YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation

Shh up-regulates YAP1 in primary CGNP cultures

SHH pathway-activated medulloblastomas are thought to arise from CGNPs in the developing cerebellum that depend on Shh signaling for their expansion during development. Therefore, we wished to determine whether the association of YAP1 with Shh signaling was conserved between cancer and developmental biology. We carried out quantitative RT–PCR analysis of mRNA extracts from primary CGNP cultures that were maintained without serum and that were treated with purified Shh protein, which sustains their proliferation, or with Shh vehicle, in whose presence these cells undergo differentiation. As shown in Figure 2A, Shh treatment resulted in rapidly increased levels of YAP1 mRNA. This increase was prevented in the presence of the Smoothened inhibitor cyclopamine, but not when cells were treated with the protein synthesis inhibitor cycloheximide, indicating that new protein synthesis is not required to up-regulate YAP1 expression. The increase in YAP1 mRNA levels was not maintained over time.

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Figure 2.

YAP1 mRNA and protein expression are up-regulated by Shh. (A) YAP1 mRNA expression in CGNPs treated as indicated was analyzed by real-time PCR and fold change is represented. There is a transient increase in YAP1 mRNA expression in Shh-treated cells with a maximum increase at 7 h. Treatment with cyclopamine (cyc) blocks YAP1 mRNA up-regulation, while treatment with cycloheximide (chx) does not. Statistically significant differences are indicated as (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (B) CGNPs were cultured in vitro for 48 h in the presence or absence of Shh, then treated with cyclopamine for 12 h when indicated. Shh treatment leads to YAP1 protein accumulation and reduced LATS1 phosphorylation. Both events are prevented in the presence of cyclopamine. (C) CGNPs were cultured in vitro for 48 h in the presence or absence of Shh ± cyclopamine and/or cycloheximide. Treatment with cyclopamine accelerates YAP1 protein degradation. (D) CGNPs cultured in the absence or presence of Shh were fixed and immunostained for YAP1 and Ki67. (Left) CGNPs expressing YAP1 are also Ki67 positive. (E) Cerebella of SW129 mice were immunostained for YAP1 at three developmental stages. (Left) At P7, YAP1 is expressed mainly in the EGL. (Middle) At P15, YAP1 is present in granule cells in the IGL, and in their processes in the molecular layer. In adult cerebella, we also detect YAP1 protein distributed sparsely throughout the IGL.

When we analyzed CGNP protein lysates by Western blotting after 48 h of Shh treatment, we detected high levels of YAP1 protein (Fig. 2B), together with a decrease in phospho-LATS (P-LATS), the kinase that phosphorylates and inactivates YAP1. To gain further insight into the mechanism through which Shh regulates YAP1, we treated CGNPs with cyclopamine for 12 h. This attenuated the Shh-induced increase in YAP1 protein and restored P-LATS1 levels (Fig. 2B). To determine whether Shh signaling regulates YAP1 stability, we carried out cycloheximide chases in the presence and absence of cyclopamine. As shown in Figure 2C, in the presence of Shh, YAP1 protein has a half-life of ∼4 h. In the presence of cyclopamine, YAP1 degradation occurs more rapidly, indicating a role for the Shh pathway in stabilizing YAP1 protein. Our results indicate that Shh mediates a transient increase in YAP1 expression and a sustained stabilization of YAP1 protein.

In the Shh-treated CGNPs, YAP1 was in cells undergoing proliferation, as determined by colocalization with the proliferation marker Ki67 (Fig. 2D; Supplemental Fig. 2B). During postnatal cerebellar development, CGNPs proliferate in the EGL. We carried out immunofluorescent staining of mouse cerebellar sections at three developmental stages: postnatal day 7 (P7), when CGNP proliferation is at its peak; P15, when proliferation is winding down; and in adulthood, when cerebellar development is complete. As shown in Figure 2E, YAP1 antibodies marked the EGL during the peak of CGNP proliferation. At P15, YAP1 was present in granule cells in the IGL, and in their processes in the molecular layer. Interestingly, we also detected YAP1 protein sparsely distributed throughout the IGL in adult mouse cerebella. Staining is specific, as determined by isotype control tests (Supplemental Fig. 3A). These results indicate that YAP1 protein is associated with CGNPs undergoing Shh-dependent proliferation in vitro and in vivo, and that YAP1 may play roles in later stages of cerebellar development as well.

Interactions with IRS1 regulate YAP1 localization

Recently, we demonstrated that Shh signaling promotes stabilization of IRS1, and that IRS1 is present in the nucleus of proliferating CGNPs (Fig. 3A, left; Parathath et al. 2008). Since we also detected YAP1 in the nucleus of CGNPs (Fig. 2D), and previous studies have implicated IRS1 in transcriptional regulation of proliferation-associated genes (Wu et al. 2008), we asked whether these two proteins interact. When we immunoprecipitated IRS1 from Shh-treated CGNPs, we detected YAP1 by Western blotting (Fig. 3A, right). Immunofluorescence analysis showed that, in the presence of Shh alone, YAP1 was present in the nucleus (Fig. 3B, top left). Interestingly, cyclopamine treatment caused YAP1 to leave the nucleus (Fig. 3B, top panel, second from left). YAP1 cytoplasmic localization was prevented by the addition of leptomycin, which inhibits the nuclear export protein CRM1 (Moroianu 1998). A role for IRS1 in promoting YAP1 nuclear localization is supported by our observation that retrovirus-mediated IRS1 expression retained YAP1 in the nucleus even in the presence of cyclopamine (Fig. 3B, bottom panel, left), while overexpression of GFP alone did not (Fig. 3B, top right). Cells that were infected with IRS1 possess nuclear YAP1 (Fig. 3B, bottom panel, second from left; Supplemental Fig. 3B). Conversely, shRNA lentivirus-mediated IRS1 knockdown prevented YAP1 nuclear accumulation (Fig. 3B, bottom panel, second from right). The knockdown efficiency was close to 80% (Fig. 3B, bottom right).

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Figure 3.

IRS1 interacts with YAP1 and regulates its nuclear accumulation. (A) Subcellular fractionation of CGNPs cultured in the presence or absence of Shh. (Left) The greatest accumulation of IRS1 protein after Shh treatment takes place in the nucleus. (Right) Immunoprecipitation of IRS1 in Shh-treated CGNPs brings down YAP1. (B, top panel, left) Immunostaining shows that YAP1 is mainly nuclear in Shh-treated CGNPs. (Top panel, second from left) In the presence of cyclopamine, YAP1 is excluded from the nucleus. (Top panel, second from right) In the presence of cyclopamine and leptomycin, YAP1 accumulates in the nucleus. Overexpressing IRS1 in the presence of cyclopamine leads to YAP1 accumulation in the nucleus (bottom panel, left), while overexpressing control viruses (GFP) does not (top panel, right). (Bottom panel, second from left) Cells that get infected with IRS1 (GFP reporter) viruses have nuclear YAP1. (Bottom panel, second from right) IRS1 knockdown prevents YAP1 from accumulating in the nucleus. (Bottom panel, right) The knockdown efficiency is shown by Western blot. (C) Immunoprecipitation of IRS1 in the Pzp53 medulloblastoma cell line coprecipitates YAP1. (Left) The YAP1:IRS1 interaction was decreased in the presence of cyclopamine. (Right) IRS1 was also detected in YAP1 immunoprecipitates. (D) Subcellular fractionation of Pzp53 cells and subsequent IRS1 immunoprecipitation. YAP1 was detected in both the nuclear and cytoplasmic precipitates. (E) Pzp53med cells were treated with leptomycin, fixed, and immunostained for YAP1 and IRS1. (Top row) In untreated cells, YAP1 and IRS1 are both nuclear and cytoplasmic. (Bottom row) In leptomycin-treated cells, there is an accumulation of both proteins in the nucleus. (F) Immunoprecipitation of CRM1 in Pzp53 cells. Both YAP1 and IRS1 were detected in the immunoprecipitate.

To further investigate whether YAP1:IRS1 interactions play roles in YAP1 nuclear localization, we turned to Pzp53med cells, a cell line derived from a Ptc^+/−/p53^−/− mouse medulloblastoma (Berman et al. 2002) that has been used to model Shh-mediated proliferation events (Corcoran and Scott 2001). As shown in Figure 3C, we detected YAP1 in the IRS1 immunoprecipitate from Pzp53med cells. When we carried out the reciprocal immunoprecipitation, we detected IRS1 in YAP1 immunoprecipitates. The interaction between IRS1 and YAP1 was reduced in the presence of cyclopamine. When we carried out subcellular fractionation, we found that IRS1 and YAP1 interacted in both the cytoplasm and the nucleus (Fig. 3D), suggesting that IRS1 may interact with both nuclear and cytoplasmic (phosphorylated) YAP1. Indeed, in IRS1 immunoprecipitates, we can detect phosphorylated YAP1 (Supplemental Fig. 4A). Consistent with a role for CRM1 in mediating IRS1 and YAP1 export to the cytoplasm, when we treated the Pzp53med cells with leptomycin, we observed an accumulation of both proteins in the nuclear fraction (Fig. 3E). Moreover, when we immunoprecipitated CRM1, the protein inhibited by leptomycin, we were able to detect both IRS1 and YAP1 (Fig. 3F). Taken together, these results suggest a model in which Shh stabilizes IRS1 (Parathath et al. 2008) and induces YAP1, and that IRS1:YAP1 interactions regulate their subcellular localization.

YAP1 interacts with TEAD1 in CGNPs and medulloblastoma cells

It has been shown recently that YAP1 interactions with the TEAD family of transcription factors are important for maintaining the neural progenitor state of cells in the developing chick spinal cord (Cao et al. 2008). Since Shh inhibits differentiation of CGNPs (Wechsler-Reya and Scott 1999), we wished to determine whether the YAP1:TEAD interaction was conserved in mammalian cerebellar neural progenitors. We first asked whether Shh treatment affected expression of TEAD1, but we found no effects of Shh treatment on TEAD1 mRNA levels in CGNPs (data not shown). Instead, as we found with YAP1, Shh promoted the accumulation of TEAD1 protein. There is no accumulation of TEAD1 in vehicle-treated cells in the presence of the proteasome inhibitor lactacystin, indicating that, even though the mRNA may be present at low levels, it is not being translated in the absence of Shh. However, in the presence of lactacystin in Shh-treated cells, there is an accumulation of TEAD1 (Fig. 4A).

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Figure 4.

TEAD1 interacts with YAP1 in CGNPs. (A) CGNPs were cultured for 48 h in the presence or absence of Shh and treated with cyclopamine (12 h) or lactacystin (6 h) as indicated. (Left) In the presence of Shh, there is an accumulation of TEAD1 protein that is blocked by cyclopamine. (Right) In the presence of lactacystin and Shh, there is an accumulation of TEAD1 compared with Shh alone. (B) Immunoprecipitation of YAP1 and TEAD1 in Pzp53 cells and CGNPs. TEAD1 was detected in YAP1 precipitates in both Pzp53 and Shh-treated CGNPs (top panel) and vice versa (bottom panel). (C) Immunoprecipitation of TEAD1 and IRS1 in Pzp53 cells and CGNPs. TEAD1 was detected in IRS1 immunoprecipitates in Pzp53 and Shh-treated CGNPs (top panel) and vice versa (bottom panel). (D) Cerebella of SW129 P7 mice were immunostained for TEAD1. (Left) TEAD1 is found in the EGL. (Right) TEAD1 and YAP1 are coexpressed in the EGL of the cerebellum. (E) CGNPs were cultured for 48 h and immunostained for TEAD1, YAP1, and Ki67. (Top left) In the absence of Shh, TEAD1 protein expression is very low. (Top right) In the presence of Shh, TEAD1 accumulates. (Bottom left) YAP1 and TEAD1 are localized in the nucleus. (Bottom right) TEAD1 is coexpressed with Ki67.

As shown in Figure 4B, when we immunoprecipitated endogenous YAP1, we could detect TEAD1 by Western blotting, and vice versa. Moreover, TEAD1 and IRS1 also interact, based on coimmunoprecipitation experiments carried out in Pzp53med cells and CGNPs (Fig. 4C). In the developing cerebellum, TEAD1 was found in the EGL (Fig. 4D, left) where it colocalizes with YAP1 (Fig. 4D, right). Consistent with our Western blot analyses, immunofluorescent staining revealed up-regulation of YAP1 and TEAD1 in Shh-treated CGNPs (Fig. 4E, top panels). In primary CGNP cultures treated with Shh, we found both YAP1 and TEAD1 in the nucleus (Fig. 4E, bottom left panel). A high percentage of cells expressing TEAD1 also express YAP1. All YAP1 and many TEAD1-positive cells express the proliferation marker Ki67 as well (Fig. 4E, bottom right; Supplemental Fig. 2B). Taken together, these results suggest that YAP1 and TEAD1 function in a complex in Shh-treated proliferating CGNPs, consistent with the study by the Gage group (Cao et al. 2008). However, we cannot rule out the possibility that TEAD1 has additional interacting partners in CGNPs. Likewise, YAP1 may function in additional complexes in Shh-stimulated CGNPs. Our future studies will identify novel YAP1 interactors in cerebellar precursors.

YAP1 is required to sustain CGNP proliferation

Shh is the obligate mitogen for CGNPs; when it is withdrawn from their culture medium, CGNPs leave the cell cycle within 6 h (Kenney and Rowitch 2000) and begin to differentiate. Downstream effectors of Shh mitogenic signaling include Gli1, Gli2, N-myc, and IRS1. It has been shown that ectopic expression of these genes can drive CGNP proliferation even in the absence of Shh (Kenney et al. 2003; Oliver et al. 2003; Parathath et al. 2008). We wished to determine whether YAP1, too, could sustain CGNP proliferation and/or synergize with exogenous Shh. We used retroviral transduction to express YAP1 in CGNPs that had been pretreated with Shh to maintain their proliferative state, then maintained them in culture with or without Shh for 24 h. Ectopic YAP1 expression significantly increased proliferation in cells grown without Shh (Fig. 5A,B), and YAP1 overexpression also elicited a 2.5-fold increase in proliferation in the presence of Shh. However, in cultures left without Shh after infection, which had a similar infection efficiency as those grown in Shh post-infection (Fig. 5C, top panel), proliferation was less than in Shh-treated cultures, indicating that YAP1 alone is not sufficient to completely recapitulate CGNP mitogenic response to Shh, perhaps due to the requirement for Shh signaling to up-regulate YAP1’s binding partner TEAD1, which is nearly absent from vehicle-treated cells (Fig. 3). In both conditions, the majority of cells expressing Ki67 are the ones expressing YAP1 (Fig. 5C, bottom). As shown in Figure 5D, we achieved high levels of YAP1 expression, and this was associated with increased levels of cyclin D1 and cyclin D2—markers for CGNP proliferation.

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Figure 5.

YAP1 overexpression induces proliferation of CGNPs. (A) CGNPs were transduced with YAP1-expressing retroviruses. Cells were immunostained for Ki67. (Left) In the absence of Shh, few cells proliferate. When YAP1 is overexpressed in the absence of Shh, proliferation increases (second from the left), although not to the same extent as in the presence of Shh (second from the right). (Right) Overexpression of YAP1 in the presence of Shh leads to increased proliferation. (B) Automated quantification of Ki67 staining in CGNPs transduced with GFP or YAP1 retroviruses. Three different fields were considered in each case. Statistically significant differences are indicated as (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (C, top panel) GFP immunostaining shows similar infection efficiency in Shh-treated cells and cells from which Shh was withdrawn after infection. (Bottom panel) YAP1 + Ki67 immunostaining shows that the majority of cells expressing Ki67 are the ones expressing YAP1. (D) Western blot showing the increase in YAP1 protein expression after viral transduction. YAP1 overexpression leads to cyclin D1 and cyclin D2 up-regulation, reflecting an increase in proliferation. (E) Ki67 immunostaining in CGNPs infected with YAP1 shRNAs shows a decrease in proliferation compared with control shRNA-infected cells. (F) Western blot showing YAP1 protein levels were reduced by 65% in cells infected with YAP1 shRNA lentiviruses. Cyclin D2 levels were dramatically decreased with only a subtle increase in cleaved caspase 3. (G) CGNPs were transduced with YAP1-expressing retroviruses. After 24 h, Gli2 mRNA expression was analyzed by real-time PCR. YAP1 overexpression induces a statistically significant induction in Gli2 mRNA levels in the absence of Shh. (*) P < 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001. (H) ChIP analysis was carried out to test the presence of YAP1 on the Gli2 promoter. Four regions containing TEAD1-binding sites were assessed. Fold enrichments normalized to the level observed at the control region are shown. Statistically significant differences are indicated as (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

YAP1 is not only sufficient to increase CGNP proliferation but is also required to sustain their proliferation, since knocking down YAP1 expression in the presence of Shh leads to a dramatic decrease in proliferation with only a subtle increase in apoptosis (Fig. 5E,F). Our observation that YAP1 is required to sustain Shh-induced CGNP proliferation is in agreement with previous studies that have shown that YAP1 is limiting for proliferation (Buttitta and Edgar 2007; Pan 2007; Zhao et al. 2007; Zeng and Hong 2008). A database search (http://www.switchdb.com/motifs) revealed four TEAD1-binding sites in the promotor of the Shh effector Gli2 (Supplemental Fig. 4B). Consistent with YAP1-mediated Gli2 induction contributing to YAP1’s proliferative effects in CGNPs, we observed increased expression of Gli2 in vehicle-treated CGNPs infected with YAP1 retroviruses. In the presence of Shh, Gli2 expression was already at maximal levels (Fig. 5G). In order to confirm a role for YAP1/TEAD1 in regulating the Gli2 promotor, we conducted a chromatin immunoprecipitation (ChIP) assay to scan for YAP1 binding in the regions that we found to contain putative TEAD1-binding sites. As shown in Figure 5H, we found statistically significant evidence that YAP1 binds to two out of the four TEAD1-binding sites that we analyzed. These results suggest that one mechanism through which YAP1 could drive CGNP proliferation is induction of Gli2, but the inability of YAP1 to fully recapitulate the Shh proliferative response indicates roles for other transcription factors in regulating expression of Gli2 and other Shh mitogenic effectors.

Culture of CGNPs

CGNP cultures were generated as described previously (Kenney and Rowitch 2000). Cells were plated on individual poly-DL-ornithine (Sigma) precoated plates or precoated glass coverslips. Where indicated, Shh (R&D Systems) was used at a concentration of 3 μg/mL, lactacystin (Calbiochem) was used at 10 μM, leptomycin (Sigma) was used at 1 nM, cycloheximide (Sigma) was used at 10 μg/mL, and Cyclopamine (R&D Systems) was used at 1 μg/mL.

Retrovirus production and infection

The YAP1 cassette was cloned from pBabe-YAP1 (Addgene) into the retroviral vector pWzl-IRES-GFP. 293 EBNA (Invitrogen) packaging cells were cotransfected with gag-pol and VSVg packaging plasmids plus pWzl-YAP1-IRES-GFP, pIG-IRS1-IRES-GFP (Parathath et al. 2008) or control pWzl-eGFP, using Fugene 6 transfection reagent (Roche). The media was changed 12 h after transfection and supernatants (8 mL) were harvested at 24 and 48 h, filtered through 0.45-μm syringe filters, and pooled. For infection, Shh-treated CGNPs were exposed to the viral supernatants for 3 h. Viral supernatant was then removed and replaced with fresh medium ± Shh as indicated. Cells were cultured for 48 h post-infection.

shRNA lentiviruses

293T packaging cells were cotransfected with VSVg and Delta 8.9 packaging plasmids plus Mission shRNA lentiviral plasmids (Sigma) targeting YAP1 (TRCN0000095864, TRCN0000095865, TRCN0000095866, TRCN0000095867, and TRCN0000095868) and IRS1 (TRCN0000105880, TRCN0000105881, TRCN0000105882, TRCN0000105883, and TRCN0000105884). eGFP shRNA was used as control vector (SHC005V). Specificity of knockdown for each construct was confirmed as described previously (Parathath et al. 2008). Viral supernatants for each construct were pooled, such that cells would be infected with five different lentivirsues targeting YAP1 or IRS1. Shh-treated CGNPs were exposed to the viruses for 3 h, then treated with fresh medium ± Shh and culture for 36 h.

RNA extraction and real-time PCR

Total RNA from CGNPs, cell lines, or tissues was extracted and purified using the MiRvana kit (Ambion). cDNA was prepared from 1 μg of total RNA by using iScript cDNA Synthesis kit (Bio-Rad). Quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). RNA expression data were acquired and analyzed using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Average results and standard errors are presented.

Primer sequences used were as follows: GAPDH, 5′-TGGAAGGACTCATGACCACA-3′; GAPDHR, 5′-TTCAGCTCAGGGATGACCTT-3′; YAP1F, 5′-CAGGAATTATTTCGGCAGGA-3′; YAP1R, 5′-CATCCTGCTCCAGTGTAGGC-3′; TEAD1F, 5′-CTCAGGACGGGAAAGACAAG-3′; TEAD1R, 5′-TTCCTTCTGGCAAGAACCTG-3′; Gli2F, 5′-GCAGACTGCACCAAGGAGTA-3′; Gli2R, 5′-CGTGGATGTGTTCATTGTTGA-3′; Gli1F, 5′-TGGACAAGTGCAGGTAAAACC-3′; Gli1R, 5′-AATCCGGTGGAGTCAGACC-3′.

Protein preparation, immunoprecipitation, subcellular fractionation, and immunoblotting

For immunoblot analysis, cells were washed once in PBS and protein extracts were prepared as described previously (Kenney and Rowitch 2000). Subcellular fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) following the manufacturer's instructions. Protein content was determined by using the Bio-Rad protein assay. Fifty micrograms of each sample were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 8% polyacrylamide gels and then transferred in 20% methanol buffer at 4°C to Immobilon polyvinylidene difluoride (Millipore) membranes. Membranes were blocked in 5% milk or 3% bovine serum albumin, and immunoblotting was carried out according to standard methods. Antibodies used for Western blotting were YAP1 (Abcam and Cell Signaling), phospho-YAP1 (Cell Signaling), TEAD1 (BD Transduction Laboratories), P-LATS1 (Cell Signaling), IRS1 (Cell Signaling), Cyclin D2 (Santa Cruz Biotechnologies), GAPDH (Cell Signaling), c-jun (Calbiochem), and β-tubulin (Sigma). Donkey anti-mouse HRP-linked secondary was from Jackson Research Laboratories, and goat anti-rabbit was from Thermo Scientific. Peroxidase activity was detected using Amershams's ECL reagents and exposing membranes to Kodak Biomax film.

For immunoprecipitation studies, 1 mg of protein extract was used in each case. Ten micrograms of antibody were incubated with protein A-sepharose for 2 h. Protein extracts were precleared with protein A-sepharose for 2 h, and then incubated with the antibody plus protein A-sepharose overnight. The precipitate was washed four times and proteins were eluted with 0.2 M glycine. Antibodies used for immunoprecipitation were IRS1 (Cell Signaling), YAP1 (Abcam), TEAD1 (BD Transduction Laboratories), CRM1 (Santa Cruz Biotechnologies), and mouse and rabbit IgG (Upstate Biotechnologies).

ChIP

TEAD1-binding sites found in 5 kb upstream of Gli2 gene were considered. ChIP was carried out with the ChIP assay kit (Millipore) according to the manufacturer's instructions. Chromatin was isolated from Pzp53 medulloblastoma cells and precipitated with YAP1 antibody (Santa Cruz Biotechnologies), control Histone 3 antibody (Abcam), and IgG isotype control (Upstate Biotechnologies). Primer sequences used for quantitative PCR measurement of immunoprecipitated promotor fragments were as follows: Binding site 1 (BS1) forward, 5′-AGCACGTAGCGCAGTAGACA-3′; BS1 reverse, 5′-GAAGGCAGGATTCCCTGTTA-3′; BS2 forward, 5′-CCTCTCCCTAAACTCCCACA-3′; BS2 reverse, 5′-TTGGCCATTTTGTCTCCTCT-3′; BS3 forward, 5′-CCCAGTGACAGACCTTTTCC-3′; BS3 reverse, 5′-AGTCTCATCCACTGCAATGCT-3′; BS4 forward, 5′-GGCTCGAAAGAGATGTGACC-3′; BS4 reverse, 5′-ATGCCTGAGGACGCTTAGAA-3′; No BS forward, 5′-CTGAGGCAGTCGAAGGAGAG-3′; No BS reverse, 5′-ACACTGGCTGCCAAAATGTA-3′. No Binding site primers were selected 10 kb upstream of Gli2 gene, in a region that does not contain TEAD1-binding sites. Threshold cycles (Ct) were determined for both immunoprecipitated DNA and a known amount of DNA from the input sample for different primer pairs. Fold enrichments were calculated by determining the immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample) and were normalized to the level observed at the control region with no binding sites.

Immunofluorescence

Frozen sections (10 μm) were dried and then boiled in 0.01 M citric acid for 15 min for antigen retrieval. For paraffin-embedded sections, tissues were first dewaxed and rehydrated prior to antigen retrieval. CGNPs were grown on poly-DL-ornithine-coated glass coverslips as described previously (Parathath et al. 2008). The cells were fixed with 4% paraformaldehyde for 20 min. Sections and cells were analyzed by immunoflorescence according to standard methods. Antibodies used for immunofluerescence were Ki67 (Vector Laboratories), IRS1 (Upstate Biotechnologies), YAP1 (Abcam and Cell Signaling), TEAD1 (BD Transduction Laboratories), CD31 (BD Transduction Laboratories), GFAP (Cell Signaling), CD15 (Abcam), Nestin (Covance), Cleaved Caspase 3 (Cell Signaling), and mouse and rabbit IgG (Upstate Biotechnologies).

Image capturing

Staining of cultured primary cells and tissue sections was visualized with a Leica DM5000B microscope and images were taken using Leica FW400 software. For quantification, TIFF images of four random fields were taken for each experimental group using the 20× objective, and average pixel intensities were measured using Volocity software.

Human tumor collection and expression analysis

Exon array profiling and data analysis with tumor subgrouping were performed as published (Northcott et al. 2009a).

We are grateful to Dolores Hambardzumyan, Rebecca Bish, Massimo Squatrito, Eric Holland, Claudio Alarcon, Alexia-Ileana Zaromytidou, Joan Massague, and Mike Overholtzer for providing advice and reagents. A.F.-L receives fellowship support from the Spanish Ministry of Education. These studies were supported by funding from the NIH (NINDS R01NS061070) and Alex's Lemonade Stand Foundation to A.M.K.