Oncogene (2009) 28, 876–885 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE HAND1 gene expression is negatively regulated by the High Mobility Group A1 proteins and is drastically reduced in human thyroid carcinomas J Martinez Hoyos1,2, A Ferraro2, S Sacchetti2, S Keller1,2, I De Martino1,2, E Borbone2, P Pallante2, M Fedele1, D Montanaro2, F Esposito1, P Cserjesi3, L Chiariotti1,2, G Troncone4 and A Fusco1,2 1Dipartimento di Biologia e Patologia Cellulare e Molecolare e/o Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta` di Medicina e Chirurgia di Napoli, Universita` degli Studi di Napoli ‘Federico II’, Naples, Italy; 2NOGEC (Naples Oncogenomic Center), CEINGE Biotecnologie Avanzate, Naples, Italy; 3Department of Cell and Molecular Biology, Tulane University, New Orleans, LA, USA and 4Dipartimento di Anatomia Patologica e Citopatologia, Facolta` di Medicina e Chirurgia, Universita` di Napoli ‘Federico II’, Naples, Italy HMGA1 proteins exert their major physiological function Introduction during embryonic development and play a critical role in neoplastictransformation. Here, we show that Hand1 The high mobility group A (HMGA) protein family gene, which codes for a transcription factor crucial for includes HMGA1a and HMGA1b, which are encoded differentiation of trophoblast giant cells and heart by the same gene through alternative splicing, and the development, is upregulated in hmga1 minus embryonic closely related HMGA2 protein (Fedele et al., 2001; stem cells. We demonstrate that HMGA1 proteins bind Fusco and Fedele, 2007). These proteins are non-histone directly to Hand1 promoter both in vitro and in vivo and architectural nuclear factors that bind the minor groove inhibit Hand1 promoter activity. We have also investi- of AT-rich DNA sequences through three short basic gated HAND1 expression in human thyroid carcinoma repeats, called ‘AT-hooks’, located at the NH2-terminal cell lines and tissues, in which HMGA proteins are region of the proteins (Thanos and Maniatis, 1995; overexpressed, with respect to normal thyroid; an inverse Reeves, 2001). The involvement of HMGA proteins in correlation between HMGA1 and HAND1 expression embryogenesis, cell proliferation, differentiation, apop- was found in all thyroid tumor histotypes. A correlation tosis and above all, cancer development has been between HAND1 gene repression and promoter hyper- extensively demonstrated (Fedele et al., 2001; Reeves, methylation was found in anaplastic carcinomas but not in 2001; Fusco and Fedele, 2007). In particular, HMGA other thyroid tumor histotypes. Therefore, we can proteins seem to play their major physiological role hypothesize that HMGA1 overexpression plays a key during embryonic development; in fact, their expression role on HAND1 silencing in differentiated thyroid is very high during embryogenesis, whereas it is very low carcinomas and that promoter hypermethylation occurs or negligible in normal adult tissues (Chiappetta et al., in later stages of thyroid tumor progression. Finally, the 1996). HMGA1 proteins have been found abundant in restoration of the HAND1 gene expression reduces the several malignant neoplasias, including colorectal, clonogenic ability of two human thyroid carcinoma- prostate, cervical, lung and thyroid carcinoma (Fedele derived cell lines, suggesting that HAND1 downregulation et al., 2001; Fusco and Fedele, 2007) and, recently, also may have a role in the process of thyroid carcinogenesis. in glioblastoma (Donato et al., 2004). Oncogene (2009) 28, 876–885; doi:10.1038/onc.2008.438; We identified HMGA1-regulated genes analysing by published online 8 December 2008 microarrays the expression profile of murine embryonic stem (ES) cells carrying two, one and no hmga1 Keywords: HMGA1; HAND1; knockout mice; ES cells; functional alleles (Martinez Hoyos et al., 2004; Fedele thyroid carcinomas et al., 2006). In this study, we focused our attention on the Hand1 gene, which showed by microarrays a 14-fold change in the hmga1-null ES in comparison to the wild-type cells (Martinez Hoyos et al., 2004). Hand1 (also named eHand/Hxt/Thing1) belongs to the Twist subfamily of Class B bHLH transcription factors. Murine Hand1 is expressed in developing heart tissue and derivatives of neural crest cells (Cserjesi et al., 1995). Extra-embryonic mRNA production has been Correspondence: Dr A Fusco, Dipartimento di Biologia e Patologia detected in the ectoplacental cone, in giant cells as well Cellulare e Molecolare, Facolta` di Medicina e Chirurgia di Napoli, as in distinct regions of the spongiotrophoblast cell layer via Pansini 5, 80131 Naples, Italy. (Cross et al., 1995). Embryos carrying a homozygous E-mail: [email protected] Received 21 May 2008; revised 16 October 2008; accepted 1 November mutation of Hand1, arrested at E7.5 with defects in 2008; published online 8 December 2008 trophoblast giant cells differentiation. Upon rescue of HAND1 regulation by HMGA1 proteins J Martinez Hoyos et al 877 the placental defect by aggregating wild-type tetraploid ES cells ES cells +/+ +/– –/– +/+ +/– –/– embryos, the fetuses died at E10.5 due to cardiac failure, 1 demonstrating that Hand1 has essential roles in both Hand1 0.5 trophoblast giant cell differentiation and cardiac mor- Hand1 0 phogenesis (Riley et al., 1998). In humans, HAND1 Hand2 HMGA1 -0.5 expression has been detected in trophoblast-like cells, log10 RQ the amniotic epithelium and in adult heart tissue, HMGA1 -1 suggesting that the protein may fulfill similar functions β-actin (Knofler et al., 2002). The mechanisms, however, which initiate and maintain HAND1 expression in extra- heart thyroid embryonic cell types are not well known. A role of +/+ +/– –/– +/+ +/– –/– HAND1 in cell proliferation and neoplastic transforma- tion has been recently envisaged because HAND1 gene Hand1 has been found silenced and hypermethylated in human β-actin gastric (Kaneda et al., 2002), pancreatic (Hagihara et al., 2004) and ovarian carcinomas (Takada et al., 2004). Figure 1 Hand1 expression in hmga1-knockout cells and tissues. Here, we demonstrate that Hand1 is upregulated in Semiquantitative (a and c) and quantitative (b) RT–PCR analyses hmga1-null ES cells, and that HMGA1 proteins bind for Hand1 and Hand2 expression were performed on RNA directly to the Hand1 promoter in vitro and in vivo extracted from wild-type ( þ / þ ), hmga1-single knockout ( þ /À) and hmga1-double knockout (À/À) ES cells and tissues. b-actin resulting in the inhibition of its activity. We also found expression was evaluated as internal control. that HAND1 was downregulated in human thyroid tumors. Such downregulation was associated with when we analysed the same tissues from the hmga2-null HMGA1 overexpression and, limited to anaplastic mice, no changes in Hand1 expression were observed carcinomas, with promoter hypermethylation. The (data not shown), indicating that Hand1 regulation was restoration of HAND1 expression led to a reduced cell HMGA1-specific. growth of two human papillary thyroid carcinoma cell It is noteworthy that no changes in the expression of lines, indicating a critical role of the HAND1 down- Hand2, another gene that belongs to the Twist subfamily regulation, likely mediated by HMGA1, in thyroid of class B bHLH transcription factors, were observed carcinogenesis. neither in ES cells, MEFs, nor in adult tissues from hmga1-andhmga2-knockout mice (Figures 1a and c). Results HMGA1 proteins bind to murine and human Hand1 promoter in vitro and in vivo Loss of HMGA1 correlates with an increased Hand1 To evaluate whether the differential Hand1 gene expression in murine ES cells,heart and thyroid tissues expression was a direct effect of HMGA1 binding to Expression profile, as assessed by microarray analysis Hand1 gene regulatory regions, we performed an of ES cells bearing one or two disrupted hmga1 alleles, electrophoretic mobility shift assay. In particular, we identified Hand1 as a candidate gene negatively regu- analysed a region spanning nucleotides from À2326 to lated by HMGA1. In fact, microarray data showed an À2296 related to the transcription start site (TSS) of the increase of 3.8- and a 14.0-fold for the heterozygous and murine Hand1 gene which contains AT-rich putative homozygous ES cells, respectively, when compared with HMGA1 binding sites. As shown in Figure 2a, a wild-type cells (Martinez Hoyos et al., 2004). Our first recombinant HMGA1 protein was able to bind directly aim was to validate the results obtained by microarray to this region. Binding specificity was demonstrated by analysis by semiquantitative and quantitative RT–PCR competition experiments showing loss of binding with (Figures 1a and b). These analyses confirmed that the addition of 200-fold molar excess of specific, Hand1 was strongly overexpressed in hmga1-knockout unlabeled oligonucleotides. Subsequently, we performed ES compared with wild-type cells. Data clearly showed binding assays with total extracts from wild-type and that regulation of Hand1 expression was HMGA1-dose- hmga1-knockout ES cells. A specific complex, with a dependent as an intermediate level of Hand1 expression mobility corresponding to the HMGA1 proteins, was was observed in the hmga1-heterozygous ES cells. present in extracts from wild-type mice whereas it was The analysis of Hand1 expression in heart and thyroid absent in extracts from homozygous hmga1-knockout tissues derived from hmga1-knockout mice also revealed ES cells. This complex was specifically displaced a negative