SHORT COMMUNICATION Cyclin D1 is a direct transcriptional target of GATA3 in neuroblastoma tumor cells JJ Molenaar 1,2 , ME Ebus 1 , J Koster 1 , E Santo 1 , D Geerts 1 , R Versteeg 1 and HN Caron 2 1 Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands and 2 Department of Pediatric Oncology, Emma Kinderziekenhuis, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Almost all neuroblastoma tumors express excess levels of Cyclin D1 (CCND1) compared to normal tissues and other tumor types. Only a small percentage of these neuroblastoma tumors have high-level amplification of the Cyclin D1 gene. The other neuroblastoma tumors have equally high Cyclin D1 expression without amplification. Silencing of Cyclin D1 expression was previously found to trigger differentiation of neuroblastoma cells. Over- expression of Cyclin D1 is therefore one of the most frequent mechanisms with a postulated function in neuro- blastoma pathogenesis. The cause for the Cyclin D1 overexpression is unknown. Here we show that Cyclin D1 overexpression results from transcriptional upregulation. To identify upstream regulators, we searched in mRNA profiles of neuroblastoma tumor series for transcription factors with expression patterns correlating to Cyclin D1. GATA3 most consistently correlated to Cyclin D1 in four independent data sets. We identified a highly conserved GATA3 binding site 27 bp upstream of the Cyclin D1 transcriptional start. Chromatin immune precipitation confirmed binding of GATA3 to the Cyclin D1 promoter. Overexpression of GATA3 induced Cyclin D1 promoter activity, which decreased after site-directed mutagenesis of the GATA3 binding site in the Cyclin D1 promoter. Silencing of GATA3 resulted in reduced Cyclin D1 promoter activity and reduced Cyclin D1 mRNA and protein levels. Moreover, GATA3 silencing caused differentiation that was similar to that caused by Cyclin D1 inhibition. These finding implicate GATA3 in Cyclin D1 overexpression in neuroblastoma. Oncogene (2010) 29, 2739–2745; doi:10.1038/onc.2010.21; published online 15 February 2010 Keywords: CCND1; ChIP; GATA3; microarray; neuro- blastoma Introduction The cell cycle is a tightly controlled process that is deregulated in all cancer cells (Hanahan and Weinberg, 2000). Several checkpoints normally prevent premature cell-cycle progression and cell division. The crucial G1 entry point is controlled by the D-type Cyclins that can activate CDK4/6 that in turn phosphorylate the pRb protein. This results in a release of the E2F transcription factor that causes transcriptional upregulation of numerous genes involved in further progression of the cell cycle (Sherr, 1996). Neuroblastomas are embryonal tumors that originate from precursor cells of the sympathetic nervous system. This tumor has a very poor prognosis and despite the low frequency it is the second cause of cancer-related deaths in children (van Noesel and Versteeg, 2004; Maris et al., 2007). The G1 checkpoint is deregulated in neuroblastoma in various ways. Sporadic CDKN2A (p16) mutations and CDK4 amplifications have been reported but most striking is the extensive overexpres- sion of Cyclin D1 (Van Roy et al., 1995; Molenaar et al., 2003; Caren et al., 2008). We have previously shown a very high expression on mRNA and protein level compared to normal tissue and even compared to other tumors with known high expression levels of Cyclin D1. Functional evaluation of Cyclin D1 in neuroblastoma showed a crucial function in maintaining the undiffer- entiated phenotype of the neuroblasts in vitro and in vivo (Molenaar et al., 2008). A genomic cause for the extensive Cyclin D1 overexpression could be identified in a minority of cases. We have previously reported high-level genomic amplification of the Cyclin D1 gene in 5 out of 202 neuroblastoma tumors and cell lines. Others also reported recurrent high-level genomic amplifications of the 11q13 region encompassing the Cyclin D1 gene (Molenaar et al., 2003; Michels et al., 2007). These clonal events clearly pointed to a crucial function for Cyclin D1 in neuroblastoma tumor genesis, but could not explain the overexpression of Cyclin D1 in about 75% of the remaining neuroblastomas, that showed Cyclin D1 expression levels reaching equal or even higher levels compared to tumors with genomic amplification. Identification of the mechanism that drives Cyclin D1 overexpression in neuroblastomas is therefore an important question. In this paper, we identify by in silico analysis the GATA3 transcription factor as a candidate regulator of Cyclin D1. We show that GATA3 binds to the Cyclin D1 promoter and that silencing of GATA3 reduced promoter activity, resulting in decreased Cyclin Received 30 June 2009; revised 9 December 2009; accepted 3 January 2010; published online 15 February 2010 Correspondence: Dr JJ Molenaar, Department of Human Genetics, M1-132, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: j.j.molenaar@amc.uva.nl Oncogene (2010) 29, 2739–2745 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc