Histone Acetyltransferase PCAF Is Required for Hedgehog-Gli- Dependent Transcription and Cancer Cell Proliferation

Author Manuscript Published OnlineFirst on August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4660 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Histone acetyltransferase PCAF is required for Hedgehog-Gli- dependent transcription and cancer cell proliferation

Martina Malatesta1,2, Cornelia Steinhauer1,2, Faizaan Mohammad1,2, Deo P. Pandey1,2, Massimo Squatrito3 and Kristian Helin1,2,4

1Biotech Research and Innovation Centre (BRIC) and 2Centre for Epigenetics, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark. 3F-BBVA Cancer Cell Biology Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, Madrid E-28029, Spain 4The Danish Stem Cell Center (Danstem), Blegdamsvej 3, DK-2200 Copenhagen, Denmark

Running title: PCAF is required for GLI-dependent transcription Keywords: PCAF/Hedgehog/GLI/Glioblastoma/Medulloblastoma/Histone acetyltransferase

Grant Support: The Danish National Advanced Technology Foundation, The Danish National Research Foundation, the Danish Medical Research Council, the Lundbeck Foundation, and the Novo Nordisk Foundation.

Corresponding author and author who should receive reprint requests:

Kristian Helin BRIC University of Copenhagen Ole Maaløes Vej 5 DK-2200 Copenhagen N E-mail: [email protected]

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4660 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

PCAF is required for GLI-dependent transcription

Abstract The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has thus emerged as an attractive approach for anticancer therapy, however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we demonstrate that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. We also found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally we observed that PCAF silencing reduces the tumor forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.

PCAF is required for GLI-dependent transcription

Introduction

The evolutionary conserved Hh signaling pathway plays an important role in development, proliferation and stem cell maintenance (1) (2). In agreement with such role, deregulation of the Hh pathway leads to several developmental syndromes and tumors of different tissues (3, 4). In mammals, Hh signaling is initiated when the secreted Hh proteins bind to and inhibit the transmembrane receptor PTCH. The interaction between Hh and PTCH releases Smoothened (SMO), a second transmembrane protein, which in turn induces the downstream components of the Hh signaling pathway and leads to the activation of the Glioma-associated oncogene (GLI) transcription factor family (5). Once activated, the GLI proteins induce the expression of genes that regulate multiple cellular functions such as cell cycle progression, proliferation and apoptosis (6, 7). GLI1 and PTCH are also activated by Hh signaling suggesting a regulation of the pathway through both a positive and a negative feedback (8). The importance of the Hh pathway in tumorigenesis was first discovered in Gorlin syndrome patients. This rare pathology is caused by an inactivating mutation in PTCH, which leads to development of tumors like basal cell carcinoma, medulloblastoma and rhabdomyosarcoma (9). Inappropriate activation of Hh signaling has also been shown to lead to development of tumors in the lung, the gastrointestinal tract and the pancreas (10-12). GLI1 was originally isolated as a highly amplified gene in human malignant glioma and subsequently found amplified in other tumor types, including liposarcoma, rhabdomyosarcoma and osteosarcoma (9, 13, 14). GLI1 is a key regulator of glioma growth and of cancer stem cell self-renewal. Moreover studies in transgenic mice have shown that ectopic expression of Gli1 is sufficient to induce basal cell carcinoma (15-17). Despite the importance of the Hh pathway in development and disease, the molecular mechanisms by which the Hh signal is leading to the activation of GLI- regulated transcription is still not completely understood. Here we show that transcriptional activation by the Hh pathway requires the histone acetyltransferase PCAF.

PCAF is required for GLI-dependent transcription

Materials and Methods

Cell Culture and Reagents Human cell lines (U87, U118, T98G and Daoy) were purchased from American Type Culture Collection and they were used at low passage numbers. A Short Tandem Repeat profile was performed by ATTC, and we tested them negative for mycoplasm. 293-ShhN and NIH3T3 cells were kindly provided by Dr. Taipale (Karolinska Institute, Sweden) and grown as previously described (18). Cells were maintained at

37°C and 5% CO2 in Dulbecco´s modified Eagle´s medium supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin and 10% FBS (Gibco) (U87, U118, T98G, DAOY and 293-ShhN), or 10% NCS (NIH3T3). GBM543 neurospheres were isolated from patients suffering from GBMs and propagated as previously described (19). Ink4a-Arf-null NSCs were isolated and grown as previously described (20). Viral transductions were performed using pLKO vectors. The different target sequences are available in Supplementary Table 1. Cells were transduced with lentiviral particles for 16 h, and selected with 2 µg/ml Puromycin (Invitrogen) 48h after transduction. Treatments using SAG (ALX-270-426-M001 Alexis), Cyclopamine (C4116 SIGMA) or AA (G5173 SIGMA) were performed 24h after cell plating or as indicated. For cell proliferation assays, cells were seeded at 2 different densities into 96 well plates (Nunc#161093) and 3 images per well were acquired every hour over a period of 3-5 days using the Incucyte Zoom (Essen Biosciences). Image analysis was performed using the Incucyte Zoom software package.

siRNA Screening and Luciferase Assay The screening was performed on a customized siRNA library against 17 different mouse acetyltransferases with each gene represented by three independent siRNA constructs (SIGMA). The different target sequences are available in Supplementary Table 1. In addition each plate contained four non-targeting controls and four siRNAs against Smo as positive controls. The automated screening was performed using a MicrolabSTAR liquid handling system (Hamilton Robotics). NIH3T3 reporter cells (21) were reverse transfected with the customized siRNA library including individual positive and negative controls using Lipofectamine 2000 (Invitrogen) following manufacturer’s instructions. After 24 hours transfection medium was exchanged to

PCAF is required for GLI-dependent transcription

conditioned medium containing Shh (derived from 293-ShhN cells) or respective culture medium and incubated for another 48 hours. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Kit (Promega) according to the manufacturer´s protocol.

Screening data analysis and statistics Individual Firefly-intensities were normalized over their corresponding Renilla- readings and further normalized to the median sample score of each plate. From this a gene-based hit list was generated using the statistical method “redundant siRNA activity” (RSA) analysis (22). The RSA method first ranked individual siRNAs according to their normalized scores and then calculated p-values for each gene based on the likelihood for this distribution of siRNA ranks to occur by chance. This calculation of p-values was based on the iterative hypergeometric distribution. The entire statistical analysis was conducted using the statistical software ‘R’.

RNA extraction and gene expression analysis Total RNA was isolated using the RNAeasy Minikit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the TaqMan Reverse Transcription kit (Applied Biosystems). qPCR was performed using SYBR Green 2 PCR Master mix (Applied Biosystems) on an ABI Prism 7300 Real-Time PCR system (Applied Biosystems) or on a LightCycler 480 System (Roche Applied Science), using the LightCycler 480 SYBR Green I Master mix (Roche Applied Science) according to the manufacturers’ instructions. Error bars represent standard deviation of three PCR amplifications for each sample. Similar results were obtained in at least three independent experiments. The qPCR primers are available in Supplementary Table 2.

Immunoblotting and immunoprecipitation To prepare whole cell extracts for immunoblotting analysis cells were lysed in high salt buffer S300P (50mM Tris-HCl, 300mM NaCl, 0.5% Igepal, 1mM EDTA, 1mM DTT, 1mM PMSF, 1 μg/μl leupeptin, 1 μg/μl aprotinin). For immunoprecipitations, protein A/G-agarose beads (GE Healthcare) were pre-coupled O/N with the indicated antibodies. Equal amounts of protein lysates (S300P buffer) were used for each

PCAF is required for GLI-dependent transcription

immunoprecipitation. Immunoprecipitates were eluted from beads and analyzed by immunoblotting with the indicated antibodies. The identity and the suppliers of the antibodies are provided in Supplementary Table 3.

Chromatin immunoprecipitation assay ChIP was performed as described (23). For each IP, 0.5–1mg of chromatin was used except for histones and histone modifications where 100 µg were used. The qPCR primers are available in Supplementary Table 2, and the antibodies in Supplementary Table 3.

Orthotopic transplantation Transformed NSCs (tNSCs, Ink4a-Arf-/- NSCs expressing constitutively active EGFR as well as luciferase) were cultured in serum free NSC medium containing growth factors (human recombinant EGF and FGFb). A total of 100,000 control and Pcaf knockdown tNSCs were diluted in 5 µl of NSC medium and orthotopically injected into the brain of immunocompromised mice (2mm lateral and 1mm anterior to bregma and 3mm beneath the skull) using a stereotactic device (24). The mice were followed daily for the development of neurological deficits.

Statistical analysis Survival curves were compared using the Log-rank test. In other experiments data were presented as mean ± SD. Two-tailed Student’s t test were used for statistical analysis. P<0.05 was considered as statistically significant.

PCAF is required for GLI-dependent transcription

Results

A siRNA screening to identify acetyltransferases involved in the Hh pathway To identify novel transcriptional regulators of GLI1, we performed a siRNA-based screening targeting the 17 different histone acetyltransferases (HATs) that have been characterized in mammalian cells (25). We used a siRNA library, generated and validated in our laboratory where each HAT was targeted by 3 different siRNA oligonucleotides (26). As a cellular system we used NIH3T3 cells containing a luciferase reporter fused with the responsive element for Gli repeated 8 times (8x 3´Gli-BS Luc) and a renilla reporter (RL) as internal control (Fig. 1A) (21). In this system, luciferase activity reflects the activity of the Hh pathway. To activate the Hh pathway we used conditioned medium containing the Hh protein Sonic Hedgehog (Shh), produced in 293T cells stably expressing this protein (Fig. 1B). As a positive control for the screening we knocked down Smo, which is one of the most upstream positive regulators of the Hh pathway, using a specific siRNA. As shown in Fig. 1B and C, efficient depletion of Smo led to an impairment of the Shh- dependent induction of the pathway. In the screening, we used three different siRNAs against each acetyltransferase, a scrambled siRNA sequence and siRNA against the positive regulator Smo. The statistical analyses of the results are described in detail in Material and Methods and in Supplementary Figure S1. As expected the downregulation of Smo led to a significant decrease in Gli-mediated transcriptional activation (Supplementary Fig. S1A, Fig. 1D). Moreover, downregulation of Ep300, Ncoa1 and Pcaf also led to a significant decrease in Gli1 activity. Thus, these 3 candidates and the two hits closest to being significant, Gtf3c4 and Kat2a, were selected for further analysis (Fig. 1D). To validate the effect of these 5 candidates we repeated the Hh luciferase assay (Supplementary Fig. S2A). In this assay, we observed a comparable negative effect on Hh pathway activation by downregulating Pcaf, Ep300 or Smo. To further corroborate this finding, we correlated the efficiency of mRNA knockdown for each of the 3 siRNAs with each of the candidate genes to the corresponding effect on the Hh luciferase readings (Supplementary Fig. S2B). Pcaf showed highly significant correlation (R2 > 0.8) between the transcript knockdown and the luciferase activity.

PCAF is required for GLI-dependent transcription

Pcaf is required for the activation of a Hedgehog pathway dependent promoter We performed additional tests to validate the role of Pcaf in the Hh pathway. As shown in Fig. 2A, the two most efficient Pcaf siRNAs from the screening (Supplementary Fig. 2B and Fig. 2B) impair the activation of the Hh pathway to the same extent as the Smo siRNA control. Moreover two independent shRNAs targeting Pcaf in different regions than those targeted by the siRNAs, used in the screening, reduce Hh signaling with comparable strength to a control shRNA directed against Gli1 (Fig. 2C). Additionally, Pcaf and Gli1 silencing abolish also the activation of the Hh pathway induced by treatment with the small molecule agonist of Smo (SAG) (Fig. 2D). Taken together, these results show that Pcaf, like Gli1, is required for the activation of a Gli-dependent promoter.

Pcaf is required for the expression of Hh target genes The activation of the Hh pathway leads to increased expression of Gli-regulated target genes (16). To test the involvement of Pcaf on modulation of Hh target genes, we determined the expression of two of these, namely Gli1 and Ptch upon depletion of Pcaf in NIH3T3 cells. Shh stimulation of NIH3T3 cells leads to an increase in Gli1 and Ptch expression of 7- and 4-fold, respectively (Fig. 3A), and this increase is significantly reduced in cells transfected with two different siRNAs against Pcaf. A similar effect was observed in Smo siRNA treated cells (Fig 3A). The specificity of this observation was further supported by the fact that stable knockdown of Pcaf or Gli1 also impaired the activation of Gli1 and Ptch by Shh (Fig 3B). Importantly the impairment of the Shh-mediated up-regulation of Gli1 and Ptch, correlates with the efficiency of Pcaf knockdown (Fig 3B). In NIH3T3 cells, Pcaf depletion did not affect DNA synthesis and cell cycle profile (Supplementary Figure 3 and data not shown), suggesting that increased expression of Hh target genes is directly mediated by Pcaf and not caused by changes in proliferation.

PCAF affects cell proliferation and expression of Hh target genes in glioblastoma and medulloblastoma cells Hh signaling has been reported to play a role in glioma and glioblastoma (GBM) as well as in medulloblastoma (MB), the most frequent childhood brain cancer (16). Indeed deregulation of the Hh-pathway resulting from PTCH inactivation or from

PCAF is required for GLI-dependent transcription

mutations in transducers of the signaling pathway can induce MB in mice and in humans (27). To test if PCAF plays a role in modulating the Hh pathway also in glioblastoma and medulloblastoma cells, we stably knocked down the acetyltransferase using two independent shRNAs in different GBM and MB cell lines. In PCAF-depleted cells, the growth is slowed down compared to control transduced cells (Fig. 4A-C right panels). Importantly, a similar effect is observed when GLI1 expression is downregulated (Fig. 4A-C right panels). Moreover, the downregulation of PCAF and GLI1 led to similar reductions in the expression of GLI1 and PTCH (Fig. 4A-C left panels), indicating a possible common mechanism by which the two proteins affect cell proliferation. It has been reported that inhibition of Hh signaling mediated by treatment with the Smo antagonist Cyclopamine, decreases cell proliferation of different GBM cells including U87 and U118 (28). In contrast, proliferation of the T98G GBM cells is not affected (28). To further investigate the specificity of PCAF towards Hh pathway, we stably depleted the acetyltransferase or GLI1 in T98G cell. Interestingly neither GLI1 nor PCAF knockdown affects GLI1 and PTCH expression (Fig 4D). We then evaluated the impact of PCAF silencing on the proliferation of human primary GBM neurospheres, propagated in the presence of EGF and bFGF. These cell cultures more closely recapitulate the phenotype and genotype of the tumors than do serum cultured cell lines (29). As shown in Fig 4E and in Supplementary Fig. 4A, PCAF stable depletion in GBM543 neurospheres leads to reduction of cell growth and affects the expression of the Hh target genes. Moreover the capability of GBM543 cells to grow as neurospheres is strongly impaired upon PCAF or GLI1 depletion (Fig. 4E and Suppl. Fig. 4B). The requirement of PCAF for cell proliferation was also tested by colony formation assays in both GBM and MB cell lines. Indeed PCAF or GLI1 depletion strongly reduces the number of U87 GBM and DAOY MB cell colonies (Supplementary Fig. 5A-B). Taken together these findings suggest a role for PCAF in regulating proliferation of GBM and MB cells through its contribution to Hh target gene expression. Previous results have shown that DAOY cells require a functional Hh pathway for cell proliferation (30), and that cyclopamine treatment leads to a reduction in Hh

PCAF is required for GLI-dependent transcription

target gene expression and increased apoptosis (31). In agreement with being required for the expression of Hh target genes (Fig. 4A), PCAF depletion also induces apoptosis (Supplementary Fig. 5C). As further evidence for the requirement of PCAF for Hh target gene expression in MB cells, we used Anacardic Acid (AA) to inhibit the acetyltransferase activity (32, 33). AA treatment of DAOY-MB cells led to a strong reduction of PTCH expression and to an increase in apoptosis, similar to the effect induced by Cyclopamine (Fig. 5A and C). Importantly, PCAF expression is not affected by AA treatment (Supplementary Fig 5D), suggesting that the chemical compound specifically inhibit the activity of PCAF. These results strongly corroborate the finding that PCAF is required for the expression of Hh target genes and consequently for cell proliferation.

PCAF interacts with GLI1 and regulates H3K9 acetylation on Hh target gene promoters To characterize the role and the mechanism of action of PCAF in regulating Hh pathway, we tested if PCAF can interact with the GLI1 transcription factor. We performed co-immunoprecipitation assays using cells in which we ectopically expressed PCAF and GLI1. As shown in Fig. 6A, PCAF can indeed bind to GLI1, and most importantly this interaction was also observed between the two endogenous proteins (Fig. 6B). PCAF is a co-activator of transcription that is recruited to the region surrounding the transcription start site of target genes (34). Furthermore PCAF has been described to acetylate histones on specific residues. In vitro the recombinant protein shows a preference towards lysine residues 9 and 14 on histone H3 (H3K9 and H3K14) and with a lower efficiency towards H4K8 and H4K16 (35-37). Deletion of Pcaf together with its family member Gcn5 in vivo leads to a dramatic reduction of H3K9 acetylation (38). To gain further insight into the molecular mechanisms underlying PCAF dependent regulation of Hh target genes, we examined if Pcaf can regulate the posttranslational modification of H3 on Gli1 and Ptch promoters. To do this, we determined the amount of acetylated H3K9 (H3K9ac) associated with the Gli1 and Ptch promoters by chromatin immunoprecipitation (ChIP)-qPCR analysis in NIH3T3 cells treated with SAG. As shown in Fig. 6C, the

PCAF is required for GLI-dependent transcription

amount of H3K9Ac, but not H4Ac, associated with the two promoters is significantly reduced in cells expressing Pcaf shRNA. Furthermore, we showed that knockdown of PCAF and GLI1 both led to a dramatic reduction of H3K9ac associated with the PTCH promoter (Fig. 6D). To further characterize the mechanism by which PCAF regulates Hh target genes, we tested if it is associated with the PTCH promoter. As shown in Fig. 6E, PCAF associates with the PTCH promoter, and the binding is dependent on the expression of GLI1.

Pcaf depletion in tumorigenic neural stem cells affects the expression of Hh target genes and impairs tumor formation In high-grade glioma the inactivation of the locus Ink4a-Arf is frequently found together with the activation of the epidermal growth factor receptor (EGFR). Moreover it has been shown that constitutively active EGFR mutants (EGFR*) are able to transform murine Ink4a-Arf-null neural stem cells (NSCs) in the mouse brain (39). To investigate the role of Pcaf in regulating Hh pathway in this context we stably knocked-down the acetyltransferase in tumorigenic NSCs (Ink4a-Arf-/-; EGFR*). As shown in Figure 7A, Pcaf depletion impairs the expression of different Hh target genes and affects tNSCs proliferation (Supplementary Fig. 6). To test the role of Pcaf for the formation of tumors in vivo, we injected 100.000 control-depleted or Pcaf depleted tNSCs into the brain of immunocompromised mice. The control depleted cells readily formed tumors, and the mice died with a median latency of 7 weeks, whereas the mice injected Pcaf-depleted tNSCs survived with no signs of tumor development (Fig 7B). Taken together these results suggest a role for PCAF in regulating tumor formation through its requirement for the expression of the Hh target genes in vivo.

Discussion In this study we have identified PCAF as an important co-factor of the Hh-GLI signaling pathway. We have shown that PCAF is required for the transcriptional activation of Hh-Gli target genes. Moreover, we have demonstrated that PCAF binds to GLI1 and that both proteins are required for the increased H3K9Ac levels on Hh target gene promoters in response to the Hh-Gli activation. Since the association of PCAF with GLI-regulated promoters is dependent on GLI1, we propose a model (Fig

PCAF is required for GLI-dependent transcription

7C), in which the activation of the Hh-Gli signaling pathway leads to GLI1-dependent recruitment of PCAF. This in turn leads to H3K9acetylation of Hh target gene promoters and their activation. Deregulation of the Hh-Gli pathway contributes to the development and maintenance of several types of tumors, including glioblastomas and medulloblastomas. Interestingly, our results show that depletion of PCAF leads to impairment of cell proliferation and induction of apoptosis in GBM and MB cell lines, whereas no effect was observed in fibroblasts. These results are in agreement with the functional requirement of Hh-Gli signaling pathway in GBM and MB, and suggest that inhibition of PCAF activity could be an attractive avenue to treat Hh/Gli- dependent tumors such as GBMs and MBs. Different inhibitors targeting the Hh pathway have been proposed as anticancer agents (Fig. 7C). SMO inhibitors, like Cyclopamine, inhibit Hh-induced tumor growth in vivo and in vitro (40, 41). Recently other small molecule antagonists of SMO have been identified and in particular one compound (Vismodegib) recently became the first FDA-approved Smo inhibitor for basal cell carcinoma (BCC) treatment (42). Vismodegib and other SMO antagonists are currently being tested in clinical trials as potential treatments for other tumors including medulloblastoma, pancreatic, ovarian and hematopoietic cancers. Use of Hh-inhibitors acting downstream of SMO have also been reported as therapeutic alternatives, including inhibitors to GLI1(43-45). Histone deacetylase inhibitors (HDACi) have been described as multifunctional anticancer agents. Their role in brain tumors has been studied, and in particular HDCAi have been shown to induce cell cycle arrest associated with p21 increase and to inhibit the growth of glioblastoma cell lines (46). Moreover multiple oral HDACi have been studied in phase I trial in pediatric patients (47). Recently a role for HDACs in Hh pathway regulation has been reported. For instance, a recent study has shown that HDAC1 can deacetylate GLI1 leading to the activation of GLI- target genes (48). Histone acetyltransferases (HATs) play essential roles in normal cellular function, however they also to contribute to the pathogenesis of different diseases, including tumorigenesis and neurodegenerative disorders. HATs, as a class of enzymes, are therefore interesting as drug targets. Recently, some natural HAT

PCAF is required for GLI-dependent transcription

inhibitors including anacardic acid (AA), garcinol and curcumin were shown to inhibit the activities of PCAF, p300 and CBP (33, 49). Other compounds have been synthesized based on these natural products, and some of these have been shown to prevent growth of cancer cells without affecting proliferation of non-malignant cells (50). As a proof-of-concept for the use of chemical inhibitors to PCAF to treat Hh- Gli dependent tumors we used AA, and showed that AA treatment leads to reduced expression of PTCH, a Gli-target gene, and a clear increase in apoptosis in DAOY- MB cells. Although we cannot exclude that AA treatments are inhibiting not only PCAF but also p300 and CBP, the observation that depletion of PCAF gives very similar effects suggests PCAF to be the main HAT in this setting. We hope that our results will inspire to the development of highly potent and specific inhibitors of PCAF for the treatment of cancer patients. Such compounds will also be valuable for a better definition of PCAF as anti-cancer drug target, and for the understanding of the biological function of PCAF in normal cells as well as in tumors.

Acknowledgements We thank Anna Fossum for assistance in FACS sorting and Michael Lees for helping with acquiring data for the proliferation experiments. We thank Dr Jussi Taipale for providing 239-ShhN and NIH3T3 cells. We acknowledge all the members of the Helin laboratory for critical and fruitful discussions.

PCAF is required for GLI-dependent transcription

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PCAF is required for GLI-dependent transcription

coactivators p300 and PCAF within nucleosomal substrates. J Biol Chem. 1999;274:1189-92. 37. Kuo MH, Brownell JE, Sobel RE, Ranalli TA, Cook RG, Edmondson DG, et al. Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines. Nature. 1996;383:269-72. 38. Jin Q, Yu LR, Wang L, Zhang Z, Kasper LH, Lee JE, et al. Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation. EMBO J. 2011;30:249-62. 39. Bachoo RM, Maher EA, Ligon KL, Sharpless NE, Chan SS, You MJ, et al. Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell. 2002;1:269-77. 40. Berman DM, Karhadkar SS, Hallahan AR, Pritchard JI, Eberhart CG, Watkins DN, et al. Medulloblastoma growth inhibition by hedgehog pathway blockade. Science. 2002;297:1559-61. 41. Chen JK, Taipale J, Cooper MK, Beachy PA. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 2002;16:2743-8. 42. Sekulic A, Migden MR, Oro AE, Dirix L, Lewis KD, Hainsworth JD, et al. Efficacy and safety of vismodegib in advanced basal-cell carcinoma. N Engl J Med. 2012;366:2171-9. 43. Lauth M, Bergstrom A, Shimokawa T, Toftgard R. Inhibition of GLI- mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A. 2007;104:8455-60. 44. Hyman JM, Firestone AJ, Heine VM, Zhao Y, Ocasio CA, Han K, et al. Small- molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade. Proc Natl Acad Sci U S A. 2009;106:14132-7. 45. Hosoya T, Arai MA, Koyano T, Kowithayakorn T, Ishibashi M. Naturally occurring small-molecule inhibitors of hedgehog/GLI-mediated transcription. Chembiochem. 2008;9:1082-92. 46. Horing E, Podlech O, Silkenstedt B, Rota IA, Adamopoulou E, Naumann U. The histone deacetylase inhibitor trichostatin a promotes apoptosis and antitumor immunity in glioblastoma cells. Anticancer Res. 2013;33:1351-60. 47. New M, Olzscha H, La Thangue NB. HDAC inhibitor-based therapies: can we interpret the code? Mol Oncol. 2012;6:637-56. 48. Canettieri G, Di Marcotullio L, Greco A, Coni S, Antonucci L, Infante P, et al. Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation. Nat Cell Biol. 2010;12:132-42. 49. Balasubramanyam K, Altaf M, Varier RA, Swaminathan V, Ravindran A, Sadhale PP, et al. Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression. J Biol Chem. 2004;279:33716-26. 50. Stimson L, Rowlands MG, Newbatt YM, Smith NF, Raynaud FI, Rogers P, et al. Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity. Mol Cancer Ther. 2005;4:1521-32.

PCAF is required for GLI-dependent transcription

Figure Legends

Figure 1. Identification of acetyltransferases involved in the Hh pathway A) Schematic representation of the cellular system used for the screening. B) Luciferase assay analysis in NIH3T3 cells transfected with a non-targeting scramble (Scr) control siRNA and with a siRNA against Smo. The Hh pathway is induced by treatment with Shh conditioned medium. C) qRT-PCR expression analysis of Smo (normalized to Rplpo levels) in cells transfected with the indicated siRNAs. The expression is monitored both at steady state and upon Shh treatment. For the above panels B-C are shown the mean and the standard deviation (SD) for three independent experiments. ***P<0.0004, ****P<0.0001. D) RSA based hit list of the acetyltransferase siRNA screen. Each gene is assigned a single logP-value based on the ranking distribution of all three siRNAs.

Figure 2. Pcaf is required for the Hh pathway activity A) Luc-assay analysis in NIH3T3 cells transfected with siRNAs against Pcaf, Smo and a scramble siRNA control (Scr). B) Western blot analysis of protein extracts of cells transfected with the indicated siRNAs using the indicated antibodies. Tubulin is used as a loading control. C) Luciferase activity measured in NIH3T3 cells transduced with Pcaf, Gli1 and Scr shRNAs. The Hh pathway is activated by Shh treatment. The corresponding expression of Pcaf and Gli1 (normalized to Rplpo levels) is determined by qRT-PCR. D) Luciferase activity analyzed in NIH3T3 cells upon stable knockdown of Pcaf, Gli1 and Scr control. The Hh pathway is induced by treatment with SAG, a compound homolog to Smo. The expression of Pcaf and Gli1 (normalized to Rplpo levels) is measured by qRT-PCR. In the above panels are represented the mean and the SD for at least three independent experiments. *P<0.01, **P<0.003, ***P<0.0006, ****P<0.0001.

Figure 3. Pcaf is required for expression of Hh target genes A) qRT-PCR analysis for expression of Hh target genes, Gli1 and Ptch (normalized to Rplpo levels), performed in NIH3T3 cells transfected with the reported siRNAs. The cells are treated with Shh conditioned medium. The expression of Pcaf and Smo (normalized to Rplpo levels) is also reported. B) qRT-PCR experiments performed in cells with a stable knockdown shRNA-mediated of Pcaf, Gli1 and Scr control. The

PCAF is required for GLI-dependent transcription

Gli-Hh pathway is activated using Shh treatment. Gli1, Ptch and Pcaf expressions are normalized to Rplpo levels. All data of the above panels are average values and SD from at least 3 experiments, each carried out in triplicate. **P<0.001, ***P<0.0002, ****P<0.0001.

Figure 4. PCAF affects proliferation and expression of Hh target genes in glioblastoma and medulloblastoma cells A-E) Left panels: qRT-PCR analysis for the expression of the endogenous Hh target genes, GLI1 and PTCH (normalized to Rplpo levels), measured upon depletion of Pcaf and Gli1. All data of the above panels (A-E) are average values and SD from at least 3 experiments, each carried out in triplicate. *P<0.01, **P<0.003, ***P<0.0005, ****P< 0.0001. A-E) Right panels: Growth curve of cells stably depleted for Gli1 and Pcaf by different shRNAs, compared to the non-targeting control shScr.

Figure 5. PCAF inhibitor and cyclopamine induce apoptosis in DAOY MB cells. A) qRT-PCR analysis to measure the expression of the endogenous PTCH (normalized to Rplpo levels), in DAOY cells treated with AA or Cyclopamine for the indicated time. B) Western blot analysis in cells treated for 24h with AA or Cyclopamine. Pcaf levels and levels of the apoptotic marker cleaved caspase3 were measured using specific antibodies. Tubulin serves as a loading control. C) Detection of apoptotic Daoy cells. Cells were treated with AA or Cyclopamine or DMSO as control for the indicated time, and stained using fluorochrome-conjugated Annexin V and 7AAD. All data from the above panels are average values and SD from 3 independent experiments. *P<0.01, **P<0.001, ***P<0.0009.

Figure 6 PCAF interacts with GLI1 and regulates H3K9 acetylation on promoters of Hh target genes A) Co-immunoprecipitation experiments in 293 cells ectopically expressing PCAF- flag and GLI1-His vectors using the indicated antibodies. B) Endogenous binding between PCAF and GLI1 in U87 cells. C) ChIP-qPCR analysis of the histone modifications H3K9ac and H4ac in NIH3T3 cells depleted for Pcaf versus control cells upon induction with SAG treatment. H3K9ac and H4ac signals are normalized to histone density using an H3-specific antibody. D-E) ChIP-qPCR analysis of the PTCH promoter in U87 cells stably depleted using shRNAs against Pcaf or Gli1

PCAF is required for GLI-dependent transcription

versus a Scr control. H3K9Ac and Pcaf specific antibodies are used for the immunoprecipitation. H3K9Ac signal is normalized to histone density using an H3 antibody. All data from the above panels are average values and SD from 3 independent experiments, each carried out in triplicate.

Fig. 7 PCAF depletion in NSCs Ink4a-Arf-/-;EGFR* affects expression of Hh target genes and impairs tumor formation. A) qRT-PCR analysis for the expression of the endogenous Hh target genes (normalized to Rplpo levels) measured upon depletion of Pcaf. Error bars represent mean and SD from 3 independent experiments. **P<0.004. B) Survival curve of mice intracranially injected with NSCs Ink/Arf-null EGFR* engineered to express an shRNA against Pcaf or against a scramble control. C) Model describing PCAF function. Upon Hh pathway activation PCAF is recruited to Hh target gene promoters through an interaction with GLI1. The association between PCAF and GLI1 leads to the H3K9Acetylation of the Hh target gene promoters resulting in an increase of the Hh target genes expression. Known and potential novel Hh inhibitors are represented in the grey boxes.

Fig 1

Luciferase 8X3´BSGli1

Shh

GLI1 Luciferase 8X3´BSGli1

B C

Smo 1.00 1.00

0.75 0.75

0.50 0.50 *** *** 0.25 0.25 **** Relative luciferase readings luciferase Relative

0.00 levels mRNA expression Relative 0.00

siScr siScr siScr siScr siSmo siSmo siSmo siSmo + Shh + Shh

p=5% siScr siMyst4 siCrebbp siMyst3

siKat5 siHat1 siClock siTaf1 siMyst1

siElp3 siNcoa2

siMyst2 siNcoa3 siKat2a siGtf3c4

siPcaf

siNcoa1 siSmo

siEp300

Relative luciferase readings0.75 1.00 0.50 0.00 0.25 Relative luciferase readings 0.00 0.25 0.50 0.75 1.00 Relative luciferase readings 0.75 1.00 0.50 0.00 0.25 shScr siScr shScr shScr shPcaf#1 siScr shScr shPcaf#1 siSmo **** + Shh shPcaf#2 **** + Shh siPcaf#1 *** + SAG

shPcaf#2 **** ****

Downloaded from shGli1 siPcaf#2 ** **** ****

shGli1 Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. **** Author ManuscriptPublishedOnlineFirstonAugust13,2013;DOI:10.1158/0008-5472.CAN-12-4660 0.75 1.00 0.50

0.00 Relative mRNA expression levels 0.25 Relative mRNA expression levels B 0.75 1.00 0.50 0.00 0.25 shScr

shScr Gl cancerres.aacrjournals.org shScr + Shh i1 shScr shGli1 siScr Gli1 ** + SAG shGli1

* siSmo *

Relative mRNA expression levels 0.00 0.25 0.50 0.75 1.00 Relative mRNA expression levels siPcaf#1 0.00 0.25 0.75 1.00 0.50 shScr shScr shScr siPcaf#2 shScr shPcaf#1 P caf Fig 2 P

shPcaf#2 * Tubulin Pcaf Smo shPcaf#2 * * Research. * Author Manuscript Published OnlineFirst on August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4660 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig 3

Gli1 Ptch Smo Pcaf 1.00 1.00 1.00 1.00

0.75 0.75 ** 0.75 0.75 *** **** *** **** **** 0.50 0.50 **** 0.50 0.50 ****

Relative mRNA Relative 0.25 mRNA Relative 0.25 mRNA Relative 0.25 **** mRNA Relative 0.25

0.00 0.00 0.00 0.00

siScr siScr siScr siScr siScr siScr siSmo siSmo siSmo siPcaf#1siPcaf#2 siPcaf#1siPcaf#2 siPcaf#1siPcaf#2 + Shh + Shh + Shh + Shh

B Gli1 Ptch Pcaf 1.00 1.00 1.00

**** 0.75 0.75 0.75 **** **** **** **** 0.50 **** 0.50 **** 0.50 ****

Relative mRNA Relative 0.25 mRNA Relative 0.25 mRNA Relative 0.25

0.00 0.00 0.00

shScr shScr shGli1 shScr shScr shGli1 shScr shPcaf#1shPcaf#2Downloaded from cancerres.aacrjournals.orgshPcaf#1shPcaf#2 onshPcaf#1 SeptembershPcaf#2 28, 2021. © 2013 American Association for Cancer + Shh + Shh +Research. Shh Author Manuscript Published OnlineFirst on August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4660 Author manuscripts have been peer reviewed and accepted forF publicationig 4 but have not yet been edited.

A DAOY GLI1 PTCH PCAF 100.0 shScr 1.00 1.00 2.00 ** ** ** 0.75 0.75 1.50 **** **** *** 0.50 0.50 1.00 50.0

Relative mRNA Relative 0.25 mRNA Relative 0.25 mRNA Relative 0.50 **

Con!uence (percentage) Con!uence shPcaf#1 *** shGli1 0.00 0.00 0.00 shPcaf#2 0.00 r r 1 1 1 f#1 c li 20.0 40.0 60.0 80.0 shScr shGli shSc shGli1 shS shG Time (hours) B shPca shPcaf#2 shPcaf#1shPcaf#2 shPcaf#shPcaf#2 U87 80.0 shScr GLI1 PTCH PCAF 1.00 1.00 1.50 60.0 *** 0.75 0.75 **** 1.00 40.0 shPcaf#1 0.50 **** 0.50 **** **** **** *** 0.50 shGli1 Relative mRNA Relative 0.25 mRNA Relative 0.25 mRNA Relative **** 20.0 Con!uence (percentage) Con!uence 0.00 0.00 0.00 shPcaf#2 0.00 50.0 100.0 shScr shGli1 shScr shGli1 shScrshGli1 shPcaf#1shPcaf#2 shPcaf#1shPcaf#2 shPcaf#1shPcaf#2 Time (hours) C U118 80.0 GLI1 PTCH PCAF shScr 1.00 1.00 1.00 60.0 * 0.75 0.75 0.75 ** ** 40.0 0.50 * 0.50 0.50 * **** 20.0 shGli1 Relative mRNA Relative 0.25 ** mRNA Relative 0.25 mRNA Relative 0.25 ****

Con!uence (percentage) Con!uence shPcaf#1 0.00 0.00 0.00 shPcaf#2 2 0.00 f#1 50.0 100.0 shScr shGli1 shScr shPcaf#1shPcaf#2 shScr shGli1 shGli1shPcaf#1 Time (hours) shPcashPcaf#2 shPcaf#

D T98G 100.0 shPcaf#1 GLI1 PTCH PCAF shScr 1.00 1.50 1.50 0.75 shGli1 shPcaf#2 1.00 1.00 50.0 0.50 **** 0.50 0.50 0.25 **** Relative mRNA Relative mRNA Relative mRNA Relative Con!uence (percentage) Con!uence **** 0.00 0.00 0.00 0.00 20.0 40.0 60.0 80.0 Scr Time (hours) sh shGli1 shScr shGli1 shScrshGli1 shPcaf#1shPcaf#2 shPcaf#1shPcaf#2 shPcaf#1shPcaf#2

E GBM543 2500 GLI1 PTCH PCAF shScr 1.00 1.00 1.00 2000 * 0.75 0.75 0.75 * ** 1500 0.50 0.50 **** 0.50 ( μ m2) Area ** 1000 Relative mRNA Relative 0.25 ** mRNA Relative 0.25 mRNA Relative 0.25 **** shGli1 **** shPcaf#1 0.00 0.00 0.00 500 shPcaf#2 2 cr 50.0 100.0 150.0 shScr shGli1 Downloaded from cancerres.aacrjournals.orgshScr on September 28,Time 2021. (hours) © 2013 American Association for Cancer shPcaf#1 shS shGli1 shGli1 shPcaf#2 shPcaf#1shPcaf#2 shPcaf#1shPcaf# Research. Author Manuscript Published OnlineFirst on August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4660 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig 5

A PTCH 1.00

0.80 **

0.60

0.40 ** *** ** Relative mRNA Relative 0.20

0.00

DMSO AA 6h AA 24h Cycl 6h Cycl 24h

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* 30.0

20.0

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0.0 Percentage Annexin V positive cellls V positive Annexin Percentage Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2013 American Association for Cancer DMSOAA 24hAA 48hAA 72h Cycl 24hCycl 48hCycl 72h Research. Author Manuscript Published OnlineFirst on August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4660 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig 6

A B

+ + + - + + + - pcDNA - + - + - + +- PCAF-Flag -- + + - - + + GLI1-His IP: IgG IP: Gli1 INPUT Gli1 Gli1

Pcaf Flag

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D E

PTCH PTCH PTCH shScr 10.0 shScr 0.60 shScr 10.0 shPcaf#2 shGli1 shPcaf#2 shGli1 8.0 8.0 0.40 6.0 6.0 % INPUT % INPUT % 4.0 INPUT % 4.0 0.20 2.0 2.0

0.00 Downloaded0.00 from cancerres.aacrjournals.org0.00 on September 28, 2021. © 2013 American Association for Cancer IgG H3K9Ac IgG H3K9Ac IgG PcafResearch. Author Manuscript Published OnlineFirst on August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4660 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig 7

Gli1 Ptch Bmi1 Pcaf 1.00 1.00 1.00 1.00 ** 0.80 0.80 0.80 0.80 ** 0.60 0.60 0.60 ** 0.60

0.40 0.40 0.40 0.40 Relative mRNA Relative mRNA Relative mRNA Relative mRNA Relative ** 0.20 0.20 0.20 0.20

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shPcaf#2 shScr

50 Percent Survival Percent

0 0 50 100 150 Days

anti-Shh Ab C Smo inhibitors

Cytoplasm SMO PTCH Compounds acting at di!erent levels within the pathway

PCAF inhibitor K9Ac K9Ac PCAF anti-Gli agents GLI1 Gli target genes

DownloadedNucleus from cancerres.aacrjournals.org on September 28, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 13, 2013; DOI: 10.1158/0008-5472.CAN-12-4660 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and cancer cell proliferation

Martina Malatesta, Cornelia Steinhauer, Faizaan Mohammad, et al.

Cancer Res Published OnlineFirst August 13, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-4660

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