Oncogene (2011) 30, 2319–2332 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Essential role of microphthalmia transcription factor for DNA replication, mitosis and genomic stability in melanoma T Strub1,4, S Giuliano2,4,TYe1, C Bonet2, C Keime1, D Kobi1, S Le Gras1, M Cormont3, R Ballotti2, C Bertolotto2 and I Davidson1 1Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS, INSERM, Universite´ de Strasbourg, Illkirch, France; 2INSERM U895 Team 1 and Department of Dermatology, CHU Nice, France and 3INSERM U895 Team 7, Nice, France Malignant melanoma is an aggressive cancer known use of internal promoters (Steingrimsson, 2008). The for its notorious resistance to most current therapies. MITF-M isoform (hereafter designated simply as The basic helix-loop-helix microphthalmia transcription MITF) is the major form produced specifically in the factor (MITF) is the master regulator determining the melanocyte lineage from an intronic promoter (Goding, identity and properties of the melanocyte lineage, and is 2000b). MITF is essential for the survival of melano- regarded as a lineage-specific ‘oncogene’ that has a blasts and postnatal melanocytes (McGill et al., 2002; critical role in the pathogenesis of melanoma. MITF Hou and Pavan, 2008), in which it also controls the promotes melanoma cell proliferation, whereas sustained expression of genes required for the melanin synthesis supression of MITF expression leads to senescence. (Bertolotto et al., 1998). By combining chromatin immunoprecipitation coupled to In addition to regulating multiple aspects of normal high throughput sequencing (ChIP-seq) and RNA sequen- melanocyte function, MITF also has a critical role in cing analyses, we show that MITF directly regulates a melanoma, in which it is required for survival, and set of genes required for DNA replication, repair and controls the proliferation, invasive and metastatic mitosis. Our results reveal how loss of MITF regulates properties of melanoma cells (Garraway et al., mitotic fidelity, and through defective replication and 2005a, b; Goodall et al., 2008; Pinner et al., 2009). repair induces DNA damage, ultimately ending in cellular Primary and metastatic melanoma lesions comprise senescence. These findings reveal a lineage-specific control populations of cells expressing high and low MITF of DNA replication and mitosis by MITF, providing levels. Cells expressing low levels of MITF show high new avenues for therapeutic intervention in melanoma. levels of the POU3F2 (BRN2) transcription factor that The identification of MITF-binding sites and gene- binds to the MITF promoter and represses its transcrip- regulatory networks establish a framework for under- tion (Goodall et al., 2008; Kobi et al. 2010). These cells standing oncogenic basic helix-loop-helix factors such as show reduced proliferation, but enhanced motility and N-myc or TFE3 in other cancers. invasiveness, whereas higher levels of MITF are asso- Oncogene (2011) 30, 2319–2332; doi:10.1038/onc.2010.612; ciated with enhanced proliferative capacity. Melanoma published online 24 January 2011 cells seem to switch between these two states during tumour progression (Hoek et al., 2008a). Recently, it has Keywords: senescence; DNA repair; cancer; metastasis also been shown that sustained MITF suppression triggers senescence of melanoma cells (Giuliano et al. 2010). Hence, depending on its expression level, MITF can have pro- or anti-proliferative effects. Introduction To better understand the function of MITF in melanoma and to more generally define the mole- Micropthalmia transcription factor (MITF) is a basic cular basis of its function in the melanocyte lineage, helix-loop-helix protein that is the master regulator we have performed chromatin immunoprecipitation determining the identity and properties of the melano- coupled to high throughput sequencing (ChIP-seq) cyte lineage (Goding, 2000a, b). The MITF locus experiments in human 501Mel cells and RNA sequen- encodes multiple isoforms from alternate splicing and cing (RNA-seq) following small interfering RNA (siRNA)-mediated MITF knockdown. Our results show direct MITF regulation of an extensive set of Correspondence: C Bertolotto, INSERM U895 Team 1 and Depart- ment of Dermatology, CHU Nice, France. genes required for DNA replication, repair and mitosis. E-mail: [email protected] or I Davidson, CNRS; INSERM; This lineage-specific control of replication and mitosis Universite´de Strasbourg, IGBMC, 1 Rue Laurent Fries, Illkirch explains how MITF promotes melanoma proliferation, Ce´dex 67404, France. and suggests a mechanism by which diminished MITF E-mail: [email protected] 4These authors contributed equally to this work. expression in melanoma lesions may favour genomic Received 22 September 2010; revised 20 October 2010; accepted 20 instability and the emergence of more aggressive October 2010; published online 24 January 2011 metastatic cells. MITF regulates proliferation T Strub et al 2320 Results MITF occupies sites at 5578 potential target genes, of which 2771 genes have occupied sites in the proximal Identification of MITF-binding sites in human 501Mel promoter (À5/ þ 2kb) and 1743 genes in the immediate cells upstream region (À1000/ þ 100 bp; Supplementary Table Human 501Mel cells are considered as having a high 1). We observed MITF occupancy of previously identi- proliferative, low invasive phenotype in vitro, are fied target genes involved in pigmentation (TYR, DCT), poorly tumourigenic when injected subcutaneously in apoptosis (BAX, BIRC7;Dyneket al.,2008),cellcycle nude mice and express high levels of MITF (Moore (cyclin-dependent kinase 4, cyclin-dependent kinase 2; et al., 2004; Goodall et al., 2008; and see Supplementary Du et al., 2004) and transformation (MET; Beuret et al., Figure 1A, lane 1). To facilitate ChIP-seq in these cells, 2007). At some genes, the major MITF-occupied site we generated clonal lines expressing 3 Â hemagglutinin does not correspond to that previously described. For (HA)-tagged MITF at levels comparable with the endo- example, there is little or no occupancy of the previously genous (Supplementary Figure 1A). Anti-HA ChIP– described proximal promoter sites in the BCL2 and quantitative PCR (qPCR) showed MITF occupancy of DIAPH1 genes (McGill et al.,2002;Carreiraet al.,2006), three previously identified binding sites in the promoters but MITF rather occupies several intronic sites, including of the tyrosinase (TYR; Bentley et al., 1994), hypoxia major sites towards the 30 end of the gene (Supplementary inducible factor 1a (HIF1A; Busca et al., 2005) and Table 1, Supplementary Figure 2B and data not shown). RAB27A (Chiaverini et al., 2008) genes, but not at the However, we do not see occupancy of the DICER1 protamine 1 (PRM1) promoter used as a negative promoter that has recently been shown to be a MITF- control (Supplementary Figure 1B). Although the anti- target gene in melanocytes (Levy et al., 2010), suggesting HA ChIP showed strong enrichment only in the cells that the repertoire of MITF-occupied genes may differ in expressing the tagged MITF, polymerase II (Pol II) was melanocytes and melanoma cells. Together these results observed at the TYR, HIF1A and RAB27A, but not the indicate that MITF occupies a large set of loci and can PRM1 promoters in both the tagged and native 501Mel potentially regulate many target genes. cells (Supplementary Figure 1C). Having demonstrated the specificity of the anti-HA ChIP, we then performed anti-HA ChIP-seq on the Identification of MITF-regulated genes in 501MEL cells tagged and native 501Mel cells along with ChIP against To determine which of the potential target genes are Pol II and mono- and tri-methylated lysine 4 of histone regulated by MITF, we used two previously described H3. After peak detection and annotation (Experi- siRNA sequences to knockdown MITF expression mental procedures in Supplementary Information), in 501Mel cells (Figure 2a). MITF knockdown resulted 1 2139 MITF-occupied loci were identified (see Supple- in a profound morphological change (Carreira et al., mentary Table 1 showing the genomic localisation of the 2006) and entry into a senescent state characterised by MITF occupied sites and their nearby transcripts). Of activation of the DNA damage response, the appea- these, 9447 sites were located within±20 kb of anno- rance of senesence-associated heterochromatic foci tated RefSeq genes, 43% of which were located within and b-galactosidase staining (Giuliano et al., 2010; and À5/ þ 10 kb of annotated transcription start sites Figure 3). Both the senescence-associated heterochro- (Figure 1a). Promoter proximal MITF-binding sites matic foci and b-galactosidase activity were promi- are not randomly located within the À5to þ 2kb region, nent in the siMITF-knockdown cells that had altered but are preferentially located around 150 bp upstream morphology, but not in the control siluciferase (siLUC) of the transcription start site, a region that is often cells or in the fraction of siMITF cells that had escaped nucleosome free in active promoters (Figure 1b). transfection and did not show the characteristic Amongst 1000 highly occupied loci, 822 comprised one morphological changes. or several E-boxes of the type 50-CACGTG-30 or To understand how loss of MITF leads to senescence, 50-CATGTG-30, showing that these are preferred we performed 30-end sequencing of RNA from siMITF sequences, but only four examples of the ‘M-box’ and control siLUC cells (see Supplementary