Multisite interaction with Sufu regulates Ci/Gli activity through distinct mechanisms in Hh signal transduction Yuhong Hana,1, Qing Shia,1, and Jin Jianga,b,2 Departments of aDevelopmental Biology and bPharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390 Edited by Gary Struhl, Columbia University College of Physicians and Surgeons, New York, NY, and approved April 16, 2015 (received for review November 11, 2014)
Hedgehog
| Sufu | Ci | Gli | CBP
T
he Hedgehog (Hh) signaling pathway controls embryogenesis and adult tissue homeostasis by regulating the Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) family of zinc-finger transcription factors (1, 2). Initially identified as a suppressor of the segmentation defects caused by the loss of the serine/threonine kinase Fused (Fu) in Drosophila (3), Suppressor of fused (Sufu) plays a conserved negative role in Hh signal transduction by inhibiting the Ci/Gli transcription factors (4–6). Moreover, mutations in human Sufu predispose to medulloblastoma and meningioma (7, 8). Much of the attention in the past has been given to the role of Sufu in the cytoplasmic sequestration of Ci/Gli (5, 9–14). In addition, Sufu is also required for the production of the repressor form of Gli in mammals (15–17), a function carried out by the kinesin-like protein Costal2 (Cos2) in Drosophila (18). However, several studies have suggested that Sufu may also function in the nucleus to inhibit the activator form of Ci/Gli. For example, in Drosophila cos2 mutant wing discs, full-length Ci accumulated in the nucleus in a latent form inhibited by Sufu (18, 19). In cultured mammalian cells, overexpression of a truncated Sufu could inhibit Gli activity without sequestering it in the cytoplasm (20). Sufu can interact with several nuclear proteins, including the Drosophila myelodysplasia/myeloid leukemia factor and transcriptional corepressor complex Sin3–SAP18 (21, 22); however, a role for these nuclear factors in the regulation of Ci/Gli activity has not been demonstrated by a loss-of-function study (17). In this study, we observed that Sufu could still inhibit a fulllength Ci lacking the previously identified N-terminal Sufu-binding motif. Following up this unexpected observation, we identified a previously unidentified and conserved Sufu-binding motif located at the C terminus of Ci/Gli. We show that both the N- and C-terminal Sufu-interacting sites are required for optimal binding of Ci/Gli to Sufu as well as for effective inhibition of Ci/Gli by Sufu. We find that the N- and C-terminal sites can mediate cytoplasmic retention and nuclear inhibition of Ci/Gli by Sufu, www.pnas.org/cgi/doi/10.1073/pnas.1421628112
respectively. Furthermore, we provide evidence that binding of Sufu to Ci impedes the recruitment of the transcriptional coactivator CBP. Results Sufu Can Inhibit a Full-Length Ci Lacking the N-Terminal Sufu-Binding Site. Previous studies indicated that Ci/Gli binds Sufu through its
N-terminal domain containing an SYGH core motif (Fig. 1A) (11, 12, 23, 24), which we named SIN (Sufu-interacting site in the N-terminal region). We were surprised to observe that the transcriptional activity of a Ci variant (Ci-PKAΔN) lacking SIN was still inhibited by Sufu (Fig. S1). Of note, Ci-PKA has three PKA sites mutated to Ala and is no longer processed into a truncated repressor (CiR) (25). By using Ci-PKA as a backbone for structure– function study, we could focus on the regulation of the activator form of Ci (CiA) by Sufu. When SIN was deleted in CiGA1, in which the Ci sequence C-terminal to its Zn-finger DNA-binding domain was replaced by the Gal4 activation domain (GA), the resulting CiGA1ΔN was less inhibited by Sufu (Fig. S1), suggesting that Sufu can inhibit Ci through a region C-terminal to its Zn-finger DNA-binding domain. Sufu Interacts with a Conserved C-Terminal Site in Ci/Gli. To determine whether Sufu could interact with Ci through a domain(s) other than SIN, we divided Ci into three fragments—Ci1–439 (amino acids 1–439), Ci440–1160 (amino acids 440–1160), and Ci1161–1397 (amino acids 1161–1397)—and examined their interaction with Sufu by coimmunoprecipitation (CoIP) assay. When coexpressed with a Flag-tagged Sufu (Fg-Sufu) in S2 cells, all
Significance Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis by regulating the Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) family of transcription factors. Abnormal Hh pathway activity causes congenital disease and cancer. As a conserved negative regulator of the Hh signaling pathway, the tumor suppressor protein Suppressor of fused (Sufu) binds and inhibits Ci/Gli, but how Sufu contacts Ci/Gli and how Sufu–Ci/Gli interaction inhibits Hh signaling activity remain poorly understood. Here we identified a conserved Sufu-binding site in the C-terminal region of Ci/Gli. Further characterization of this Sufu-binding site provided insight into how Sufu blocks Ci/Gli activation in the nucleus. Understanding the mechanism by which Sufu regulates Ci/Gli activity is important for developing therapeutic treatment of cancers caused by abnormal Hh pathway activation. Author contributions: Y.H., Q.S., and J.J. designed research; Y.H. and Q.S. performed research; Y.H., Q.S., and J.J. analyzed data; and Q.S. and J.J. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
Y.H. and Q.S. contributed equally to this work.
2
To whom correspondence should be addressed. Email: [email protected].
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The tumor suppressor protein Suppressor of fused (Sufu) plays a conserved role in the Hedgehog (Hh) signaling pathway by inhibiting Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. Here we show that Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in addition to a previously identified N-terminal site (SIN), and that both SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. We show that Sufu can sequester Ci/Gli in the cytoplasm through binding to SIN while inhibiting Ci/Gli activity in the nucleus depending on SIC. We also find that binding of Sufu to SIC and the middle region of Ci can impede recruitment of the transcriptional coactivator CBP by masking its binding site in the C-terminal region of Ci. Indeed, moving the CBP-binding site to an “exposed” location can render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, our study identifies a previously unidentified and conserved Sufubinding motif in the C-terminal region of Ci/Gli and provides mechanistic insight into how Sufu inhibits Ci/Gli activity in the nucleus.
Fig. 1. Sufu binds Ci through both the N- and C-terminal domains. (A) A diagram of Ci domain structure and sequence alignment of SIN (blue bar) and SIC (green bar) within Ci and human (h) Gli proteins. ZF and CBP indicate the positions of the zinc-finger DNA-binding domain and CBP-binding domain, respectively. The SYGH motif is highlighted by a dashed box, and conserved residues in SIN and SIC are colored in blue and green, respectively. (B) Western blots (Left) and quantification (Right) of coimmunoprecipitation experiments from lysates of S2 cells coexpressing Fg-Sufu and various Myc-tagged Ci fragments (asterisks). The arrow indicates IgG. Data are means ± SD from two independent experiments. IB, immunoblotting; IP, immunoprecipitation. (C) Western blots of GST pull-down experiments. One or 5 μg of GST or GST fusion proteins was used as bait and coincubated with equal amounts of cell lysates from S2 cells expressing Fg-Sufu. (D) Western blots (Top) and quantification (Bottom) of coimmunoprecipitation experiments from lysates of S2 cells coexpressing Fg-Sufu and the indicated Myc-tagged Ci proteins. TCL, total cell lysates. Data are means ± SD from two independent experiments. (E) Western blots of a competition experiment. Equal amounts of immunopurified Fg-Sufu were incubated with cell extracts from S2 cells expressing a fixed amount of Myc-Ci1161–1397 and increasing amounts of Myc-Ci1–439. The arrow indicates IgG. (F) Western blots of coimmunoprecipitates from lysates of S2 cells coexpressing Fg-Sufu or Fg-SufuD154R with Myc-Ci1–439 or Myc-Ci1161–1397.
three Ci fragments coimmunoprecipitated Fg-Sufu, with Ci1–439 exhibiting 10-fold higher affinity than Ci440–1160 or Ci1161–1397 (Fig. 1B). Sequence alignment revealed a C-terminally conserved sequence motif present in Ci (amino acids 1370–1397), Gli2, and Gli3 (Fig. 1A). To determine whether this conserved sequence mediates the interaction between Ci and Sufu, we deleted it from Ci1161–1397 to generate Ci1160–1370, and found that Ci1160–1370 failed to bind Sufu (Fig. 1B). GST pull-down experiments revealed that GST-Ci1370–1397 pulled down Fg-Sufu derived from S2 cell extracts but 10-fold less effectively compared with GSTCi1–439 (Fig. 1C), suggesting that Ci1370–1397 contains a lowaffinity Sufu-binding site, which we name SIC (Sufu-interacting site in the C terminus). Simultaneous Binding to SIN and SIC Promotes Strong Sufu–Ci Association. We next determined how SIN and SIC contribute to
the overall binding of Sufu to Ci. Accordingly, we generated Myctagged Ci-PKA and its variants lacking either SIN (Ci-PKAΔN), SIC (Ci-PKAΔC), or both (Ci-PKAΔNΔC) (Fig. 2A), and compared their binding affinity toward Sufu in S2 cells using the CoIP assay. As shown in Fig. 1D, deletion of either SIN or SIC resulted in approximately fivefold reduction in Sufu binding, whereas their combined deletion nearly abolished Sufu binding. In a competition assay, we found that increasing the amount of Ci1–439 did not affect the association between Sufu and Ci1161–1397; instead, Sufu coimmunoprecipitated both Ci fragments (Fig. 1E), suggesting that SIN and SIC may bind different regions of Sufu instead of competing for the same binding pocket. Consistent with this notion, a Sufu variant (SufuD154R), which contains a point mutation at D154 implicated in contacting the SYGH motif (26, 27), exhibited diminished binding to Ci1–440 but interacted with Ci1161–1397 similar to the wild-type Sufu (Fig. 1F). Taken together, these observations 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1421628112
suggest that Sufu may simultaneously interact with both SIN and SIC, and that this multisite interaction greatly increases the overall binding affinity. Both SIN and SIC Contribute to Sufu-Mediated Inhibition of Ci. Having established that Sufu can bind Ci through both SIN and SIC, we next explored the functional significance of these Sufu-binding domains in mediating Ci inhibition. We first compared the activity of Ci-PKA, Ci-PKAΔN, Ci-PKAΔC, and Ci-PKAΔNΔC in the absence or presence of coexpressed Fg-Sufu using the ptc-luc reporter assay in S2 cells. As shown in Fig. 2B, Ci-PKA was suppressed by Sufu more effectively than Ci-PKAΔN and Ci-PKAΔC, whereas Ci-PKAΔNΔC exhibited little if any suppression by Sufu, suggesting that both SIN and SIC contribute to the Sufu-mediated inhibition of Ci. To determine the relative contribution of SIN and SIC to Sufu-mediated inhibition of Ci in vivo, UAS transgenes expressing individual Ci variants were introduced into flies at the same genomic locus using the phiC31 integration system to ensure similar levels of transcription of the transgenes (28). Wing discs expressing these transgenes using MS1096, a wing-specific Gal4 driver, in the absence or presence of a UAS-Sufu transgene were immunostained with Ci and Patched (Ptc) antibodies to monitor the levels of fulllength Ci (derived from both transgenic and endogenous expression) and Hh pathway activity, respectively. Quantification of full-length Ci levels in control and Ci-PKA–expressing wing discs indicated that exogenously derived Ci reached levels four- to fivefold higher than the endogenous levels in anterior (A)compartment cells distant from the anterior–posterior (A/P) boundary after subtracting the background signal, whereas in A-compartment cells near the A/P boundary, Ci-PKA–expressing wing discs contained full-length Ci at levels approximately twofold higher than the control discs (Fig. S2 A–C). Real-time Han et al.
SIN and SIC Mediate Sufu Inhibition Through Distinct Mechanisms.
Fig. 2. Both the N- and C-terminal Sufu-binding sites can contribute to Ci inhibition. (A) A diagram of full-length (FL), ΔN, ΔC, and ΔNΔC variants of Ci-PKA. SIN and SIC are indicated by blue and green bars, respectively. (B) ptcluc reporter assays in S2 cells transfected with Ci-PKA FL, ΔN, ΔC, and ΔNΔC alone or with Sufu. Data are means ± SD from three independent experiments. (C–K′) Late third-instar wing discs of wild type (C and C′) or expressing the indicated UAS-Ci transgenes either alone (D–G′) or together with a UASSufu transgene (H–K′) under the control of the MS1096 Gal4 driver were immunostained with anti-Ci (red) and anti-Ptc (green) antibodies to monitor the levels of full-length Ci derived from both transgenic and endogenous expression, and Hh pathway activity, respectively. Arrowheads and arrows indicate A and P compartments, respectively (H′–K′).
quantitative PCR revealed that Sufu mRNA levels in Sufuoverexpressing wing discs were approximately threefold higher than in control discs (Fig. S2D). CoIP experiments using wing disc extracts indicated that both Ci-PKAΔN and Ci-PKAΔC bound approximately fivefold less endogenous Sufu compared with Ci-PKA, whereas Ci-PKAΔNΔC exhibited no detectable binding to less endogenous Sufu (Fig. S3). In control wing discs, Hh is expressed in the posterior (P) compartment whereas Ci is expressed in the A compartment. Hh moves to the A compartment to stabilize Ci and activate target genes such as ptc in A-compartment cells near the A/P boundary (Fig. 2 C and C′). When Ci is ectopically expressed in P-compartment cells using the MS1096 Gal4 driver, it can ectopically activate downstream Hh target genes, and this ectopic activity is subject to suppression by Sufu, providing an additional place to assay Sufu–Ci regulatory interactions. Both full-length and Han et al.
We next determined how SIN and SIC mediate Ci inhibition by Sufu. Consistent with previous findings that blocking nuclear export promotes Ci nuclear accumulation (11, 30), when expressed alone in S2 cells, Myc-Ci-PKA, Myc-Ci-PKAΔN, and Myc-Ci-PKAΔC were accumulated mainly in the nucleus after the transfected cells were treated with the nuclear export inhibitor LMB (Fig. 3 A–C). Coexpression of Fg-Sufu retained Myc-Ci-PKA and Myc-Ci-PKAΔC but not Myc-Ci-PKAΔN in the cytoplasm (Fig. 3 E–G′). Furthermore, Fg-SufuD154R, which failed to bind SIN but exhibited normal binding to SIC (Fig. 1F), failed to retain Myc-Ci-PKA in the cytoplasm (Fig. 3 H and H′). Quantification of nuclear–cytoplasmic distribution of the various Ci constructs is shown in Fig. S5. When expressed in wing discs, Myc-Ci-PKAΔN exhibited more nuclear localization than Myc-Ci-PKA in A-compartment cells away from the A/P boundary, whereas Myc-Ci-PKAΔC behaved similarly to Myc-Ci-PKA (Fig. S6). Taken together, these observations suggest that Sufu–SIN interaction is essential for Sufu-mediated cytoplasmic retention of Ci. Despite failing to sequester Myc-Ci-PKA in the cytoplasm, Fg-SufuD154R still inhibited its transcriptional activity (Fig. 3J), suggesting that SufuD154R could inhibit Ci activity in the nucleus. To confirm this, we generated a constitutively nuclear form of Ci, HA-Nuc-Ci-PKA, in which an SV40 nuclear localization signal (NLS) was inserted at the N terminus of Ci (19) and a major nuclear export signal (NES) was mutated (31). When expressed in S2 cells, HA-Nuc-Ci-PKA was accumulated largely in the nucleus even in the presence of Fg-Sufu (Fig. 3 D, I, and I′ and Fig. S5); however, HA-Nuc-Ci-PKA activity was still sensitive to FgSufu–mediated inhibition (Fig. 3K). Whereas deleting SIN (ΔN) did not affect SufuD154R-mediated inhibition of Ci-PKA or Sufumediated inhibition of Nuc-Ci-PKA, deletion of either SIC (ΔC) or both SIN and SIC (ΔNΔC) abolished these inhibitions (Fig. 3 J and K), suggesting that Sufu can inhibit Ci activity in the nucleus depending on SIC. Interaction Between Sufu and the C-Terminal Half of Ci Interferes with CBP Recruitment. We next determined the mechanism by
which Sufu inhibits Ci in the nucleus. It has been shown that Ci activates its target genes by binding to the Drosophila CBP (dCBP) through a C-terminal binding domain between amino acids 1020 and 1160 (32). Because of the proximity between the PNAS Early Edition | 3 of 6
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truncated Ci-PKA induced ectopic expression of ptc along the A/P axis (Fig. 2 D′–G′). Coexpression of Sufu completely suppressed the ectopic ptc expression induced by Ci-PKA in both A- and P-compartment cells (Fig. 2H′ compared with Fig. 2D′). Coexpression of Sufu completely suppressed the ectopic ptc expression induced by Ci-PKAΔN or Ci-PKAΔC in A-compartment cells but only partially suppressed ectopic ptc expression induced by Ci-PKAΔN or Ci-PKAΔC in P-compartment cells (Fig. 2 I′ and J′ compared with Fig. 2 E′ and F′). By contrast, coexpression of Sufu did not block the ectopic ptc expression induced by Ci-PKAΔNΔC in either A- or P-compartment cells (Fig. 2K′ compared with Fig. 2G′). Hence, both SIN and SIC can contribute to Sufu-mediated inhibition of Ci. We noted that Ci-PKAΔNΔC induced ectopic ptc expression at lower levels compared with other Ci transgenes (Fig. 2G′ compared with Fig. 2 D–F′), likely due to its instability, because wing discs expressing Ci-PKAΔNΔC exhibited weaker Ci staining than those expressing other Ci transgenes (Fig. 2G compared with Fig. 2 D–F). Previous studies indicated that Sufu protects Ci/Gli from HIB/SPOP-mediated degradation (17, 29). Because Ci-PKAΔNΔC did not bind Sufu, it could be degraded by HIB more rapidly than other Ci variants. In support of this notion, we found that inactivation of HIB by RNAi stabilized Ci-PKAΔNΔC in wing discs, leading to elevated ptc expression at levels similar to those induced by Ci-PKA (Fig. S4).
Ci621–1020, but not to either Ci440–1370 or Ci1020–1397 (Fig. 4B), suggesting that simultaneous binding of Sufu to SIC and Ci621–1020 is likely to be required to impede dCBP recruitment. If Sufu inhibits Ci in the nucleus by impeding dCBP recruitment, we reasoned that moving the dCBP-binding domain (dCBP-BD) to an “exposed” location in Ci should overcome the inhibition by Sufu in the nucleus. Therefore, we fused a dCBP-BD to the C terminus of either Ci-PKA or Nuc-Ci-PKA after addition of a flexible linker protein, MBP, to separate SIC from the exogenously added dCBPBD (Fig. 4C). In the meantime, the endogenous dCBP-BD was deleted from these fusion proteins to generate CiΔCBP-MBP-CBP or Nuc-Ci-ΔCBP-MBP-CBP, respectively (Fig. 4C). We also generated several Ci constructs, including Ci-MBP, CiΔCBP-MBP, NucCi-MBP, and Nuc-CiΔCBP-MBP as controls (Fig. 4C). As shown in Fig. 4, addition of MBP to the C terminus of either Ci-PKA or NucCi-PKA did not affect their activity as well as Sufu- or SufuD154Rmediated inhibition (Fig. 4C, compare groups 2 and 6 with 1 and 5). Both CiΔCBP-MBP-CBP and Nuc-CiΔCBP-MBP-CBP were as active as Ci-PKA//Ci-PKA-MBP and Nuc-Ci-PKA/Nuc-Ci-PKA-MBP whereas neither CiΔCBP-MBP nor Nuc-CiΔCBP-MBP was active, suggesting that the activity of CiΔCBP-MBP-CBP and Nuc-CiΔCBP-MBP-CBP depends on the exogenously added dCBP-BD. Importantly, CiΔCBPMBP-CBP and Nuc-CiΔCBP-MBP-CBP were no longer suppressed by SufuD154R (Fig. 4C, groups 4 and 8). Furthermore, although Sufu still inhibited CiΔCBP-MBP-CBP, likely by sequestering it in the cytoplasm (Fig. 4C, group 4), Sufu failed to inhibit Nuc-CiΔCBPMBP-CBP (Fig. 4C, group 8). Taken together, these results further support the notion that Sufu can inhibit Ci in the nucleus by impeding dCBP recruitment. Fig. 3. SIN and SIC can regulate Ci by distinct mechanisms. (A–I′) Representative confocal images of S2 cells transfected with Myc-tagged Ci-PKA, Ci-PKAΔN, or Ci-PKAΔC or HA-tagged Nuc-Ci-PKA either alone or together with Flag-tagged Sufu or SufuD154R and immunostained with antibodies against Myc or HA (red) and Flag (green) and phalloidin for cell membrane (blue). Of note, transfected cells were treated with 20 ng/mL LMB for 3 h before immunostaining. (J and K) ptc-luc reporter assays in S2 cells transfected with the indicated Ci and Sufu constructs. Data are means ± SD from three independent experiments.
dCBP-binding domain and SIC, we speculated that binding of Sufu to SIC may impede the recruitment of dCBP. Therefore, we carried out CoIP experiments using nuclear extracts prepared from S2 cells transfected with HA-Nuc-Ci-PKA and Myc-tagged dCBP (Myc-dCBP) with or without cotransfection of Fg-Sufu. In the absence of Fg-Sufu, Myc-dCBP formed a complex with HA-Nuc-Ci-PKA; however, the association between Myc-dCBP and HA-Nuc-Ci-PKA was blocked by Fg-Sufu (Fig. 4A, lanes 1 and 2). Deletion of SIN (Nuc-Ci-PKAΔN) only slightly affected this blockage (Fig. 4A, lanes 3 and 4). By contrast, deletion of either SIC (Nuc-Ci-PKAΔC) or both SIN and SIC (Nuc-Ci-PKAΔNΔC) abolished the Sufu-mediated inhibition of dCBP–Ci association (Fig. 4A, lanes 5–8), suggesting that binding of Sufu to SIC may be essential for preventing dCBP–Ci association. Because SIC is an intrinsically weak Sufu-binding site (Fig. 1C), we speculated that SIC might cooperate with a Sufu-binding site located in the middle region of Ci to bind Sufu when the function of SIN was compromised. Indeed, Ci440–1397, which contains both SIC and the middle Sufu-binding site (Fig. 1B), exhibited approximately fivefold higher binding affinity to Sufu than Ci440–1160 and Ci1161–1397, both of which contain only one Sufu-binding site (Fig. S7A). Further mapping revealed that Sufu interacted with a Ci fragment (Ci621–1020) between the zinc-finger DNA-binding domain and the CBP-binding domain (Fig. S7B), consistent with the finding of a previous study showing that a Ci fragment containing amino acids 832–1187 interacted with Sufu in a yeast two-hybrid assay (33). Importantly, Fg-Sufu inhibited the binding of dCBP to Ci440–1397, which contains both SIC and 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1421628112
A Conserved Role of SIC in Sufu-Mediated Inhibition of Gli2. In the vertebrate Hh pathway, Gli2 and Gli3 are the primary transcription factors. We noticed that the sequence of SIC is highly
Fig. 4. Interaction of Sufu with the C-terminal half of Ci can impede CBP binding. (A and B) Western blots of coimmunoprecipitation experiments from lysates of S2 cells expressing the indicated constructs. Asterisks in B indicate HA-tagged Ci fragments. (C) ptc-luc reporter assays (Left) in S2 cells transfected with the indicated Ci constructs (diagrams are shown; Right) without or with Sufu or SufuD154R. Data are means ± SD from three independent experiments.
Han et al.
Fig. 5. A conserved role of SIC in mediating Sufu binding and nuclear inhibition of Gli2. (A) Western blots of coimmunoprecipitation experiment from lysates of NIH 3T3 cells expressing the indicated Gli2 and Sufu constructs. (B) Gli-luc reporter assays in NIH 3T3 cells transfected with the indicated Gli2 and Sufu constructs. Data are means ± SD from three independent experiments. (C) Representative confocal images of NIH 3T3 cells transfected with an the indicated mouse Myc-Gli2 and Sufu constructs and immunostained with an anti-Myc antibody (Left) and DNA dye Hoechst that labels the nucleus (Nuc) (Right).
conserved in both Gli2 and Gli3 (Fig. 1A). Because Gli2 contributes mostly to Gli activator activity, we focused on Gli2 using the approaches similar to what we have applied to Ci. Accordingly, we generated Myc-tagged full-length Gli2 and its variants with either SIN (Gli2ΔN), SIC (Gli2ΔC), or both (Gli2ΔNΔC) deleted. These constructs were cotransfected with a Flag-tagged mouse Sufu (mSufu) into NIH 3T3 cells, followed by CoIP experiments or Gli-luc reporter assays. Similar to Ci, deletion of either SIN or SIC from Gli2 greatly reduced its binding to mSufu, and their combined deletion nearly abolished the Gli2– mSufu association (Fig. 5A). Furthermore, both SIN and SIC contributed to mSufu-mediated inhibition of Gli2 activity, as deletion of either SIN or SIC only partially alleviated whereas their combined deletion almost completely blocked the mSufumediated inhibition (Fig. 5B). Immunostaining of the transfected cells with an antibody against Myc and a nuclear marker revealed that Myc-Gli2 was primarily localized in the nucleus when expressed alone but was sequestered in the cytoplasm when mSufu was cotransfected (Fig. 5C and Fig. S8). However, cytoplasmic retention of Gli2 by mSufu was abolished by the deletion of SIN (ΔN + mSufu; Fig. 5C and Fig. S8) but was unaffected by the deletion of SIC (ΔC + mSufu; Fig. 5C and Fig. S8). Consistent with a previous study (26), mSufuD159R, which is the counterpart of Drosophila SufuD154R, exhibited reduced binding to Gli2 (Fig. 5A) and failed to sequester Gli2 in the cytoplasm (Fig. 5C and Fig. S8). Nevertheless, mSufuD159R still inhibited the activity of Gli2 (Fig. 5B). Furthermore, this inhibition was abolished by deletion of SIC (ΔC or ΔNΔC) but was unaffected by deletion of SIN (ΔN). Taken together, these results suggest that Sufu can sequester Gli2 in the cytoplasm through binding to SIN but inhibit Gli2 activity in the nucleus through binding to SIC. Han et al.
Discussion As a conserved negative regulator of intracellular Hh signaling, Sufu binds and inhibits Ci/Gli. A conserved Sufu-binding site with an SYGH core motif (SIN) was identified in the N-terminal region of Ci/Gli, and structural information has recently been obtained on how Sufu binds this conserved site (26, 27); however, how Sufu interacts with full-length Ci/Gli remains a mystery, because the crystal structures of the Sufu–Gli complexes only contain a small peptide flanking the SYGH motif. Here we identified a conserved Sufu-binding site (SIC) at the C terminus of Ci/Gli. We provided evidence that Sufu may contact SIN and SIC simultaneously, which could be essential for effective Sufu– Ci/Gli association under physiological conditions. In addition, both SIN and SIC appear to be required for optimal inhibition of Ci/Gli but may regulate Ci/Gli through distinct mechanisms, with SIN primarily mediating cytoplasmic retention and SIC contributing to nuclear inhibition of Ci/Gli. Previous studies have suggested that Sufu can impede Ci/Gli nuclear localization and inhibit Ci/Gli transcriptional activity in the nucleus (5, 9–12, 19, 20); however, how Sufu exerts this dual regulation still remains poorly understood. Our recent study revealed that binding of Sufu to SIN may inhibit the binding of Kapβ2 to a NLS of the PY family located in the N-terminal region of Ci/Gli (13). Another recent study also provided evidence that binding of Sufu to Gli1 can preclude the binding of importin β1 (14). Taken together, these studies suggest that binding of Sufu to SIN may inhibit Ci/Gli nuclear import by masking its NLSs. Here we provide evidence that binding of Sufu to SIC can inhibit Ci transcriptional activity by impeding the recruitment of dCBP (Fig. 6). Indeed, deletion of SIC but not SIN abolished Sufu-mediated inhibition of Ci in the nucleus (Fig. 3K). By contrast, deletion of SIN but not SIC affected Sufu-mediated cytoplasmic retention of Ci (Fig. 3 E–G and Figs. S5 and S6). We found that both SIN and SIC were required for optimal binding of Sufu to Ci, because deletion of either site in the fulllength Ci background resulted in approximately fivefold reduction in Sufu binding affinity (Fig. 1 and Fig. S3). Therefore, we speculate that in anterior-compartment cells distant from the A/P boundary, where there is no Hh and the levels of full-length Ci (CiF) are low, Sufu may bind CiF by simultaneously contacting both SIN and SIC to prevent nuclear import of CiF. In A-compartment cells 5–10 cells away from the A/P boundary, where low to intermediate levels of Hh block Ci processing to increase the levels of CiF and promote nuclear translocation of CiF (11, 30),
Fig. 6. Diagrams of Ci bound by Sufu or CBP. The N- and C-terminal Sufubinding sites (SIN and SIC) and the CBP-binding domain are indicated by blue, green, and red bars, respectively. See text for details.
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Intriguingly, Zhang et al. reported that mSufuD159R failed to inhibit Gli1 activity (26), which we confirmed in this study (Fig. S9). Although a previous study indicated that Sufu could interact with the C-terminal half of Gli1, the binding site was not mapped (34). Sequence alignment revealed that the C-terminal sequence of Gli1 diverges significantly from those of Gli2/3 and Ci (Fig. S9), suggesting that Gli1 may contain a less potent SIC, which could explain why mSufuD159R failed to inhibit Gli1.
SIN–Sufu interaction may be compromised, likely due to Hhinduced modification of Ci–Sufu. Under such conditions, SIC may cooperate with the middle region of Ci (Ci621–1020) to bind Sufu to mask the dCBP-binding site (Fig. 6). This could explain why low to medium levels of Hh can promote CiF nuclear translocation but fail to fully activate CiF. In A-compartment cells immediately abutting the A/P boundary, where peak levels of Hh convert CiF into CiA, both N- and C-terminal interactions may be compromised, allowing dCBP to be recruited to CiF (Fig. 6). In the mammalian Hh pathway, full-length Gli3 interacts with CBP and the MED12–Mediator complex through its C-terminal transactivation domain (35, 36). Gli1 and Gli2 also contain extended transactivation domains in their C-terminal regions (37). Therefore, it is conceivable that binding of Sufu to Gli proteins may also preclude coactivator binding. However, our study does not exclude the possibility that Sufu inhibits Ci/Gli in the nucleus 1. Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15(6): 801–812. 2. Briscoe J, Thérond PP (2013) The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14(7):416–429. 3. Préat T (1992) Characterization of Suppressor of fused, a complete suppressor of the fused segment polarity gene of Drosophila melanogaster. Genetics 132(3):725–736. 4. Ohlmeyer JT, Kalderon D (1998) Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 396(6713):749–753. 5. Ding Q, et al. (1999) Mouse Suppressor of fused is a negative regulator of Sonic hedgehog signaling and alters the subcellular distribution of Gli1. Curr Biol 9(19): 1119–1122. 6. Svärd J, et al. (2006) Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway. Dev Cell 10(2): 187–197. 7. Taylor MD, et al. (2002) Mutations in SUFU predispose to medulloblastoma. Nat Genet 31(3):306–310. 8. Aavikko M, et al. (2012) Loss of SUFU function in familial multiple meningioma. Am J Hum Genet 91(3):520–526. 9. Méthot N, Basler K (1999) Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96(6):819–831. 10. Kogerman P, et al. (1999) Mammalian Suppressor-of-Fused modulates nuclearcytoplasmic shuttling of Gli-1. Nat Cell Biol 1(5):312–319. 11. Wang G, Amanai K, Wang B, Jiang J (2000) Interactions with Costal2 and Suppressor of fused regulate nuclear translocation and activity of Cubitus interruptus. Genes Dev 14(22):2893–2905. 12. Dunaeva M, Michelson P, Kogerman P, Toftgard R (2003) Characterization of the physical interaction of Gli proteins with SUFU proteins. J Biol Chem 278(7):5116–5122. 13. Shi Q, Han Y, Jiang J (2014) Suppressor of fused impedes Ci/Gli nuclear import by opposing Trn/Kapβ2 in Hedgehog signaling. J Cell Sci 127(Pt 5):1092–1103. 14. Szczepny A, et al. (2014) Overlapping binding sites for importin β1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization. Biochem J 461(3):469–476. 15. Kise Y, Morinaka A, Teglund S, Miki H (2009) Sufu recruits GSK3beta for efficient processing of Gli3. Biochem Biophys Res Commun 387(3):569–574. 16. Humke EW, Dorn KV, Milenkovic L, Scott MP, Rohatgi R (2010) The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins. Genes Dev 24(7):670–682. 17. Chen MH, et al. (2009) Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved. Genes Dev 23(16):1910–1928. 18. Zhang W, et al. (2005) Hedgehog-regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of Cubitus interruptus. Dev Cell 8(2):267–278. 19. Wang G, Jiang J (2004) Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev Biol 268(2): 493–505.
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through additional mechanisms such as recruiting a corepressor complex. Indeed, while our manuscript was under review, Lin et al. reported that Sufu inhibited Gli in the nucleus by recruiting p66β (38). We speculate that Sufu may inhibit Ci/Gli more effectively in the nucleus by simultaneously excluding coactivators and recruiting corepressors. Experimental Procedures Standard procedures for Drosophila genetics and tissue-culture experiments were used. Drosophila stocks, transgenes, and DNA constructs and detailed procedures for cell culture, transfection, cell fractionation, immunostaining, immunoprecipitation, Western blot analysis, GST pull-down assays, and luciferase reporter assays are described in SI Experimental Procedures. ACKNOWLEDGMENTS. We thank Bing Wang for assistance and the Developmental Studies Hybridoma Bank for reagents. This work was supported by grants from the NIH (GM061269, GM067045) and Welch Foundation (I-1603) (to J.J.).
20. Barnfield PC, Zhang X, Thanabalasingham V, Yoshida M, Hui CC (2005) Negative regulation of Gli1 and Gli2 activator function by Suppressor of fused through multiple mechanisms. Differentiation 73(8):397–405. 21. Fouix S, et al. (2003) Over-expression of a novel nuclear interactor of Suppressor of fused, the Drosophila myelodysplasia/myeloid leukaemia factor, induces abnormal morphogenesis associated with increased apoptosis and DNA synthesis. Genes Cells 8(11):897–911. 22. Cheng SY, Bishop JM (2002) Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex. Proc Natl Acad Sci USA 99(8): 5442–5447. 23. Monnier V, Dussillol F, Alves G, Lamour-Isnard C, Plessis A (1998) Suppressor of fused links fused and Cubitus interruptus on the Hedgehog signalling pathway. Curr Biol 8(10):583–586. 24. Méthot N, Basler K (2000) Suppressor of fused opposes Hedgehog signal transduction by impeding nuclear accumulation of the activator form of Cubitus interruptus. Development 127(18):4001–4010. 25. Wang G, Wang B, Jiang J (1999) Protein kinase A antagonizes Hedgehog signaling by regulating both the activator and repressor forms of Cubitus interruptus. Genes Dev 13(21):2828–2837. 26. Zhang Y, et al. (2013) Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci. Nat Commun 4:2608. 27. Cherry AL, et al. (2013) Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation. Acta Crystallogr D Biol Crystallogr 69(Pt 12):2563–2579. 28. Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci USA 104(9):3312–3317. 29. Zhang Q, et al. (2006) A Hedgehog-induced BTB protein modulates Hedgehog signaling by degrading Ci/Gli transcription factor. Dev Cell 10(6):719–729. 30. Wang QT, Holmgren RA (2000) Nuclear import of Cubitus interruptus is regulated by Hedgehog via a mechanism distinct from Ci stabilization and Ci activation. Development 127(14):3131–3139. 31. Seong KH, et al. (2010) Inhibition of the nuclear import of Cubitus interruptus by Roadkill in the presence of strong Hedgehog signal. PLoS ONE 5(12):e15365. 32. Akimaru H, et al. (1997) Drosophila CBP is a co-activator of cubitus interruptus in hedgehog signalling. Nature 386(6626):735–738. 33. Croker JA, Ziegenhorn SL, Holmgren RA (2006) Regulation of the Drosophila transcription factor, Cubitus interruptus, by two conserved domains. Dev Biol 291(2): 368–381. 34. Merchant M, et al. (2004) Suppressor of fused regulates Gli activity through a dual binding mechanism. Mol Cell Biol 24(19):8627–8641. 35. Dai P, et al. (1999) Sonic Hedgehog-induced activation of the Gli1 promoter is mediated by GLI3. J Biol Chem 274(12):8143–8152. 36. Zhou H, Kim S, Ishii S, Boyer TG (2006) Mediator modulates Gli3-dependent Sonic hedgehog signaling. Mol Cell Biol 26(23):8667–8682. 37. Hui CC, Angers S (2011) Gli proteins in development and disease. Annu Rev Cell Dev Biol 27:513–537. 38. Lin C, et al. (2014) Regulation of Sufu activity by p66β and Mycbp provides new insight into vertebrate Hedgehog signaling. Genes Dev 28(22):2547–2563.
Han et al.
Hedgehog
| Sufu | Ci | Gli | CBP
T
he Hedgehog (Hh) signaling pathway controls embryogenesis and adult tissue homeostasis by regulating the Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) family of zinc-finger transcription factors (1, 2). Initially identified as a suppressor of the segmentation defects caused by the loss of the serine/threonine kinase Fused (Fu) in Drosophila (3), Suppressor of fused (Sufu) plays a conserved negative role in Hh signal transduction by inhibiting the Ci/Gli transcription factors (4–6). Moreover, mutations in human Sufu predispose to medulloblastoma and meningioma (7, 8). Much of the attention in the past has been given to the role of Sufu in the cytoplasmic sequestration of Ci/Gli (5, 9–14). In addition, Sufu is also required for the production of the repressor form of Gli in mammals (15–17), a function carried out by the kinesin-like protein Costal2 (Cos2) in Drosophila (18). However, several studies have suggested that Sufu may also function in the nucleus to inhibit the activator form of Ci/Gli. For example, in Drosophila cos2 mutant wing discs, full-length Ci accumulated in the nucleus in a latent form inhibited by Sufu (18, 19). In cultured mammalian cells, overexpression of a truncated Sufu could inhibit Gli activity without sequestering it in the cytoplasm (20). Sufu can interact with several nuclear proteins, including the Drosophila myelodysplasia/myeloid leukemia factor and transcriptional corepressor complex Sin3–SAP18 (21, 22); however, a role for these nuclear factors in the regulation of Ci/Gli activity has not been demonstrated by a loss-of-function study (17). In this study, we observed that Sufu could still inhibit a fulllength Ci lacking the previously identified N-terminal Sufu-binding motif. Following up this unexpected observation, we identified a previously unidentified and conserved Sufu-binding motif located at the C terminus of Ci/Gli. We show that both the N- and C-terminal Sufu-interacting sites are required for optimal binding of Ci/Gli to Sufu as well as for effective inhibition of Ci/Gli by Sufu. We find that the N- and C-terminal sites can mediate cytoplasmic retention and nuclear inhibition of Ci/Gli by Sufu, www.pnas.org/cgi/doi/10.1073/pnas.1421628112
respectively. Furthermore, we provide evidence that binding of Sufu to Ci impedes the recruitment of the transcriptional coactivator CBP. Results Sufu Can Inhibit a Full-Length Ci Lacking the N-Terminal Sufu-Binding Site. Previous studies indicated that Ci/Gli binds Sufu through its
N-terminal domain containing an SYGH core motif (Fig. 1A) (11, 12, 23, 24), which we named SIN (Sufu-interacting site in the N-terminal region). We were surprised to observe that the transcriptional activity of a Ci variant (Ci-PKAΔN) lacking SIN was still inhibited by Sufu (Fig. S1). Of note, Ci-PKA has three PKA sites mutated to Ala and is no longer processed into a truncated repressor (CiR) (25). By using Ci-PKA as a backbone for structure– function study, we could focus on the regulation of the activator form of Ci (CiA) by Sufu. When SIN was deleted in CiGA1, in which the Ci sequence C-terminal to its Zn-finger DNA-binding domain was replaced by the Gal4 activation domain (GA), the resulting CiGA1ΔN was less inhibited by Sufu (Fig. S1), suggesting that Sufu can inhibit Ci through a region C-terminal to its Zn-finger DNA-binding domain. Sufu Interacts with a Conserved C-Terminal Site in Ci/Gli. To determine whether Sufu could interact with Ci through a domain(s) other than SIN, we divided Ci into three fragments—Ci1–439 (amino acids 1–439), Ci440–1160 (amino acids 440–1160), and Ci1161–1397 (amino acids 1161–1397)—and examined their interaction with Sufu by coimmunoprecipitation (CoIP) assay. When coexpressed with a Flag-tagged Sufu (Fg-Sufu) in S2 cells, all
Significance Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis by regulating the Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) family of transcription factors. Abnormal Hh pathway activity causes congenital disease and cancer. As a conserved negative regulator of the Hh signaling pathway, the tumor suppressor protein Suppressor of fused (Sufu) binds and inhibits Ci/Gli, but how Sufu contacts Ci/Gli and how Sufu–Ci/Gli interaction inhibits Hh signaling activity remain poorly understood. Here we identified a conserved Sufu-binding site in the C-terminal region of Ci/Gli. Further characterization of this Sufu-binding site provided insight into how Sufu blocks Ci/Gli activation in the nucleus. Understanding the mechanism by which Sufu regulates Ci/Gli activity is important for developing therapeutic treatment of cancers caused by abnormal Hh pathway activation. Author contributions: Y.H., Q.S., and J.J. designed research; Y.H. and Q.S. performed research; Y.H., Q.S., and J.J. analyzed data; and Q.S. and J.J. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
Y.H. and Q.S. contributed equally to this work.
2
To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1421628112/-/DCSupplemental.
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The tumor suppressor protein Suppressor of fused (Sufu) plays a conserved role in the Hedgehog (Hh) signaling pathway by inhibiting Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. Here we show that Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in addition to a previously identified N-terminal site (SIN), and that both SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. We show that Sufu can sequester Ci/Gli in the cytoplasm through binding to SIN while inhibiting Ci/Gli activity in the nucleus depending on SIC. We also find that binding of Sufu to SIC and the middle region of Ci can impede recruitment of the transcriptional coactivator CBP by masking its binding site in the C-terminal region of Ci. Indeed, moving the CBP-binding site to an “exposed” location can render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, our study identifies a previously unidentified and conserved Sufubinding motif in the C-terminal region of Ci/Gli and provides mechanistic insight into how Sufu inhibits Ci/Gli activity in the nucleus.
Fig. 1. Sufu binds Ci through both the N- and C-terminal domains. (A) A diagram of Ci domain structure and sequence alignment of SIN (blue bar) and SIC (green bar) within Ci and human (h) Gli proteins. ZF and CBP indicate the positions of the zinc-finger DNA-binding domain and CBP-binding domain, respectively. The SYGH motif is highlighted by a dashed box, and conserved residues in SIN and SIC are colored in blue and green, respectively. (B) Western blots (Left) and quantification (Right) of coimmunoprecipitation experiments from lysates of S2 cells coexpressing Fg-Sufu and various Myc-tagged Ci fragments (asterisks). The arrow indicates IgG. Data are means ± SD from two independent experiments. IB, immunoblotting; IP, immunoprecipitation. (C) Western blots of GST pull-down experiments. One or 5 μg of GST or GST fusion proteins was used as bait and coincubated with equal amounts of cell lysates from S2 cells expressing Fg-Sufu. (D) Western blots (Top) and quantification (Bottom) of coimmunoprecipitation experiments from lysates of S2 cells coexpressing Fg-Sufu and the indicated Myc-tagged Ci proteins. TCL, total cell lysates. Data are means ± SD from two independent experiments. (E) Western blots of a competition experiment. Equal amounts of immunopurified Fg-Sufu were incubated with cell extracts from S2 cells expressing a fixed amount of Myc-Ci1161–1397 and increasing amounts of Myc-Ci1–439. The arrow indicates IgG. (F) Western blots of coimmunoprecipitates from lysates of S2 cells coexpressing Fg-Sufu or Fg-SufuD154R with Myc-Ci1–439 or Myc-Ci1161–1397.
three Ci fragments coimmunoprecipitated Fg-Sufu, with Ci1–439 exhibiting 10-fold higher affinity than Ci440–1160 or Ci1161–1397 (Fig. 1B). Sequence alignment revealed a C-terminally conserved sequence motif present in Ci (amino acids 1370–1397), Gli2, and Gli3 (Fig. 1A). To determine whether this conserved sequence mediates the interaction between Ci and Sufu, we deleted it from Ci1161–1397 to generate Ci1160–1370, and found that Ci1160–1370 failed to bind Sufu (Fig. 1B). GST pull-down experiments revealed that GST-Ci1370–1397 pulled down Fg-Sufu derived from S2 cell extracts but 10-fold less effectively compared with GSTCi1–439 (Fig. 1C), suggesting that Ci1370–1397 contains a lowaffinity Sufu-binding site, which we name SIC (Sufu-interacting site in the C terminus). Simultaneous Binding to SIN and SIC Promotes Strong Sufu–Ci Association. We next determined how SIN and SIC contribute to
the overall binding of Sufu to Ci. Accordingly, we generated Myctagged Ci-PKA and its variants lacking either SIN (Ci-PKAΔN), SIC (Ci-PKAΔC), or both (Ci-PKAΔNΔC) (Fig. 2A), and compared their binding affinity toward Sufu in S2 cells using the CoIP assay. As shown in Fig. 1D, deletion of either SIN or SIC resulted in approximately fivefold reduction in Sufu binding, whereas their combined deletion nearly abolished Sufu binding. In a competition assay, we found that increasing the amount of Ci1–439 did not affect the association between Sufu and Ci1161–1397; instead, Sufu coimmunoprecipitated both Ci fragments (Fig. 1E), suggesting that SIN and SIC may bind different regions of Sufu instead of competing for the same binding pocket. Consistent with this notion, a Sufu variant (SufuD154R), which contains a point mutation at D154 implicated in contacting the SYGH motif (26, 27), exhibited diminished binding to Ci1–440 but interacted with Ci1161–1397 similar to the wild-type Sufu (Fig. 1F). Taken together, these observations 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1421628112
suggest that Sufu may simultaneously interact with both SIN and SIC, and that this multisite interaction greatly increases the overall binding affinity. Both SIN and SIC Contribute to Sufu-Mediated Inhibition of Ci. Having established that Sufu can bind Ci through both SIN and SIC, we next explored the functional significance of these Sufu-binding domains in mediating Ci inhibition. We first compared the activity of Ci-PKA, Ci-PKAΔN, Ci-PKAΔC, and Ci-PKAΔNΔC in the absence or presence of coexpressed Fg-Sufu using the ptc-luc reporter assay in S2 cells. As shown in Fig. 2B, Ci-PKA was suppressed by Sufu more effectively than Ci-PKAΔN and Ci-PKAΔC, whereas Ci-PKAΔNΔC exhibited little if any suppression by Sufu, suggesting that both SIN and SIC contribute to the Sufu-mediated inhibition of Ci. To determine the relative contribution of SIN and SIC to Sufu-mediated inhibition of Ci in vivo, UAS transgenes expressing individual Ci variants were introduced into flies at the same genomic locus using the phiC31 integration system to ensure similar levels of transcription of the transgenes (28). Wing discs expressing these transgenes using MS1096, a wing-specific Gal4 driver, in the absence or presence of a UAS-Sufu transgene were immunostained with Ci and Patched (Ptc) antibodies to monitor the levels of fulllength Ci (derived from both transgenic and endogenous expression) and Hh pathway activity, respectively. Quantification of full-length Ci levels in control and Ci-PKA–expressing wing discs indicated that exogenously derived Ci reached levels four- to fivefold higher than the endogenous levels in anterior (A)compartment cells distant from the anterior–posterior (A/P) boundary after subtracting the background signal, whereas in A-compartment cells near the A/P boundary, Ci-PKA–expressing wing discs contained full-length Ci at levels approximately twofold higher than the control discs (Fig. S2 A–C). Real-time Han et al.
SIN and SIC Mediate Sufu Inhibition Through Distinct Mechanisms.
Fig. 2. Both the N- and C-terminal Sufu-binding sites can contribute to Ci inhibition. (A) A diagram of full-length (FL), ΔN, ΔC, and ΔNΔC variants of Ci-PKA. SIN and SIC are indicated by blue and green bars, respectively. (B) ptcluc reporter assays in S2 cells transfected with Ci-PKA FL, ΔN, ΔC, and ΔNΔC alone or with Sufu. Data are means ± SD from three independent experiments. (C–K′) Late third-instar wing discs of wild type (C and C′) or expressing the indicated UAS-Ci transgenes either alone (D–G′) or together with a UASSufu transgene (H–K′) under the control of the MS1096 Gal4 driver were immunostained with anti-Ci (red) and anti-Ptc (green) antibodies to monitor the levels of full-length Ci derived from both transgenic and endogenous expression, and Hh pathway activity, respectively. Arrowheads and arrows indicate A and P compartments, respectively (H′–K′).
quantitative PCR revealed that Sufu mRNA levels in Sufuoverexpressing wing discs were approximately threefold higher than in control discs (Fig. S2D). CoIP experiments using wing disc extracts indicated that both Ci-PKAΔN and Ci-PKAΔC bound approximately fivefold less endogenous Sufu compared with Ci-PKA, whereas Ci-PKAΔNΔC exhibited no detectable binding to less endogenous Sufu (Fig. S3). In control wing discs, Hh is expressed in the posterior (P) compartment whereas Ci is expressed in the A compartment. Hh moves to the A compartment to stabilize Ci and activate target genes such as ptc in A-compartment cells near the A/P boundary (Fig. 2 C and C′). When Ci is ectopically expressed in P-compartment cells using the MS1096 Gal4 driver, it can ectopically activate downstream Hh target genes, and this ectopic activity is subject to suppression by Sufu, providing an additional place to assay Sufu–Ci regulatory interactions. Both full-length and Han et al.
We next determined how SIN and SIC mediate Ci inhibition by Sufu. Consistent with previous findings that blocking nuclear export promotes Ci nuclear accumulation (11, 30), when expressed alone in S2 cells, Myc-Ci-PKA, Myc-Ci-PKAΔN, and Myc-Ci-PKAΔC were accumulated mainly in the nucleus after the transfected cells were treated with the nuclear export inhibitor LMB (Fig. 3 A–C). Coexpression of Fg-Sufu retained Myc-Ci-PKA and Myc-Ci-PKAΔC but not Myc-Ci-PKAΔN in the cytoplasm (Fig. 3 E–G′). Furthermore, Fg-SufuD154R, which failed to bind SIN but exhibited normal binding to SIC (Fig. 1F), failed to retain Myc-Ci-PKA in the cytoplasm (Fig. 3 H and H′). Quantification of nuclear–cytoplasmic distribution of the various Ci constructs is shown in Fig. S5. When expressed in wing discs, Myc-Ci-PKAΔN exhibited more nuclear localization than Myc-Ci-PKA in A-compartment cells away from the A/P boundary, whereas Myc-Ci-PKAΔC behaved similarly to Myc-Ci-PKA (Fig. S6). Taken together, these observations suggest that Sufu–SIN interaction is essential for Sufu-mediated cytoplasmic retention of Ci. Despite failing to sequester Myc-Ci-PKA in the cytoplasm, Fg-SufuD154R still inhibited its transcriptional activity (Fig. 3J), suggesting that SufuD154R could inhibit Ci activity in the nucleus. To confirm this, we generated a constitutively nuclear form of Ci, HA-Nuc-Ci-PKA, in which an SV40 nuclear localization signal (NLS) was inserted at the N terminus of Ci (19) and a major nuclear export signal (NES) was mutated (31). When expressed in S2 cells, HA-Nuc-Ci-PKA was accumulated largely in the nucleus even in the presence of Fg-Sufu (Fig. 3 D, I, and I′ and Fig. S5); however, HA-Nuc-Ci-PKA activity was still sensitive to FgSufu–mediated inhibition (Fig. 3K). Whereas deleting SIN (ΔN) did not affect SufuD154R-mediated inhibition of Ci-PKA or Sufumediated inhibition of Nuc-Ci-PKA, deletion of either SIC (ΔC) or both SIN and SIC (ΔNΔC) abolished these inhibitions (Fig. 3 J and K), suggesting that Sufu can inhibit Ci activity in the nucleus depending on SIC. Interaction Between Sufu and the C-Terminal Half of Ci Interferes with CBP Recruitment. We next determined the mechanism by
which Sufu inhibits Ci in the nucleus. It has been shown that Ci activates its target genes by binding to the Drosophila CBP (dCBP) through a C-terminal binding domain between amino acids 1020 and 1160 (32). Because of the proximity between the PNAS Early Edition | 3 of 6
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truncated Ci-PKA induced ectopic expression of ptc along the A/P axis (Fig. 2 D′–G′). Coexpression of Sufu completely suppressed the ectopic ptc expression induced by Ci-PKA in both A- and P-compartment cells (Fig. 2H′ compared with Fig. 2D′). Coexpression of Sufu completely suppressed the ectopic ptc expression induced by Ci-PKAΔN or Ci-PKAΔC in A-compartment cells but only partially suppressed ectopic ptc expression induced by Ci-PKAΔN or Ci-PKAΔC in P-compartment cells (Fig. 2 I′ and J′ compared with Fig. 2 E′ and F′). By contrast, coexpression of Sufu did not block the ectopic ptc expression induced by Ci-PKAΔNΔC in either A- or P-compartment cells (Fig. 2K′ compared with Fig. 2G′). Hence, both SIN and SIC can contribute to Sufu-mediated inhibition of Ci. We noted that Ci-PKAΔNΔC induced ectopic ptc expression at lower levels compared with other Ci transgenes (Fig. 2G′ compared with Fig. 2 D–F′), likely due to its instability, because wing discs expressing Ci-PKAΔNΔC exhibited weaker Ci staining than those expressing other Ci transgenes (Fig. 2G compared with Fig. 2 D–F). Previous studies indicated that Sufu protects Ci/Gli from HIB/SPOP-mediated degradation (17, 29). Because Ci-PKAΔNΔC did not bind Sufu, it could be degraded by HIB more rapidly than other Ci variants. In support of this notion, we found that inactivation of HIB by RNAi stabilized Ci-PKAΔNΔC in wing discs, leading to elevated ptc expression at levels similar to those induced by Ci-PKA (Fig. S4).
Ci621–1020, but not to either Ci440–1370 or Ci1020–1397 (Fig. 4B), suggesting that simultaneous binding of Sufu to SIC and Ci621–1020 is likely to be required to impede dCBP recruitment. If Sufu inhibits Ci in the nucleus by impeding dCBP recruitment, we reasoned that moving the dCBP-binding domain (dCBP-BD) to an “exposed” location in Ci should overcome the inhibition by Sufu in the nucleus. Therefore, we fused a dCBP-BD to the C terminus of either Ci-PKA or Nuc-Ci-PKA after addition of a flexible linker protein, MBP, to separate SIC from the exogenously added dCBPBD (Fig. 4C). In the meantime, the endogenous dCBP-BD was deleted from these fusion proteins to generate CiΔCBP-MBP-CBP or Nuc-Ci-ΔCBP-MBP-CBP, respectively (Fig. 4C). We also generated several Ci constructs, including Ci-MBP, CiΔCBP-MBP, NucCi-MBP, and Nuc-CiΔCBP-MBP as controls (Fig. 4C). As shown in Fig. 4, addition of MBP to the C terminus of either Ci-PKA or NucCi-PKA did not affect their activity as well as Sufu- or SufuD154Rmediated inhibition (Fig. 4C, compare groups 2 and 6 with 1 and 5). Both CiΔCBP-MBP-CBP and Nuc-CiΔCBP-MBP-CBP were as active as Ci-PKA//Ci-PKA-MBP and Nuc-Ci-PKA/Nuc-Ci-PKA-MBP whereas neither CiΔCBP-MBP nor Nuc-CiΔCBP-MBP was active, suggesting that the activity of CiΔCBP-MBP-CBP and Nuc-CiΔCBP-MBP-CBP depends on the exogenously added dCBP-BD. Importantly, CiΔCBPMBP-CBP and Nuc-CiΔCBP-MBP-CBP were no longer suppressed by SufuD154R (Fig. 4C, groups 4 and 8). Furthermore, although Sufu still inhibited CiΔCBP-MBP-CBP, likely by sequestering it in the cytoplasm (Fig. 4C, group 4), Sufu failed to inhibit Nuc-CiΔCBPMBP-CBP (Fig. 4C, group 8). Taken together, these results further support the notion that Sufu can inhibit Ci in the nucleus by impeding dCBP recruitment. Fig. 3. SIN and SIC can regulate Ci by distinct mechanisms. (A–I′) Representative confocal images of S2 cells transfected with Myc-tagged Ci-PKA, Ci-PKAΔN, or Ci-PKAΔC or HA-tagged Nuc-Ci-PKA either alone or together with Flag-tagged Sufu or SufuD154R and immunostained with antibodies against Myc or HA (red) and Flag (green) and phalloidin for cell membrane (blue). Of note, transfected cells were treated with 20 ng/mL LMB for 3 h before immunostaining. (J and K) ptc-luc reporter assays in S2 cells transfected with the indicated Ci and Sufu constructs. Data are means ± SD from three independent experiments.
dCBP-binding domain and SIC, we speculated that binding of Sufu to SIC may impede the recruitment of dCBP. Therefore, we carried out CoIP experiments using nuclear extracts prepared from S2 cells transfected with HA-Nuc-Ci-PKA and Myc-tagged dCBP (Myc-dCBP) with or without cotransfection of Fg-Sufu. In the absence of Fg-Sufu, Myc-dCBP formed a complex with HA-Nuc-Ci-PKA; however, the association between Myc-dCBP and HA-Nuc-Ci-PKA was blocked by Fg-Sufu (Fig. 4A, lanes 1 and 2). Deletion of SIN (Nuc-Ci-PKAΔN) only slightly affected this blockage (Fig. 4A, lanes 3 and 4). By contrast, deletion of either SIC (Nuc-Ci-PKAΔC) or both SIN and SIC (Nuc-Ci-PKAΔNΔC) abolished the Sufu-mediated inhibition of dCBP–Ci association (Fig. 4A, lanes 5–8), suggesting that binding of Sufu to SIC may be essential for preventing dCBP–Ci association. Because SIC is an intrinsically weak Sufu-binding site (Fig. 1C), we speculated that SIC might cooperate with a Sufu-binding site located in the middle region of Ci to bind Sufu when the function of SIN was compromised. Indeed, Ci440–1397, which contains both SIC and the middle Sufu-binding site (Fig. 1B), exhibited approximately fivefold higher binding affinity to Sufu than Ci440–1160 and Ci1161–1397, both of which contain only one Sufu-binding site (Fig. S7A). Further mapping revealed that Sufu interacted with a Ci fragment (Ci621–1020) between the zinc-finger DNA-binding domain and the CBP-binding domain (Fig. S7B), consistent with the finding of a previous study showing that a Ci fragment containing amino acids 832–1187 interacted with Sufu in a yeast two-hybrid assay (33). Importantly, Fg-Sufu inhibited the binding of dCBP to Ci440–1397, which contains both SIC and 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1421628112
A Conserved Role of SIC in Sufu-Mediated Inhibition of Gli2. In the vertebrate Hh pathway, Gli2 and Gli3 are the primary transcription factors. We noticed that the sequence of SIC is highly
Fig. 4. Interaction of Sufu with the C-terminal half of Ci can impede CBP binding. (A and B) Western blots of coimmunoprecipitation experiments from lysates of S2 cells expressing the indicated constructs. Asterisks in B indicate HA-tagged Ci fragments. (C) ptc-luc reporter assays (Left) in S2 cells transfected with the indicated Ci constructs (diagrams are shown; Right) without or with Sufu or SufuD154R. Data are means ± SD from three independent experiments.
Han et al.
Fig. 5. A conserved role of SIC in mediating Sufu binding and nuclear inhibition of Gli2. (A) Western blots of coimmunoprecipitation experiment from lysates of NIH 3T3 cells expressing the indicated Gli2 and Sufu constructs. (B) Gli-luc reporter assays in NIH 3T3 cells transfected with the indicated Gli2 and Sufu constructs. Data are means ± SD from three independent experiments. (C) Representative confocal images of NIH 3T3 cells transfected with an the indicated mouse Myc-Gli2 and Sufu constructs and immunostained with an anti-Myc antibody (Left) and DNA dye Hoechst that labels the nucleus (Nuc) (Right).
conserved in both Gli2 and Gli3 (Fig. 1A). Because Gli2 contributes mostly to Gli activator activity, we focused on Gli2 using the approaches similar to what we have applied to Ci. Accordingly, we generated Myc-tagged full-length Gli2 and its variants with either SIN (Gli2ΔN), SIC (Gli2ΔC), or both (Gli2ΔNΔC) deleted. These constructs were cotransfected with a Flag-tagged mouse Sufu (mSufu) into NIH 3T3 cells, followed by CoIP experiments or Gli-luc reporter assays. Similar to Ci, deletion of either SIN or SIC from Gli2 greatly reduced its binding to mSufu, and their combined deletion nearly abolished the Gli2– mSufu association (Fig. 5A). Furthermore, both SIN and SIC contributed to mSufu-mediated inhibition of Gli2 activity, as deletion of either SIN or SIC only partially alleviated whereas their combined deletion almost completely blocked the mSufumediated inhibition (Fig. 5B). Immunostaining of the transfected cells with an antibody against Myc and a nuclear marker revealed that Myc-Gli2 was primarily localized in the nucleus when expressed alone but was sequestered in the cytoplasm when mSufu was cotransfected (Fig. 5C and Fig. S8). However, cytoplasmic retention of Gli2 by mSufu was abolished by the deletion of SIN (ΔN + mSufu; Fig. 5C and Fig. S8) but was unaffected by the deletion of SIC (ΔC + mSufu; Fig. 5C and Fig. S8). Consistent with a previous study (26), mSufuD159R, which is the counterpart of Drosophila SufuD154R, exhibited reduced binding to Gli2 (Fig. 5A) and failed to sequester Gli2 in the cytoplasm (Fig. 5C and Fig. S8). Nevertheless, mSufuD159R still inhibited the activity of Gli2 (Fig. 5B). Furthermore, this inhibition was abolished by deletion of SIC (ΔC or ΔNΔC) but was unaffected by deletion of SIN (ΔN). Taken together, these results suggest that Sufu can sequester Gli2 in the cytoplasm through binding to SIN but inhibit Gli2 activity in the nucleus through binding to SIC. Han et al.
Discussion As a conserved negative regulator of intracellular Hh signaling, Sufu binds and inhibits Ci/Gli. A conserved Sufu-binding site with an SYGH core motif (SIN) was identified in the N-terminal region of Ci/Gli, and structural information has recently been obtained on how Sufu binds this conserved site (26, 27); however, how Sufu interacts with full-length Ci/Gli remains a mystery, because the crystal structures of the Sufu–Gli complexes only contain a small peptide flanking the SYGH motif. Here we identified a conserved Sufu-binding site (SIC) at the C terminus of Ci/Gli. We provided evidence that Sufu may contact SIN and SIC simultaneously, which could be essential for effective Sufu– Ci/Gli association under physiological conditions. In addition, both SIN and SIC appear to be required for optimal inhibition of Ci/Gli but may regulate Ci/Gli through distinct mechanisms, with SIN primarily mediating cytoplasmic retention and SIC contributing to nuclear inhibition of Ci/Gli. Previous studies have suggested that Sufu can impede Ci/Gli nuclear localization and inhibit Ci/Gli transcriptional activity in the nucleus (5, 9–12, 19, 20); however, how Sufu exerts this dual regulation still remains poorly understood. Our recent study revealed that binding of Sufu to SIN may inhibit the binding of Kapβ2 to a NLS of the PY family located in the N-terminal region of Ci/Gli (13). Another recent study also provided evidence that binding of Sufu to Gli1 can preclude the binding of importin β1 (14). Taken together, these studies suggest that binding of Sufu to SIN may inhibit Ci/Gli nuclear import by masking its NLSs. Here we provide evidence that binding of Sufu to SIC can inhibit Ci transcriptional activity by impeding the recruitment of dCBP (Fig. 6). Indeed, deletion of SIC but not SIN abolished Sufu-mediated inhibition of Ci in the nucleus (Fig. 3K). By contrast, deletion of SIN but not SIC affected Sufu-mediated cytoplasmic retention of Ci (Fig. 3 E–G and Figs. S5 and S6). We found that both SIN and SIC were required for optimal binding of Sufu to Ci, because deletion of either site in the fulllength Ci background resulted in approximately fivefold reduction in Sufu binding affinity (Fig. 1 and Fig. S3). Therefore, we speculate that in anterior-compartment cells distant from the A/P boundary, where there is no Hh and the levels of full-length Ci (CiF) are low, Sufu may bind CiF by simultaneously contacting both SIN and SIC to prevent nuclear import of CiF. In A-compartment cells 5–10 cells away from the A/P boundary, where low to intermediate levels of Hh block Ci processing to increase the levels of CiF and promote nuclear translocation of CiF (11, 30),
Fig. 6. Diagrams of Ci bound by Sufu or CBP. The N- and C-terminal Sufubinding sites (SIN and SIC) and the CBP-binding domain are indicated by blue, green, and red bars, respectively. See text for details.
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DEVELOPMENTAL BIOLOGY
Intriguingly, Zhang et al. reported that mSufuD159R failed to inhibit Gli1 activity (26), which we confirmed in this study (Fig. S9). Although a previous study indicated that Sufu could interact with the C-terminal half of Gli1, the binding site was not mapped (34). Sequence alignment revealed that the C-terminal sequence of Gli1 diverges significantly from those of Gli2/3 and Ci (Fig. S9), suggesting that Gli1 may contain a less potent SIC, which could explain why mSufuD159R failed to inhibit Gli1.
SIN–Sufu interaction may be compromised, likely due to Hhinduced modification of Ci–Sufu. Under such conditions, SIC may cooperate with the middle region of Ci (Ci621–1020) to bind Sufu to mask the dCBP-binding site (Fig. 6). This could explain why low to medium levels of Hh can promote CiF nuclear translocation but fail to fully activate CiF. In A-compartment cells immediately abutting the A/P boundary, where peak levels of Hh convert CiF into CiA, both N- and C-terminal interactions may be compromised, allowing dCBP to be recruited to CiF (Fig. 6). In the mammalian Hh pathway, full-length Gli3 interacts with CBP and the MED12–Mediator complex through its C-terminal transactivation domain (35, 36). Gli1 and Gli2 also contain extended transactivation domains in their C-terminal regions (37). Therefore, it is conceivable that binding of Sufu to Gli proteins may also preclude coactivator binding. However, our study does not exclude the possibility that Sufu inhibits Ci/Gli in the nucleus 1. Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15(6): 801–812. 2. Briscoe J, Thérond PP (2013) The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14(7):416–429. 3. Préat T (1992) Characterization of Suppressor of fused, a complete suppressor of the fused segment polarity gene of Drosophila melanogaster. Genetics 132(3):725–736. 4. Ohlmeyer JT, Kalderon D (1998) Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 396(6713):749–753. 5. Ding Q, et al. (1999) Mouse Suppressor of fused is a negative regulator of Sonic hedgehog signaling and alters the subcellular distribution of Gli1. Curr Biol 9(19): 1119–1122. 6. Svärd J, et al. (2006) Genetic elimination of Suppressor of fused reveals an essential repressor function in the mammalian Hedgehog signaling pathway. Dev Cell 10(2): 187–197. 7. Taylor MD, et al. (2002) Mutations in SUFU predispose to medulloblastoma. Nat Genet 31(3):306–310. 8. Aavikko M, et al. (2012) Loss of SUFU function in familial multiple meningioma. Am J Hum Genet 91(3):520–526. 9. Méthot N, Basler K (1999) Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96(6):819–831. 10. Kogerman P, et al. (1999) Mammalian Suppressor-of-Fused modulates nuclearcytoplasmic shuttling of Gli-1. Nat Cell Biol 1(5):312–319. 11. Wang G, Amanai K, Wang B, Jiang J (2000) Interactions with Costal2 and Suppressor of fused regulate nuclear translocation and activity of Cubitus interruptus. Genes Dev 14(22):2893–2905. 12. Dunaeva M, Michelson P, Kogerman P, Toftgard R (2003) Characterization of the physical interaction of Gli proteins with SUFU proteins. J Biol Chem 278(7):5116–5122. 13. Shi Q, Han Y, Jiang J (2014) Suppressor of fused impedes Ci/Gli nuclear import by opposing Trn/Kapβ2 in Hedgehog signaling. J Cell Sci 127(Pt 5):1092–1103. 14. Szczepny A, et al. (2014) Overlapping binding sites for importin β1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization. Biochem J 461(3):469–476. 15. Kise Y, Morinaka A, Teglund S, Miki H (2009) Sufu recruits GSK3beta for efficient processing of Gli3. Biochem Biophys Res Commun 387(3):569–574. 16. Humke EW, Dorn KV, Milenkovic L, Scott MP, Rohatgi R (2010) The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins. Genes Dev 24(7):670–682. 17. Chen MH, et al. (2009) Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved. Genes Dev 23(16):1910–1928. 18. Zhang W, et al. (2005) Hedgehog-regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of Cubitus interruptus. Dev Cell 8(2):267–278. 19. Wang G, Jiang J (2004) Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms. Dev Biol 268(2): 493–505.
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through additional mechanisms such as recruiting a corepressor complex. Indeed, while our manuscript was under review, Lin et al. reported that Sufu inhibited Gli in the nucleus by recruiting p66β (38). We speculate that Sufu may inhibit Ci/Gli more effectively in the nucleus by simultaneously excluding coactivators and recruiting corepressors. Experimental Procedures Standard procedures for Drosophila genetics and tissue-culture experiments were used. Drosophila stocks, transgenes, and DNA constructs and detailed procedures for cell culture, transfection, cell fractionation, immunostaining, immunoprecipitation, Western blot analysis, GST pull-down assays, and luciferase reporter assays are described in SI Experimental Procedures. ACKNOWLEDGMENTS. We thank Bing Wang for assistance and the Developmental Studies Hybridoma Bank for reagents. This work was supported by grants from the NIH (GM061269, GM067045) and Welch Foundation (I-1603) (to J.J.).
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