Repression of the Melanocyte-Specific Promoter of the Microphthalmia-Associated Transcription Factor by the Adenoviral E 1 A 12 S Oncoprotein ( E 1 A / microphthalmia / MITF / melanoma / repression )

Melanocytes express MITF, which is crucial for the development of the melanocyte lineage and is overexpressed in malignant melanomas. Adenoviral E1A protein-expressing melanocytes are unpigmented, with the expression of MITF being silenced. We tested here a direct repression of the melanocyte-specific MITF promoter by E1A and its mutants. We found that the extreme N-terminus and conserved region 1 are required for repression. In contrast, the motif in conserved region 2 (a.a. 122ñ126), as well as amino acids 26ñ35 at the N-terminus, are not necessary. As these two later motifs mediate E1A binding to the retinoblastoma protein or to the transcriptional co-activator TRRAP, respectively, and are important for transformation by E1A in cooperation with other oncogenes, the results suggest that the transformationdefective E1A can still efficiently repress the MITF promoter. The CREB binding motif-mutated promoter had lower activity, but was also repressed by the same E1A mutants in human melanoma cells. The E1A protein is known to also exert an antitumour activity, which is associated with its transcription repression function and the ability to induce apoptosis, and is a potential antimelanoma agent. Since recent data suggest that MITF may be a survival factor for melanoma cells, the E1A mutants described here might constitute a good targeting agent for antimelanoma therapy. The adenovirus E1A gene encodes nuclear proteins that are necessary for productive viral infection. Two splicing variants, the E1A 12S and 13S proteins, share the nonconserved N-terminus and conserved regions (CR) 1 and 2. However, unlike the E1A 13S protein, E1A 12S does not contain the zinc finger activation domain CR3. A prominent feature of E1A proteins is the ability to repress transcription. The transcription repressory function underlies many biological effects of E1A such as immortalization of primary rodent cells, block of differentiation, and cooperation with E1B or oncogenic ras in malignant transformation (Gallimore and Turnell, 2001; Frisch and Mymryk, 2002). Mouse melanocytes are one of the cell lineages that can be immortalized upon expression of E1A and these immortalized melanocytes show characteristics of totally dedifferentiated cells with lineage-specific markers being transcriptionally silenced (Dotto et al., 1989; Yavuzer et al., 1995; Halaban et al., 1996). A basichelix-loop-helix-leucine-zipper transcription factor microphthalmia (MITF, for microphthalmia-associated transcription factor) is essential for the development and differentiation of the melanocyte lineage from the neural crest (Opdecamp et al., 1997; reviewed by Goding, 2000). MITF binds to the E-box present in promoters of several pigment cell-specific genes and melanoma antigens (tyrosinase, tyrosinase-related proteins [TRP] 1 and 2, MLANA, SILV, and melastatin), and activates their transcription (Bentley et al., 1994; Bertolotto et al., 1998; Du et al., 2003; Miller et al., 2004). Whereas melanocytes express the melanocyte isoform MITF-M, several other tissues like osteoclasts, mast cells, and retinal pigmented cells express other tissue-specific MITF isoforms differing from MITF-M in the first exon, while the downstream exons are common to all isoforms (Udono et al., 2000; Shibahara et al., 2001). Each of these isoforms (-A, -B-, -E, -M, and -H) utilizes its own tissue-specific promoter for transcription, suggesting a lineage-specific transcriptional regulation. Melanocyte-specific isoform MITF-M is expressed exclusively in melanocytes and its promoter has been shown to be regulated by four transcription factors: CREB, LEF-1, Pax3 and Sox10 (Watanabe et al., 1998; Potterf et al., 2000; Bondurand et al., 2000; Lee et al., 2000; Takeda et al., 2000; Verastegui et al., Repression of the Melanocyte-Specific Promoter of the Microphthalmia-Associated Transcription Factor by the Adenoviral E1A 12S Oncoprotein ( E1A / microphthalmia / MITF / melanoma / repression ) B. DRDOV¡, J. VACHTENHEIM Laboratory of Molecular Biology, University Hospital, 3rd Medical Faculty, Charles University Prague, Czech Republic Received May 5, 2004. Accepted June 16, 2004. The authors acknowledge the support of grant (NC/6804-3) and institutional research project (MZ00000064211) from the Ministry of Health, Czech Republic. Corresponding author: Ji ̄Ì Vachtenheim, Laboratory of Molecular Biology, University Hospital, Clinic of Pneumology, 3rd Medical Faculty, Charles University Prague, Budinova 2, 180 00 Prague 8, Czech Republic. Tel. : (+420) 266082272; Fax: (+420) 284840840; e-mail: jivach@upn.anet. cz Abbreviations: CBP ñ CREB binding protein, CR ñ conserved region, CREB ñ cAMP response element binding protein, MITF ñ microphthalmia-associated transcription factor, PKA ñ protein kinase A, Rb ñ retinoblastoma, wt ñ wild-type. Folia Biologica (Praha) 50, 159-166 (2004) 2000; Yasumoto et al., 2002). Moreover, MITF-M can potentiate its own expression through the interaction with LEF-1 (Saito et al., 2002). Recently, it has been suggested that the Sox10 binding site, besides the canonical CRE motif, is also required for the cAMP pathway-dependent, forskolin or melanocyte-stimulating hormone-activated transcription of MITF-M in melanocytes (Huber et al., 2003). The β-catenin signalling, which is often upregulated in malignant melanocytes, promotes the survival of melanoma cells and requires MITF as a downstream effector (Widlund et al., 2002). Together, the reported data indicate the importance of the cAMP/PKA and Wnt/β-catenin/LEF-1 pathways in MITF expression and melanocyte differentiation. Additionally, recent data suggest that MITF might be a critical survival factor not only for embryonic melanocytes, but also for melanoma cells (McGill et al., 2002; Widlund et al., 2003). This is supported by a previous observation that MITF expression is retained in virtually all malignant melanoma specimens (King et al., 1999). The E1A protein, paradoxically, has been shown to possess a tumour suppressor activity in human tumour cells, including melanoma (Deng et al., 2002; Frisch and Mymryk, 2002; Voigtlander et al., 2002). Apparently, this activity results from the ability of E1A to repress transcription of genes associated with the malignant phenotype. However, it has not been studied whether the downregulation of MITF is required for the E1A-mediated tumour repression in melanoma. Therefore, as a first step in addressing this issue, we studied here a direct repression of the melanocyte-specific MITF promoter by the E1A 12S protein. We show that larger deletions of the N-terminus or CR1 domain inactivate the repressive activity of E1A. However, smaller deletions affecting binding of the retinoblastoma (Rb) protein or TRRAP coactivator leave E1A fully functional. We further show that the CRE sitemutated promoter responds differently to E1A in human and mouse melanoma cells. Whereas this mutant promoter could be further downregulated by E1A in human melanoma cell lines, no effect of E1A was seen in malignant mouse melanocytes. Our results together suggest the usefulness of E1A mutants in targeting the melanocyte survival protein MITF in human melanoma cells. Material and Methods