3, and Spop. (B) Western blots of lysates derived from HEK 293T cells expressing Flag-tagged Gli3 singly or in combination with Flag-tagged Spop, Sufu, or Ext2 probed with anti-Flag antibodies. Lack of apparent Gli3 processing in cultured cells has been previously reported (B Wang et al. 2000). Coexpression of Gli2 or Gli3 with Sufu notably enhanced Gli2 and Gli3 protein levels. In contrast, coexpression of Gli2 or Gli3 with Spop (but not the control protein Ext2) significantly reduces Gli2 and Gli3 protein levels, which can then be restored when Sufu is coexpressed. We noticed that reduction in Gli2 protein levels is not as dramatic as Gli3 when Spop is coexpressed. Gli1 or Ext2 protein levels are unaffected when Spop is overexpressed (data not shown). α-Tubulin serves as the loading control (data not shown). (C) Western blot of immunoprecipitated Gli1, Gli2, and Gli3 (epitope-tagged with one copy of Flag) to detect polyubiquitinated Gli proteins. Spop promotes ubiquitination of Gli2 and Gli3 but not Gli1; Gli2 and Gli3 ubiquitination is abolished when Sufu is coexpressed. (WB) Western blot. (D) Western blot of immunoprecipitated Gli1, Gli2, and Gli3 (epitope-tagged with one copy of Flag) to detect physical interaction with Spop (epitope-tagged with one copies of HA) from HEK 293T lysates. Spop physically associates with Gli2 and Gli3 but not Gli1. (in) Input; (IP) immunoprecipitation. (E) Western blots of lysates derived from wild-type and Sufu−/− MEFs and wild-type and Sufu−/− MEFs expressing Spop shRNA probed with anti-Gli2 and anti-Gli3 antibodies. Efficient knockdown of Spop was verified by semiquantitative RT–PCR (data not shown). Gli2 and Gli3 levels are partially restored in Sufu−/− MEFs when Spop is knocked down, consistent with a model in which Sufu and Spop antagonize each other in regulating Gli2 and 3 (but not Gli1) protein levels. Lack of complete rescue of Gli protein levels could be attributed to the presence of additional mammalian Spop homologs (e.g., Spop-like and Tdpoz proteins) (Huang et al. 2004). (FL) Full-length; (R) repressor.
Figure 6.
Figure 6.
Mouse Sufu has positive and negative roles in regulating Hh signaling. (A) Hh activity assays using the 8xGliBS-luc reporter in Sufu−/− MEFs transfected with varying quantities of Sufu. Addition of increasing amounts of Sufu to Sufu−/− MEFs reduces Hh responsiveness in the absence of exogenous Shh, while promoting Hh activation in the presence of Shh. The numbers indicate the ratios of Hh responsiveness in the presence and absence of exogenous Shh (e.g., the ratio is 1.27 when no Sufu is added and is 22.43 when 240 ng of Sufu is added). Similar results were seen in two additional Sufu−/− cell lines and with the Smo agonist purmorphamine instead of ShhN (data not shown). Error bars are s.d. (B) Western blots of MEF lysates derived from wild type (wt), Sufu−/−, Ptch1−/−, or Ptch1−/− expressing Sufu shRNA probed with anti-Gli2 and anti-Gli3 antibodies. Gli2 and Gli3 protein levels are greatly reduced in Ptch1−/− MEFs when Sufu is knocked down. (C) Immunofluorescence of Ptch1−/− MEFs stably expressing Sufu shRNA using antibodies against acetylated tubulin (labeling the primary cilium) (red) and various Hh pathway components including Smo, Gli2, and Gli3 (green). Smo, Gli2, and Gli3 localize to the primary cilium in Ptch1−/− MEFs in the absence of exogenous Hh stimulation, consistent with maximal Hh pathway activation. While Smo localization to the primary cilium is unaffected in Ptch1−/− MEFs when Sufu is knocked down, ciliary localization of Gli2 and Gli3 in Ptch1−/− MEFs is abolished when Sufu is eliminated, suggesting compromised Hh pathway activation. (D) Hh reporter assays using the 8xGliBS-luc reporter in Ptch1−/− MEFs and Ptch1−/− MEFs expressing Sufu shRNA. Sufu knockdown leads to reduced Hh pathway activity in Ptch1−/− MEFs. (E) Hh reporter assays using the 8xGliBS-luc reporter in Sufu−/− and Ptch1−/− MEFs and Ptch1−/− MEFs expressing Sufu shRNA in the presence of various Hh antagonists that inhibit Smo function (Chen et al. 2002a,b). Hh pathway activation in Ptch1−/− MEFs is efficiently knocked down in the presence of Hh antagonists, but these Smo inhibitors have no effect on Hh pathway activity in Sufu−/− MEFs. When Sufu is knocked down in Ptch1−/− MEFs, these cells become partially insensitive to Hh antagonists.
Figure 7.
Figure 7.
A model of mammalian Hh signaling. Sufu plays a pivotal role in controlling Gli protein levels. Sufu protects full-length Gli2 and Gli3 proteins from Spop-mediated ubiquitination and complete degradation by the proteasome. In this way, Sufu functions as an adaptor to preserve a pool of Gli2 and Gli3 that can be readily converted into Gli activators and repressors. This aspect of Hh signaling is evolutionarily conserved and independent of the primary cilium. In contrast, the primary cilium is required for generating Gli repressors via limited proteolysis in the absence of Hh signaling and converting full-length Gli proteins into activators through unknown mechanisms upon Hh pathway activation. These events occurs downstream from Smo, which translocates to the primary cilium when Hh ligand binds to Ptch1 and removes it from the cilium. How other Hh pathway components or ciliary proteins particulate in cilium-dependent and cilium-independent activity needs to be investigated further.
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References

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