ORIGINAL ARTICLE Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated in cancer 1 C Britschgi 1 , M Rizzi 1 , TJ Grob 1 , MP Tschan 2 , B Hu¨ gli 1 , VA Reddy 2 , A-C Andres 3 , BE Torbett 2 , A Tobler 4 and MF Fey 5 1 Experimental Oncology/Hematology, Inselspital and Department of Clinical Research, University of Bern, Bern, Switzerland; 2 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA; 3 The Tiefenau Laboratories, Department of Clinical Research, University of Bern, Bern, Switzerland; 4 Hematology, Inselspital and Department of Clinical Research, University of Bern, Bern, Switzerland and 5 Medical Oncology, Inselspital and Department of Clinical Research, University of Bern, Bern, Switzerland The tumor suppressor gene hypermethylated in cancer 1 (HIC1), located on human chromosome 17p13.3, is frequently silenced in cancer by epigenetic mechanisms. Hypermethylated in cancer 1 belongs to the bric a` brac/ poxviruses and zinc-finger family of transcription factors and acts by repressing target gene expression. It has been shown that enforced p53 expression leads to increased HIC1 mRNA, and recent data suggest that p53 and Hic1 cooperate in tumorigenesis. In order to elucidate the regulation of HIC1 expression, we have analysed the HIC1 promoter region for p53-dependent induction of gene expression. Using progressively truncated luciferase repor- ter gene constructs, we have identified a p53-responsive element (PRE) 500bp upstream of the TATA-box contain- ing promoter P0 of HIC1, which is sequence specifically bound by p53 in vitro as assessed by electrophoretic mobility shift assays. We demonstrate that this HIC1 p53- responsive element (HIC1.PRE) is necessary and sufficient to mediate induction of transcription by p53. This result is supported by the observation that abolishing endogenous wild-type p53 function prevents HIC1 mRNA induction in response to UV-induced DNA damage. Other members of the p53 family, notably TAp73b and DNp63a, can also act through this HIC1.PRE to induce transcription of HIC1, and finally, hypermethylation of the HIC1 promoter attenuates inducibility by p53. Oncogene (2006) 25, 2030–2039. doi:10.1038/sj.onc.1209240; published online 21 November 2005 Keywords: HIC1; p53; transcriptional target; HIC1.PRE; p73 Introduction The gene hypermethylated in cancer 1(HIC1), located in a region telomeric to TP53 on the short arm of chromosome 17 (17p13.3), encodes for a sequence- specific transcriptional repressor with five C2H2 zinc- fingers, and an N-terminal broad complex, tramtrack, and bric a` brac/poxviruses and zinc-finger protein– protein interaction domain (Wales et al., 1995; Deltour et al., 1999; Pinte et al., 2004b). Enforced expression of HIC1 alters cellular differentiation and reduces clono- genic growth of cultured cancer cells, pointing to a possible tumor suppressive role (Wales et al., 1995). In mouse knockout studies, heterozygous disruption of Hic1 leads to the formation of cancers in a gender- specific way, with heterozygous male mice developing carcinomas, and females predominantly lymphomas and sarcomas (Chen etal., 2003). Interestingly, in this model the remaining Hic1 allele was epigenetically silenced through hypermethylation of its promoter region, but not mutated. These studies underscore the importance of maintaining sufficient HIC1 protein levels in cells via controlled regulation. Consistent with the mouse-model findings, the HIC1 gene has been shown to be epigenetically silenced in a wide variety of human malignancies, such as breast cancer (Fujii et al., 1998), colorectal cancer (Ahuja etal., 1997) and hematopoietic neoplasms (Issa et al., 1997). The human HIC1 genomic region contains three promoters that give rise to different alternatively spliced HIC1 transcripts (Pinte et al., 2004a). The minor TATA-box containing promoter P0 is associated with exon 1b, whereas an upstream major GC-rich promoter P1 is associated with exon 1a. Exon 1c as well as internally spliced exons 1d and 1e are transcribed from P2, another upstream promoter containing GC boxes. Of the alternative first exons, only exon 1b is potentially coding and the common open reading frame of HIC1 resides within the large exon 2 coding region. The complexity of the HIC1 promoter and transcriptional organization might imply that the cell tightly controls regulation of HIC1 expression. However, little has been reported on transcriptional control of HIC1. A previous report has implicated potential p53 regulation of HIC1 based on demonstration of a putative p53 binding site located approximately 4kb upstream of the exon 1a TATA-box (Wales et al., 1995) and increased HIC1 transcript expression over basal levels upon ectopic p53 Received 1 June 2005; revised 3 October 2005; accepted 7 October 2005; published online 21 November 2005 Correspondence: Dr MF Fey, Medical Oncology, Inselspital and Department of Clinical Research, University of Bern, Bern 3010, Switzerland. E-mail: martin.fey@insel.ch Oncogene (2006) 25, 2030–2039 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc