Mutations in actrt1 and its enhancer rna elements lead to aberrant activation of the Hedgehog signaling pathway in inherited and sporadic basal cell carcinomas

Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas.

Elodie Bal^1,2&, Hyun-Sook Park³, Zakia Belaid-Choucair^1,4,5*, Hülya Kayserili^6,7*_, Magali Naville^8,9,10**, Marine Madrange^1,2**, Christopher Gordon^1,11, Elena Chiticariu³, Smail Hadj-Rabia^1,2,12, Nicolas Cagnard¹³, Francois Kuonen³, Daniel Bachmann³, Marcel Huber³, Cindy Le Gall^1,2, Francine Côté^1,4,5, Sylvain Hanein^1,2, Rasim Özgür Rosti^7,14, Ayca Dilruba Aslanger⁷, Quinten Waisfisz¹⁵, Christine Bodemer^1,2,12_, Olivier Hermine^1,4,5,16,17, Fanny Morice-Picard¹⁸, Bruno Labeille¹⁹, Frédéric Caux²⁰, Juliette Mazereeuw-Hautier²¹, Nicole Philip²², Nicolas Levy²², Alain Taieb¹⁸, Marie-Françoise Avril²³, Denis Headon²⁴, Gabor Gyapay²⁵, Thierry Magnaldo²⁶, Sylvie Fraitag²⁷, Hugues Roest Crollius^8,9,10, Pierre Vabres²⁸, Daniel Hohl^3, Arnold Munnich^1,2, Asma Smahi^1,2&.

*, ** These authors contributed equally to the present work.

^& Corresponding authors: [e-mail], [e-mail]

¹ Paris-Descartes University, Sorbonne Paris Cité, F-75006 Paris, France

² Laboratory “Genetic and pathophysiological bases of autoinflammatory diseases”, IMAGINE Institute, INSERM UMR 1163, F-75015 Paris, France.

³ Department of Dermatology, Lausanne University Hospital, Hôpital de Beaumont, CH-1011 Lausanne, Switzerland

⁴ Department of Hematology, Hôpital Necker Enfants-Malades, F-75015 Paris, France;

⁵ Laboratory “Molecular mechanisms of hematologic disorders and therapeutic implications”, IMAGINE Institute, INSERM UMR 1163, CNRS ERL 8254, F-75015 Paris, France.

⁶ Medical Genetics Department,   Koç University School of Medicine (KUSOM) 34010 İstanbul Turkey.

⁷ Medical Genetics Department, İstanbul University; İstanbul Medical Faculty, 34093 Istanbul Turkey. .

⁸ Ecole Normale Supérieure, Institut de Biologie de l’ENS, IBENS, F-75005 Paris, France.

⁹ CNRS UMR8197, F-75005 Paris, France.

¹⁰ INSERM U1024, F-75005 Paris, France.

¹¹ Laboratory “Embryology and genetics of human malformation”, Institut IMAGINE, INSERM UMR 1163, F-75015 Paris, France.

¹² Department of Dermatology, Hôpital Necker Enfants-Malades, F-75015 Paris, France.

¹³ Plateforme Bio-informatique, Structure Fédérative de Recherche Necker, INSERM US24/CNRS, UMS 3633, F-75015 Paris, France

¹⁴ Laboratory of Genome Maintenance, The Rockefeller University, New York, New York 10065, USA 

     

¹⁵ Clinical Genetics VU Medical Center Amsterdam 1007, the Netherlands.
¹⁶ GR-Ex Laboratory of Excellence, F-75015 Paris, France.

¹⁷ Centre Référence Nationale pour les Mastocytoses, Hôpital Necker-Enfants Malades, F-75015 Paris, France.

¹⁸ Department of Dermatology, Centre de Référence des Maladies Rares de la Peau, Hôpital Saint André, F-33000 Bordeaux, France.

¹⁹ Department of Dermatology, CHU Hôpital Nord, F-42055 Saint-Etienne, France.

²⁰ Department of Dermatology, Hôpital Avicenne, F-93009 Bobigny, France.

²¹ Department of Dermatology, Centre de Référence des Maladies Rares de la Peau, Hôpital Larrey, F-31059 Toulouse, France.

²² Department of Genetic, Hôpital de la Timone, F-13005 Marseille, France.

²³ Department of Dermatology, Hôpital Cochin, F-75014 Paris, France.

²⁴ Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, EH25 9RG, UK.
²⁵ Genoscope (CEA), CNRS UMR 8030, University of Evry, F-91000 Evry, France

²⁶ Institute for Research on Cancer and Aging, CNRS UMR 7284, INSERM U1081, UNS, F-06000, Nice, France.

²⁷ Department of Pathological Anatomy, Hôpital Necker Enfants-Malades, F-75015 Paris, France.

²⁸ Department of Dermatology, Centre Hospitalier Universitaire, Hôpital du Bocage, F-21079 Dijon, France.

Basal cell carcinoma (BCC), the most common human cancer, results from aberrant activation of the Hedgehog signaling pathway¹. Although most cases of BCC are sporadic, some forms are inherited as in Bazex-Dupré-Christol syndrome (BDCS), an X-linked, dominant, cancer-prone genodermatosis². We have identified mutations in the ACTRT1 gene, coding for actin-related protein T1 (Arp-T1) in 2/6 BDCS families. High-throughput sequencing in the four remaining families revealed germline mutations in non-coding sequences surrounding ACTRT1. These mutations were located in transcribed enhancer RNAs^3-5 (eRNA) and were shown to impair enhancer activity and ACTRT1 expression. Arp-T1 was found to directly bind to the Gli1 promoter, thus inhibiting its expression while loss of Arp-T1 led to activation of the Hedgehog pathway in BDCS patients. Moreover, ACTRT1 reduced in vitro and in vivo proliferation of cell lines with aberrant activation of the Hedgehog signaling pathway. In summary, our study identifies a disease mechanism in BCCs involving mutations in regulatory non-coding elements, and shows that ACTRT1 has tumor suppressor functions in cancer.

Bazex-Dupré-Christol syndrome (BDCS, MIM 301845) is an X-linked, dominant predisposition to BCCs^2,6-8 (Supplementary Fig. 1a). By studying six affected families (Fig. 1a), we mapped the BDCS gene to a 7.5 Mb region in Xq25-q26.2 (Supplementary Fig. 1b,c) and identified an insertion in the ACTRT1 gene encoding actin-related protein T1 (Arp-T1) segregating with the disease in 2/6 families (c.547_548 InsA, p.M183NfsX17 in families C and D, Supplementary Fig. 1d,e). The mutant cDNA encodes a 25 kDa truncated protein (Supplementary Fig. 1f). Yet, no mutations in the coding region of ACTRT1 were found in the other four families linked to the same interval. No rearrangements in the candidate region were identified by high-density tiling-path comparative genomic hybridization array.

Immunohistochemical, RT-PCR and Western blot analyses of control skin samples detected expression of Arp-T1 in epidermal layers and skin appendages involved in BDCS but not in dermal connective tissues (Supplementary Fig. 2a-d). Interestingly, immunohistochemical analyses failed to detect any specific Arp-T1 staining in BCC tumors from BDCS patients and only a weak signal was detected in unaffected epidermis (Supplementary Fig 2e). Immunofluorescence (Fig. 1b,c) and qRT-PCR assays of epidermis (Fig. 1d) revealed low Arp-T1/ACTRT1 expression in all BDCS patients regardless of the presence or absence of mutations in the ACTRT1 gene, suggesting that the remaining cases involved hitherto unknown ACTRT1 regulatory elements.

Conserved non-coding elements (CNEs), which are known to control expression of neighboring genes^9,10, are concentrated in gene deserts¹¹. Given that the ACTRT1 gene is located in a 2.6 Mb gene desert, we studied 17 CNEs on both sides of ACTRT1 (Supplementary Fig. 3a). Sanger sequencing detected a g.127372937A>T variation of CNE12 in families E-F only (CNE12 chrX: 127371674-127374249, Supplementary Fig. 3b, c). Comparative genomic approaches aimed at predicting regulatory DNA sequences are known to have limitations, as regulatory elements are not necessarily conserved across species^12-14. We therefore performed systematic array-based capture and high-throughput sequencing of the 7.5 Mb candidate region and used a specific genome browser to select candidate variants (Supplementary Fig. 4a). We selected three additional candidate variants in family A (A1 g.125960325A>T, A2 g.125959394C>G, and A3 g.126494053 InsT) and two in family B (B1 g.127061005G>C, and B2 g.127968123T>C; Supplementary Fig. 4b, c). To identify putative disease-causing variants among candidates, we performed (i) a chromatin signature analysis to identify variations that mapped to active regulatory regions^15,16, (ii) enhancer luciferase reporter assays to identify sequences capable of activating transcription¹⁷ and (iii) in situ hybridization to identify regions that are transcribed in skin¹⁸.

Chromatin immunoprecipitation (ChIP) and quantitative targeted PCR assays were performed on protein-DNA complexes extracted from normal human epidermis. Interestingly, an enhancer signature was only found for A2, B2 and CNE12 sequences, based on enrichment in H3K27ac and H3K4me1 marks and absence of the H3K4me3 mark (Fig. 1e). In these assays even very small differences in efficiencies between primer sets constitute a limitation for comparing data obtained with different primers. A luciferase reporter assay demonstrated that only the wild-type sequences surrounding the A2, B2 and CNE12 variations had enhancer activity in HaCat keratinocytes (Fig. 1f). This activity was reduced when mutated constructs were used (Fig. 1g). Given that a subset of enhancers RNAs (eRNAs) are broadly transcribed^3,4 and that the A2, B2 and CNE12 sequences bind RNA PolII⁵ (Fig. 1e), we used RT-PCR and in situ hybridization to study transcription at the candidate loci. Consistent with the ACTRT1 expression pattern, the A2, B2 and CNE12 sequences were specifically expressed in epidermis and its appendages (Supplementary Fig. 5a-d). Interestingly, no staining was detected when the corresponding patient skin biopsies were tested, demonstrating the dramatic impact of the A2, B2 and CNE12 variations on eRNA expression and/or stability (Supplementary Fig. 5b).

Lastly, in order to provide conclusive in vitro evidence for a link between these enhancer sequences and ACTRT1 expression, we used the CRISPR-Cas9 technology to disrupt the three enhancer regions¹⁹. We generated keratinocytes containing indels in A2, B2 or CNE12 enhancer regions by independently introducing six single guide-RNAs (Supplementary Fig. 6, 7, 8). Disruption of enhancers resulted in a relative decrease in ACTRT1 expression, which was correlated with editing efficiency (Fig. 1h and Supplementary Fig. 9). We also observed that enhancer mutagenesis increased keratinocytes proliferation (Supplementary Fig. 10). Taken together, our results suggest that BDCS is caused by loss-of-function mutations either altering the ACTRT1 coding region or affecting specific enhancers located in transcribed, non-coding regions.

Further immunohistological studies of sporadic BCC cases, showed that no Arp-T1 signal was detected in BCC tissues from 51/60 unrelated sporadic cases while normal Arp-T1 staining was detected in BCC tissue from 5 patients with Xeroderma Pigmentosum (Supplementary Fig. 11a). Further sequencing of a series of 20 BCC samples identified 3 mutations in the coding sequence of ACTRT1 (Supplementary Fig. 11b) and one mutation in the B2 enhancer (hg19: 127, 968, 192 G>A) located 59 bp downstream to the mutation found in BDCS family B (hg19: 127, 968, 123 T>C). These results emphasize the genetic heterogeneity of these skin tumors and suggest that both inherited and sporadic BCCs are associated with loss of function mutations at the ACTRT1 locus.

In order to elucidate the consequences of loss-of-function ACTRT1 mutations, we performed comparative transcriptomic analyses of BDCS patient skin samples and identified 1771 deregulated genes. These genes were mostly involved in regulation of cell cycle progression and cell death, survival or migration (Fig. 2a) . Since constitutive activation of the Hedgehog pathway is found in more than 70% of BCCs^1,20,21, particular attention was given to Hedgehog activated transcription factors. Indeed, among the deregulated genes, 56 were directly controlled by the Hedgehog transcription factors Gli1 and Gli2 (Fig. 2b). Consistently, qPCR showed that Hedgehog target genes were over-expressed in skin samples of BDCS patients carrying either an ACTRT1 mutation (c.547_548 InsA) or the CNE12 variant (Fig. 2c). Transactivation assays using Gli1 binding sites upstream of a luciferase reporter gene showed that wild-type but not truncated Arp-T1 constructs inhibited the Hedgehog pathway (Fig. 3a). Moreover, wild-type but not truncated Arp-T1 inhibited Gli1 expression after transfection in primary keratinocytes and stimulation with the Smoothened (Smo) agonist, purmorphamine (Fig. 3b). Taken together, these results strongly suggest a role for Arp-T1 in regulating the activity of the Hedgehog signaling pathway.

Arp-T1 belongs to the actin-related protein family²². In the nucleus, actin-related proteins are essential elements of the macromolecular machinery that controls nucleosome remodeling, histone acetylation, histone variant exchange, transcription and DNA repair^23,24. Using ultra-thin sections of normal epidermis processed for transmission electron microscopy, we detected Arp-T1 in both nucleus and cytoplasm (Supplementary Fig. 12a). Western blot analyses of subcellular protein fractionations showed that Arp-T1 was mostly localized in the nucleus and bound to chromatin following stimulation of the Hedgehog pathway using purmorphamine (Fig. 3c, Supplementary Fig. 12b). Conversely, the truncated Arp-T1 protein was absent from the chromatin-bound fraction of transfected HEK293T (Fig. 3c). Gli1 transcriptional activity and Hedgehog signaling are reportedly controlled by chromatin regulators, such as tumor-suppressors Brg1^25,26 and Snf5²⁷, two components of the mammalian SWI/SNF chromatin remodeling complex which directly bind Gli1 regulatory domains. To determine whether Arp-T1 binds to Gli1-promoters (Fig. 3d and Supplementary Fig. 13), we performed ChIP with an anti-Arp-T1 antibody in control primary keratinocytes. An anti-RNA polII antibody was used as a positive control for transcriptional activation. Interestingly, a 5 hour stimulation of keratinocytes with purmorphamine triggered an increase in Gli1 expression (Fig. 3e) correlating with an enrichment of RNA PolII (but not Arp-T1) to Gli1-promoter regions (Fig. 3f). A longer stimulation of keratinocytes with purmorphamine (25 hr), resulted in an enrichment of Arp-T1 in Gli1 promoters (Fig. 3f) concomitant with a decrease in Gli1 expression (Fig. 3e). Taken together our results support a negative control of Arp-T1 on Gli1expression and a pivotal role of Gli1 inhibition for the tumor suppressor activity mediated by Snf5, Brg1 and also Arp-T1.

In order to investigate the tumor-suppressor activity of ACTRT1 in vivo and the impact of its mutations we stably expressed wild type or mutant constructs in the long-term human BCC cell line UW-BCC1-T2²⁸ (Fig. 4a) characterized by enhanced activation of Hedgehog signaling pathway. Interestingly, the wild type construct significantly decreased cell proliferation (Fig. 4b), accompanied by inhibition of Gli1 and Gli2 expression (Fig. 4c). Conversely, the mutated ACTRT1 cDNA (c.547_548 InsA) only partially inhibited cell proliferation and had a less pronounced effect in reducing Gli1 and Gli2 expression. Similarly, xenograft tumors on AGR129 nude mice showed that wild type ACTRT1 but not the truncated variant attenuated tumor development (Fig. 4d-f) and Ki67 expression (Supplementary Fig. 14). These results add evidence to the tumor suppressor role of ACTRT1 in limiting BCC development in vivo.

Interestingly, a rare deletion at the ACTRT1 locus has been reported in 17/63 BRCA1/2-negative familial cases of early-onset hereditary breast cancer²⁹. Hypothesizing that, in addition to BCC, ACTRT1 may have a more general tumor suppressor activity, we selected two cancer cell lines where Hedgehog signaling pathway is aberrantly active (U2OS osteosarcoma cell line^30,31 and MDA-MB231 breast cancer cell line^32,33), especially as Arp-T1 is highly expressed in glandular breast lobules, weakly in osteocytes (Supplementary Fig. 15), but absent in the two cancer cell lines (Fig. 4g, j). In addition, using the specific Gli inhibitor GANT61, we confirmed the Gli-dependent growth of U2OS and MDA-MB231 cells³³ (Supplementary Fig. 15). Stable expression of ACTRT1 in these cell lines inhibited in vitro proliferation (Fig. 4g, j) and reduced Gli1 and Gli2 expression (Fig. 4h, k). Consistently, xenograft experiments in NMRI nude mice showed that ACTRT1 prevented growth of injected U2OS cells (Fig. 4i) and decreased development of xenograft tumors derived from MDA-MB231 cells (Fig. 4l). Previously, Gli1 inhibition has been shown to decrease growth and migration of MDA-MB231 by increasing apoptosis and decreasing cell proliferation³². Consistently, wild-type ACTRT1 increased the number of apoptotic cells and procaspase-3 and cleaved caspase-3 expression in MDA-MB231 tumors (Supplementary Fig. 16 a-c), decreased Ki67 expression (Supplementary Fig. 17), and reduced the metastatic potential of MDA-MB231 cells in vitro and in vivo (Supplementary Fig. 18 a-e).

Germline mutations in the ACTRT1 coding sequence and its surrounding non-coding elements constitute a hitherto unreported causative mechanism of inherited predisposition to BCC. Here, we report for the first time the involvement of the ACTRT1 gene in human disease. We suggest potential functions of Arp-T1 and mechanisms by which it could regulate Gli1 expression based on our results and some models in the literature describing the Arp family. First of all, our findings demonstrated that Arp-T1, but not the truncated protein identified in families with BDCS , binds to chromatin, suggesting that Arp-T1 performs nuclear functions. Nuclear Arps belong to the four main chromatin remodelers complex (CRC), INO80, SRCAP, BAF or human SWI/SNF and TIP60/TRRAP^23,34. Arp5 and Arp8 can bind to core histones, to facilitate interaction of the CRC complexes with nucleosomes^23,34. Mutations in the genes encoding the major mammalian SWI/SNF (BAF) CRC subunits are present in over 20% of human cancers.³⁵ Snf5 (the core component of SWI/SNF CRC) and Brg1 (the ATPase subunit), are bona fide tumor suppressors responsible for various types of cancer^25-27. Interestingly, both proteins act through direct inhibition of Gli genes^26,27, the way Arp-T1 does. Further studies are needed to determine if Arp-T1 acts specifically at the level of regulatory elements, through recognizing specific histone marks, repositioning nucleosomes, histone exchange or binding to transcription factors involved in Gli1 repression. Also, we cannot exclude that Arp-T1 could interact with key proteins involved in various Hedgehog interconnected pathways related to tumor proliferation and progression such as the p16-RB pathway, WNT signaling pathway, and Polycomb pathway as it was demonstrated for Snf5^36,37.

The discovery of distal regulatory non-coding elements known as enhancers with critical functions in gene expression has added a new dimension to transcriptional regulation. eRNAs are potent transcription units and their alteration can impact biological processes involved in human diseases, including cancer^38,39. They cover a broad spectrum of molecular and cellular functions by implementing different modes of action. Evidence has confirmed that long non-coding RNAs (lncRNAs) contribute to cancer initiation and progression by regulating gene transcription⁴⁰. For instance, a link between lncRNAs and the SWI/SNF complexes has been reported in various tumoral conditions⁴¹. Another mechanism of eRNAs in gene regulation results from their interaction with cohesin and mediator complexes to stabilize enhancer:promoter looping causing chromatin stabilization and gene expression⁴². They are also involved in the recruitment of RNA polymerase II to gene promoters and facilitate the access of specific transcription factors to enhancer sequences⁴³. Besides DNA methylation and histone modifications, the role of non-coding RNAs in epigenetic control has recently emerged. Their alterations result then in epigenomic reprogramming during tumor initiation and progression⁴⁴. Intensive investigation is needed to place Arp-T1 and its non-coding regulatory elements in such complex mechanisms of gene regulation and cancer development. More broadly, our findings shed light on the functional relevance of genomic alterations in non-coding regions, and their contribution to tumor development. Indeed, the clinical integration of non-coding RNAs as functionally-relevant elements in conjunction with additional predictive biomarkers could improve the management of cancer patients.

ONLINE METHODS

Patients and samples. A total of 48 patients (from 6 BDCS families) and many of their unaffected relatives underwent a comprehensive clinical examination. All affected individuals had two or more of the following signs upon clinical examination or in their personal medical history: hypotrichosis, facial milia, follicular atrophoderma and BCC. All individuals provided their written, informed consent for genetic studies. The study was approved by the local investigational review board (Hôpital Necker-Enfants Malades, Paris, France). DNA was extracted from peripheral blood lymphocytes by conventional phenol-chloroform purification. Paraffin-embedded skin tumor samples were obtained from 6 patients who underwent surgical excision of biopsy-confirmed BCCs. Frozen skin samples were obtained from 2 patients. In addition, 81 paraffin-embedded skin tumor samples were obtained from the specimen collection at Necker Hospital’s Pathology Department (French Ministry of Research reference number: DC-2009-955).

Targeted high-throughput sequencing. Next-generation sequencing with targeted enrichment of the 7,633,224 bp interval spanning the disease gene was performed in four affected family members and 1 unaffected family member at the Genoscope facility (Evry, France). A custom-sequence capture array (Roche NimbleGen, Madison, WI, USA) encompassing the region chrX:123576802-131210128 bp was used to hybridize shotgun fragment libraries obtained from selected subjects. Massively parallel sequencing was performed on this enriched library using a Solexa sequencer (Illumina, San Diego, CA, USA). Sequence data were aligned with the hg19 reference version of the human genome. On average, 29,485,436 sequencing reads were obtained, with an average length of 200 bp. Largely, 95% of all reads could be mapped, and 77% overlapped with the enriched regions. Largely, 93% of the enriched regions were covered entirely. The mean coverage for the five family members was 246X.

ChIP-qPCR. The ChIP assay was performed on frozen epidermis for chromatin signature or on primary keratinocytes for the study of Arp-T1 recruitment to Gli1 promoter, using an EZ-Magna ChIP™ A/G kit (Millipore Corporation, Billerica, MA, USA). Briefly, epidermis (1mg) or keratinocytes (1x10⁷ cells) were treated with 1% formaldehyde for 10 min. The crosslinked chromatin was then sonicated to yield a mean size of 300-500 bp. For chromatin signature, the DNA fragments were immunoprecipitated with anti-H3K27 (Millipore Corporation, 17-683), anti-H3K4me1 (Millipore Corporation, 07-436), anti-H3K4me3 (Millipore Corporation, 17-614), anti-RNA PolII (Millipore Corporation, 05-623B) and anti-IgG antibodies (provided with the EZ-Magna ChIP™ A/G kit). For the study of Arp-T1 recruitment to Gli1 promoter, the DNA fragments were immunoprecipitated with anti-Arp-T1 (PA5-31691, Immunogen sequence AA 174-376, Thermo Scientific, Rockford, IL, USA), anti-RNA PolII and anti-IgG antibodies. Sequences of interest were amplified by quantitative PCR using the Power SYBR Green PCR Master Mix (PE Applied Biosystems, Carlsbad, CA, USA). Reactions were performed in triplicate on an ABI Prism 7000 machine (PE Applied Biosystems). Input recovery was calculated using a ChIP-qPCR data analysis calculation shell (Sigma Aldrich, Saint Louis, MO, USA). Oligonucleotides used in these analyses are listed in Supplementary Table 1. For chromatin signature, the efficiency of primers was calculated on sonicated DNA (Input) after several dilutions and was comparable between the studied sequences (A1: 87 %, A2: 87 %, A3: 90 %, B1: 90 %, B2: 91 %, CNE12: 101 %).

Deletion of enhancer region using CRISPR-associated RNA-guided endonuclease Cas9. A lentiviral-based single vector (LentiCRISPR v2) that simultaneously deliver Cas9, single guide RNA (sgRNA) and puromycin selection marker engineered by the Zhang Laboratory was purchased from Addgene (http://www.addgene.org/). Guide RNA sequences were designed using online tool (crispr.mit.edu, Supplementary Table 1). Cloning of guide sequence into LentiCRISPR v2 vector was performed according to protocols available from Zhang Laboratory (www.genome-engineering.org). To produce lentivirus particles, LentiCRISPR v2-containing specific guide RNA sequence was co-transfected into HEK293T cells by using jetPRIME^TM reagent (Polyplus Transfection Inc., New York, NY, USA) with packaging vectors psPAX2, pMD2.G and pRSV-REV. Infectious lentiviruses were harvested at 24 and 48hr post-transfection and filtered through 0.8 μm-pore cellulose acetate filters. Recombinant lentiviruses were concentrated by ultracentrifugation (2 hr at 20,000 g) and resuspended in HBSS buffer. Primary keratinocytes were transduced with virus particles and selected with puromycin (1µg/mL) for 2 days. Deletion of targeted regions was determined using online tool Tide-calculator (https://tide.nki.nl) after PCR amplification and Sanger sequencing.

Transcriptomic analyses. Microarray experiments were performed on Affymetrix GeneChip® Human Transcriptome Array 2.0 (a genome wide array with 70523 probe sets). Raw data CEL files were imported in R/Bioconductor using the Oligo package. Expression levels were calculated using the RMA algorithm from the affy package and flags were computed using a custom algorithm within R. Assuming that a maximum of 80% of genes are expressed, we selected the 20% lowest values for each microarray as background. A threshold was fixed at two standard deviations over the mean of the background. All probes which normalized intensity measures were lower than the computed threshold, were flagged 0 instead of 1. An unsupervised analysis step was done by clustering prior to any supervised statistical comparison to unveil natural groups among the tested samples and detect potential outliers. For each comparison, the list were created, filtering probes flagged as « background » for at the most half of the samples according to flagged measurement in the relevant chips. Group comparisons were done using Student’s t test and lists were filtered at pvalues <= 5% and fold change >= 1.2. Heat maps analysis have been created using custom R script and the Java TreeView software. Functional analyses of the resulting lists of genes were performed with Ingenuity Pathway Analysis (http://www.ingenuity.com).

Tumor growth assay using UW-BCC1-T2 cells. Animal experiments were performed in accordance with the Swiss guidelines and regulations for the care and use of laboratory animals. Adult (5-7 weeks of age) AGR129 (IFN-α/β receptor, IFN-γ receptor and RAG-2 deficient) male mice (SPF housed homozygous 129/C57BL/6, B6.129S2-Rag2tm1.1Cgn-Ifnar1tm1Agt-Ifngr1tm1Agt/J according to the nomentrature: http://www.informatics.jax.org/mgihome/nomen/) were used as host animals for grafted tumors⁴⁰. Establishment of UW-BCC1 was described in Noubissi et al. (2014)²⁸. To improve both tumor take and growth in AGR129, UW-BCC1-T2 cells were isolated from two successive in vivo passages. Primary tumors were initiated by the subcutaneous injection of UW-BCC1-T2–ACTRT1, UW-BCC1-T2- ACTRT1 547_548 InsA, or UW-BCC1-T2-Blank cells (2X106 cells in 100µl of PBS) into both the right and left lateral flanks of AGR129 mice. Each group injected was composed of five mice. Tumor growth was monitored weekly by measuring the tumor volume using a caliper. Tumor volume (V) was calculated as V = π/6 × a × b2, where a is the longer and b is the shorter of two orthogonal diameters. Mice were killed by cervical dislocation, and the tumors were excised and embedded in paraffin. Eight-micrometer sections were stained with hematoxilin-eosin reagent and Ki67 (Dako, M7249, clone MIB-1, mouse, 1 in 100) antisera.

 

Tumor growth assay using MDA-MB231 and U2OS cells. Six-week-old athymic NMRI female nude mice (foxn1nu/foxn1nu, Elevage Janvier, Le Genest-Saint-Isle, France) were housed in filtered-air laminar flow cabinets and handled under aseptic conditions. Procedures involving animals were approved by the INSERM’s Institutional Animal Care and Use Committee. MDA-MB231-ACTRT1, U2OS-ACTRT1, MDA-MB231-Blank or U2OS-Blank cells (10⁶ cells per injection in 50µL of PBS1x and 50µL of Matrigel from Becton-Dickinson, Franklin Lake, NJ, USA) were injected subcutaneously into the lateral flank of each animal (two injections per mouse and five mice per group, chosen as a statistically robust sample size in accordance with the Institutional Animal Care and Use Committee’s recommendation). Every week, tumor size was measured (using calipers) in a blind manner by two investigators (neither of whom had performed the injections). The tumor volume (V) was calculated using the equation l × w × d, where l is the length, w is the width and d is the depth. Mice were killed by cervical dislocation, and the tumors were excised, weighed, fixed in 5% acetic acid/10% formaldehyde in 37% ethanol, and embedded in paraffin. Four-micrometer sections were stained with hematoxylin-eosin reagent for histological analysis or with the following primary antibodies for immunohistochemical analysis: anti-Ki67 (KI67PCE, Leica, Solms, Germany) or anti-cytokeratin antibodies (clone 34Βe12, Dako, Carpinteria, CA, USA). The apoptosis assay was performed using an In Situ Cell Death Detection Kit Fluorescein (Roche Diagnostics Gmbh, Mannheim, Germany).

Statistical analysis. Results were expressed as the mean ± S.D. Statistical significance was determined using unpaired, two-sample equal variance t-tests. All data were normally distributed, and the variance was similar in all compared groups. The threshold for statistical significance was set to p<0.05.

Additional methods. The procedures are described in detail in the Supplementary Methods.

Data-availability statement. Transcriptomic data are available in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5597.

COMPETING FINANCIAL INTERESTS

The authors declare no conflicts of interest.

REFERENCES

¹ Epstein, E. H., Basal cell carcinomas: attack of the hedgehog. Nat Rev Cancer 8 (10), 743 (2008).

² Vabres, P. et al., The gene for Bazex-Dupre-Christol syndrome maps to chromosome Xq. J Invest Dermatol 105 (1), 87 (1995).

³ Natoli, G. and Andrau, J. C., Noncoding transcription at enhancers: general principles and functional models. Annu Rev Genet 46, 1 (2012).

⁴ Lam, M. T., Li, W., Rosenfeld, M. G., and Glass, C. K., Enhancer RNAs and regulated transcriptional programs. Trends Biochem Sci 39 (4), 170 (2014).

⁵ Mousavi, K., Zare, H., Koulnis, M., and Sartorelli, V., The emerging roles of eRNAs in transcriptional regulatory networks. RNA Biol 11 (2), 106 (2014).

⁶ Bazex, A., Dupre, A., and Christol, B., [Follicular atrophoderma, baso-cellular proliferations and hypotrichosis]. Ann Dermatol Syphiligr (Paris) 93 (3), 241 (1966).

⁷ Rapelanoro, R., Taieb, A., and Lacombe, D., Congenital hypotrichosis and milia: report of a large family suggesting X-linked dominant inheritance. Am J Med Genet 52 (4), 487 (1994).

⁸ Herges, A., Stieler, W., and Stadler, R., [Bazex-Dupre-Christol syndrome. Follicular atrophoderma, multiple basal cell carcinomas and hypotrichosis]. Hautarzt 44 (6), 385 (1993).

⁹ Visel, A., Rubin, E. M., and Pennacchio, L. A., Genomic views of distant-acting enhancers. Nature 461 (7261), 199 (2009).

¹⁰ Nelson, A. C. and Wardle, F. C., Conserved non-coding elements and cis regulation: actions speak louder than words. Development 140 (7), 1385 (2013).

¹¹ Nobrega, M. A., Ovcharenko, I., Afzal, V., and Rubin, E. M., Scanning human gene deserts for long-range enhancers. Science 302 (5644), 413 (2003).

¹² Fisher, S. et al., Conservation of RET regulatory function from human to zebrafish without sequence similarity. Science 312 (5771), 276 (2006).

¹³ Blow, M. J. et al., ChIP-Seq identification of weakly conserved heart enhancers. Nat Genet 42 (9), 806 (2010).

¹⁴ Yang, S. et al., Functionally conserved enhancers with divergent sequences in distant vertebrates. BMC Genomics 16, 882 (2015).

¹⁵ Visel, A. et al., ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457 (7231), 854 (2009).

¹⁶ Andersson, R. et al., An atlas of active enhancers across human cell types and tissues. Nature 507 (7493), 455 (2014).

¹⁷ Inoue, F. and Ahituv, N., Decoding enhancers using massively parallel reporter assays. Genomics 106 (3), 159 (2015).

¹⁸ Wu, H. et al., Tissue-specific RNA expression marks distant-acting developmental enhancers. PLoS Genet 10 (9), e1004610 (2014).

¹⁹ Korkmaz, G. et al., Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat Biotechnol 34 (2), 192 (2016).

²⁰ Xie, J. et al., Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391 (6662), 90 (1998).

²¹ Gailani, M. R. et al., The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas. Nat Genet 14 (1), 78 (1996).

²² Heid, H. et al., Novel actin-related proteins Arp-T1 and Arp-T2 as components of the cytoskeletal calyx of the mammalian sperm head. Exp Cell Res 279 (2), 177 (2002).

²³ Oma, Y. and Harata, M., Actin-related proteins localized in the nucleus: from discovery to novel roles in nuclear organization. Nucleus 2 (1), 38 (2011).

²⁴ Kristo, I. et al., Actin, actin-binding proteins, and actin-related proteins in the nucleus. Histochem Cell Biol 145 (4), 373 (2016).

²⁵ Medina, P. P. et al., Frequent BRG1/SMARCA4-inactivating mutations in human lung cancer cell lines. Hum Mutat 29 (5), 617 (2008).

²⁶ Zhan, X. et al., Dual role of Brg chromatin remodeling factor in Sonic hedgehog signaling during neural development. Proc Natl Acad Sci U S A 108 (31), 12758 (2011).

²⁷ Jagani, Z. et al., Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog-Gli pathway. Nat Med 16 (12), 1429 (2010).

²⁸ Noubissi, F. K. et al., Role of CRD-BP in the growth of human basal cell carcinoma cells. J Invest Dermatol 134 (6), 1718 (2014).

²⁹ Krepischi, A. C. et al., Germline DNA copy number variation in familial and early-onset breast cancer. Breast Cancer Res 14 (1), R24 (2012).

³⁰ Lo, W. W. et al., Involvement and targeted intervention of dysregulated Hedgehog signaling in osteosarcoma. Cancer 120 (4), 537 (2014).

³¹ Yang, W. et al., Targeting hedgehog-GLI-2 pathway in osteosarcoma. J Orthop Res 31 (3), 502 (2013).

³² Kwon, Y. J. et al., Gli1 enhances migration and invasion via up-regulation of MMP-11 and promotes metastasis in ERalpha negative breast cancer cell lines. Clin Exp Metastasis 28 (5), 437 (2011).

³³ Benvenuto, M. et al., In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors. Oncotarget 7 (8), 9250 (2016).

³⁴ Dion, V., Shimada, K., and Gasser, S. M., Actin-related proteins in the nucleus: life beyond chromatin remodelers. Curr Opin Cell Biol 22 (3), 383 (2010).

³⁵ St Pierre, R. and Kadoch, C., Mammalian SWI/SNF complexes in cancer: emerging therapeutic opportunities. Curr Opin Genet Dev 42, 56 (2017).

³⁶ Wilson, B. G. et al., Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation. Cancer Cell 18 (4), 316 (2010).

³⁷ Kohashi, K. and Oda, Y., Oncogenic roles of SMARCB1/INI1 and its deficient tumors. Cancer Sci 108 (4), 547 (2017).

³⁸ Orom, U. A. et al., Long noncoding RNAs with enhancer-like function in human cells. Cell 143 (1), 46 (2010).

³⁹ Gupta, R. A. et al., Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464 (7291), 1071 (2010).

⁴⁰ Bartonicek, N., Maag, J. L., and Dinger, M. E., Long noncoding RNAs in cancer: mechanisms of action and technological advancements. Mol Cancer 15 (1), 43 (2016).

⁴¹ Tang, Y. et al., Linking long non-coding RNAs and SWI/SNF complexes to chromatin remodeling in cancer. Mol Cancer 16 (1), 42 (2017).

⁴² Yang, L. et al., lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature 500 (7464), 598 (2013).

⁴³ Zabidi, M. A. and Stark, A., Regulatory Enhancer-Core-Promoter Communication via Transcription Factors and Cofactors. Trends Genet 32 (12), 801 (2016).

⁴⁴ Wei, J. W., Huang, K., Yang, C., and Kang, C. S., Non-coding RNAs as regulators in epigenetics (Review). Oncol Rep 37 (1), 3 (2017).

FIGURE LEGENDS

Figure 1: Genetic analysis of BDCS families

1. 
   Pedigrees of six BDCS families (including three families reported previously, A-C²). Mutations associated with BDCS syndrome are indicated under each pedigree. Circles represent females and squares males. A filled shape indicates a BDCS sufferer and an open shape an unaffected individual.
   
2. 
   Immunofluorescence double staining of skin biopsies taken from a healthy control and six BDCS patients with anti-cytokeratin 10 (K10; Alexa Fluor 546 in green) and anti-Arp-T1 antibodies (Alexa Fluor 488 in red). Cell nuclei were stained with DAPI (blue). Scale bar = 10µm
   
3. 
   Quantification of relative expression of Arp-T1 (normalized to cytokeratin 10) using immunofluorescence and confocal microscopy. The quantification was performed in duplicate. Data are shown as mean ± SD. *: p<0.05; **: p<0.01.
   
4. 
   Q-RT-PCR analysis of ACTRT1 mRNA expression in BDCS patient (n=2) and control (n=3) skin biopsies. Results were normalized against PGK-1. Experiments were performed in triplicate and expression is shown as mean ± SD. ***: p<0.001. Representative of two independent experiments.
   
5. 
   Levels of indicated histone marks in the sequences surrounding the candidate variants in families A and B, and the CNE12 variant, in control epidermis (representative of two independent experiments on two control samples). The chromatin signature of the GAPDH promoter was simultaneously assessed, as a control. Note that the sequence of the GAPDH promoter was enriched with H3K4me3, a promoter-specific histone mark. The wild-type CNE12, A2 and B2 sequences were not tagged with H3K4me3 but they were enriched with the enhancer-specific markers H3K27AC and H3K4me1. In contrast, the wild type A1, A3, and B2 were not enriched in any specific histone markers. Data are expressed as the mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001.
   
6. 
   Enhancer luciferase reporter assay based on the wild type sequences surrounding candidate variants in HaCat keratinocytes. Enhancer activity was observed with the wild-type A2, B2 and CNE12 sequences. No significant luciferase activity was observed with wild –type A1, A3 and B sequences compared to empty vector (EV) control. Luciferase activity was normalized to Renilla. The experiment was performed in triplicate and representative of three independent experiments. Data are expressed as the mean ± SD. **: p<0.01; ***: p<0.001.
   
7. 
   Enhancer luciferase reporter assays demonstrating the impact of variants A2 (g.125959394C>G), B2 (g.127968123T>C) and CNE12 (g.127372937A>T) on enhancer activity in HaCat keratinocytes. Luciferase activity was normalized to Renilla. EV: empty vector. The experiment was performed in triplicate and results are representative of three independent experiments. Data are expressed as the mean ± SD. *: p<0.05; **: p<0.01.
   
8. 
   Schematic representation of target sites of single-guide RNAs (sgRNA) around the A2, B2 and CNE12 variations. sgRNAs that create indels in enhancer regions are shown in red, and sgRNAs that failed to delete enhancer regions are shown in black. The editing efficiency in genomic DNA (gDNA cut) was measured using TIDE software (see Supplementary Fig. 6, 7, 8). ACTRT1 mRNA expression was measured in RT-PCR assays.The experiment was performed in triplicate and results are representative of two independent experiments. Data are expressed as the mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001.
   

Figure 2: Transcriptomic analyses on skin biopsies from BDCS patients

1. 
   Heat maps showing genes deregulated in BDCS patient skin from families D and Ecompared to control skin biopsies by trancriptomic analyses. The differentially expressed genes are mostly involved in cell proliferation, death, survival and migration.
   
2. 
   Ingenuity Pathway Analysis (www.ingenuity.com) of genes deregulated in BDCS patients’ skin biopsies. 56 genes/proteins are directly linked to the Hedgehog pathway transcription factor Gli1 and Gli2.
   
3. 
   Q-RT-PCR analysis of mRNA expression of selected targets of the Sonic Hedgehog pathway in skin from BDCS patients (n=2) and controls (n=3). Results were normalized to PGK-1. Q-PCR was performed in triplicate and results are representative of three independent experiments. Data are expressed as the mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001.
   

Figure 3: Functional impact of the Arp-T1 protein on the Sonic Hedgehog signaling pathway

1. 
   Luciferase activity of a GLI-luciferase reporter (8x3'Gli-BS-delta51-LucII) in HaCat keratinocytes transfected with wild type or 547_548InsA mutant ACTRT1 after a 24hr stimulation with purmorphamine (3µM).As a negative control for Hedgehog activation, a construct containing mutations in Gli binding sites (8x3'Gli-BS-mutS4-delta51-LucII) was used. Luciferase activity was normalized to Renilla. EV: empty vector; NS: non-significant. The experiment was performed in triplicate and representative of three independent experiments. Data are expressed as the mean ± SD. **: p<0.01; ***: p<0.001.
   
2. 
   Q-RT-PCR analysis of Gli1 mRNA levels normalized to PGK-1 in primary keratinocytes after transfection of an empty vector (EV), wild type or 547_548InsA ACTRT1 variant, following 5hr stimulation with purmorphamine (3µM) or cotrol.Q-PCR was performed in triplicate and representative of two independent experiments. Data are expressed as the mean ± SD. *: p<0.05; ***: p<0.001; NS: non-significant.
   
3. 
   Western blot analyses of cytoplasmic, membrane, soluble nuclear and chromatin-bound proteins from HEK293T cells transfected with Flag-wild-type ACTRT1 or the mutant Flag-ACTRT1 (547_548 InsA) following 24h stimulation with DMSO or purmorphamine. The nuclear marker histone deacetylase 2 (HDAC2), cytoplasmic marker heat-shock protein 90 (HSP90), membrane marker calreticulin and chromatin marker H3K27ACwere used to assess protein loading and fraction purity. The plot to the right shows quantification of the fraction of FLAG tagged protein in each cellular compartment in the different conditions, determined from band intensities in the Western blots.
   
4. 
   Schematic representation of the human Gli1 promoter region, showing the locations of primers used for ChIP experiments (in f), relative to transcriptional start site (TSS: black arrow). Gli1 promoter region includes 5’ flanking sequence (5’UTR), an untranslated exon (1), and a part of the first intron (see Supplementary Fig. 12 for details on chromatin marks in the Gli1 promoter region in NHEK keratinocytes). Untranslated exon sequences are represented by a white square; translated exon sequences are represented in grey. Numbering above the line indicates nucleotide location relative to the transcription start site.
   
5. 
   Time course of Gli1 mRNA expression in primary keratinocytes after stimulation with purmorphamine (3µM) at indicated times. Results were normalized to PGK-1. Q-PCR was performed in triplicate and representative of two independent experiments. Data are expressed as the mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001.
   
6. 
   ChIP-qPCR analysis performed with primers binding to locations depicted in d, showing the fold enrichment of RNA polymeraseII and Arp-T1 at the Gli1 promoters in primary keratinocytes at indicated times of stimulation with purmorphamine (3µM). Primers for RPL10A promoter were used as a negative control for Arp-T1 binding. Q-PCR was performed in triplicate and representative of two independent experiments Data are expressed as the mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001.
   

Figure 4: ACTRT1 functions as a tumor suppressor gene in vivo

1. 
   Western blot analysis of Arp-T1 expression in UW-BCC1-T2 cells transfected with wild type or mutant ACTRT1 (547_548InsA) constructs. Blank indicates transfection with empty vector. .
   
2. 
   Proliferation of UW-BCC1-T2 cells expressing wild type or mutant ACTRT1 (547_548InsA) constructs, in a BrdU incorporation assays (each point n=12.Blank indicates transfection with empty vector. Data are expressed as the mean ± SD. **: p<0.01.
   
3. 
   Q-RT-PCR analysis of Gli1 and Gli2 mRNA expression normalized to Rpl13a in UW-BCC1-T2 cells stably expressing wild-type ACTRT1, ACTRT1 547_548InsA or empty vector. Q-PCR was performed in triplicate and representative of 3 independent experiments. Data are expressed as the mean ± SD. **: p<0.01; ***: p<0.001.
   
4. 
   Tumor volume of UW-BCC1-T2 cells stably expressing wild-type or mutant ACTRT1 (n=5 mice per group, 2 tumors per mouse) subcutaneously injected in AGR129 mice measured at indicated time points. Data are expressed as the mean ± SD. **: p<0.01.
   
5. 
   Tumor volumes measured at final time point from (d). Data are expressed as the mean ± SD. **: p<0.01.
   
6. 
   Mice injected with UW-BCC1-T2 cells were sacrificed and dissected, and tumors were photographed (5 tumors representative of the 10 tumors in each group).
   
7. 
   Time course of the proliferation of U2OS cells expressing ACTRT1 constructs, in a methylthiazoltetrazolium assay (performed in triplicate; one of three independent experiments is shown). Data are expressed as the mean ± SD. ***: p<0.001. Inset: Western blot analysis of Arp-T1 expression in two different clones expressing wild-type ACTRT1. Actin was used as a loading control.
   
8. 
   Q-RT-PCR analysis of Gli1 and Gli2 mRNA expression normalized to PGK-1 in U2OS cells stably expressing wild-type ACTRT1 or empty vector. Q-PCR was performed in triplicate and representative of two independent experiments. Data are expressed as the mean ± SD. **: p<0.01; ***: p<0.001.
   
9. 
   Time-course analysis of U2OS tumor volume in NMRI nude mice. Data are expressed as the mean ± SD (n = 10 per group, ***: p<0.001 for each time point). Mice were injected subcutaneously with either U2OS control cells or U2OS cells stably expressing ACTRT1 (n=5 mice per group, 2 tumors per mouse). Mice were sacrificed and dissected, and tumors were photographed (Inset: Assembly of 5 representative tumors of each group).
   
10. 
    Time course of the proliferation of MDA-MB231 cells expressing ACTRT1, in a methylthiazoltetrazolium assay (performed in triplicate; one of three independent experiments is shown). Data are expressed as the mean ± SD. *: p<0.05; **: p<0.01; ***: p<0.001. Inset: Western blot analysis of ACTRT1 expression in various cell clones. Actin was used as a loading control.
    
11. 
    Q-RT-PCR analysis of Gli1 and Gli2 mRNA expression normalized to PGK-1 in MDA-MB231 cells stably expressing wild-type ACTRT1 or empty vector. Q-PCR was performed in triplicate and representative of two independent experiments. Data are expressed as the mean ± SD. **: p<0.01.
    

l) Time course analysis of MDA-MB231 tumor volume in NMRI nude mice. Data are expressed as the mean ± SD (n = 10 per group). ***: p<0.001. Mice were injected subcutaneously with either MDA-MB231 control cells or MDA-MB231 cells stably expressing ACTRT1 (n=5 mice per group, 2 tumors per mouse). Mice were sacrificed and dissected, and tumors were photographed (Inset: Assembly of 5 tumors representative of each group).