Oncogene (2008) 27, 1263–1272 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE The histone deacetylase inhibitor trichostatin A induces GADD45c expression via Oct and NF-Y binding sites MR Campanero1, A Herrero2 and V Calvo2 1Instituto de Investigaciones Biome´dicas, CSIC-UAM, Arturo Duperier, Madrid, Spain and 2Departamento de Bioquı´mica e Instituto de Investigaciones Biome´dicas Alberto Sols, UAM-CSIC, Facultad de Medicina de la Universidad Auto´noma de Madrid-UAM, Madrid, Spain The GADD45c protein is a potential tumor suppressor leukemia cells in response to genotoxic stress or whose expression is reduced in several tumors. However, interleukin (IL)-6 (Takekawa and Saito, 1998; Zhang very little is known about the regulation of its expression. et al., 1999), in hemangioblasts in response to oncostatin We have determined that the most relevant region of its M (Nakayama et al., 1999), in neurons in response to promoter lies between nucleotides À112 and À54, relative nerve growth factor depletion (Kojima et al., 1999) and to the transcription start site. Putative Oct and NF-Y in T cells in response to IL-2 or IL-12 plus IL-18 elements were found in this region and factors belonging (Beadling et al., 1993; Lu et al., 2004). to these families interacted with these elements in vitro Gadd45 proteins have very similar amino acid and with the promoter in vivo. Mutation of these elements sequences (Takekawa and Saito, 1998; Zhang et al., reduced the basal activity of the promoter, suggesting that 1999) and have been implicated in cell cycle arrest (Fan both sites are essential for basal expression. These factors et al., 1999; Zhang et al., 1999; Jin et al., 2002), DNA interact with chromatin modifying proteins and we found repair (Smith et al., 2000), innate immunity (Lu et al., that histone deacetylase 1 or silencing mediator for 2001; Yang et al., 2001), maintenance of genomic retinoid and thyroid hormone receptor overexpression stability (Hollander et al., 1999) and apoptosis (Take- reduced the basal activity of the promoter. In contrast, kawa and Saito, 1998; Zhang et al., 1999, 2001). Gadd45 forced expression of the histone acetylase protein PCAF proteins display a complex array of physical interactions or cell treatment with the HDAC inhibitor trichostatin A with cellular proteins involved in cell cycle regulation increased GADD45c mRNA levels and induced and the response to extrinsic stress, including p21, Cdc2/ GADD45c promoter activity through its Oct and NF-Y cyclinB1 and components of the JNK and p38 stress- elements. Moreover, ectopic expression of a dominant- induced kinase pathways (Liebermann and Hoffman, negative version of NF-YA strongly inhibited trichostatin 2003). In particular, Gadd45g regulates apoptosis in A-induced activation of the promoter. Our data strongly several cell types (Takekawa and Saito, 1998; Zhang suggest that inhibition of deacetylase activity could et al., 2001; Chung et al., 2003) and cytokine production potentially be used for treatment of tumors where in T cells (Lu et al., 2001). Moreover, Gadd45g GADD45c expression is reduced. negatively regulates progression through the cell cycle. Oncogene (2008) 27, 1263–1272; doi:10.1038/sj.onc.1210735; This effect is likely mediated by its capacity to inhibit published online 27 August 2007 cyclinB1-cdk1 activity (Vairapandi et al., 2002). Gadd45g expression is reduced in several tumors, Keywords: GADD45g; HDAC inhibitor; TSA; Oct; NF-Y such as pituitary adenomas (Zhang et al., 2002), anaplastic thyroid cancer (Chung et al., 2003), hepato- cellular carcinoma (Sun et al., 2003) and lymphoma and nasopharyngeal carcinoma cell lines (Ying et al., 2005) Introduction in comparison to normal tissues. A low Gadd45g expression could be functionally relevant to tumor The GADD45 family of genes, including GADD45a, growth because transient transfection of Gadd45g GADD45b and GADD45g, is induced by genotoxic induced growth arrest and/or apoptosis in a number of stress and a subset of cytokines in many different cell tumor cell lines (Takekawa and Saito, 1998; Zhang types (Liebermann and Hoffman, 2003). GADD45g et al., 2001, 2002; Chung et al., 2003; Jiang and Wang, (CR6/OIG37/GRP17) expression is induced in myeloid 2004; Ying et al., 2005). These observations suggest that GADD45g can be considered as a functional tumor sup- pressor gene. Although several studies have addressed the Correspondence: Dr V Calvo, Departamento de Bioquı´ mica, Facultad transcriptional regulation of the GADD45a and b genes de Medicina, Universidad Auto´ noma de Madrid, Arzobispo Morcillo (Zhan et al., 1994; Constance et al., 1996; Marhin et al., 4, 28029 Madrid, Spain. E-mail: [email protected] 1997; De Smaele et al., 2001; Jin et al., 2001; Takahashi Received 6 November 2006; revised 21 June 2007; accepted 17 July 2007; et al., 2001), very little is known about the regulation of the published online 27 August 2007 GADD45g gene. In fact, to the best of our knowledge, Induction of GADD45c by HDAC inhibitor MR Campanero et al 1264 there is only a single study addressing GADD45g gene regulation where it was suggested that transcription - 818 factors C/EBPb and d might be involved in GADD45g promoter activation during differentiation of M1 cells - 482 (Jung et al., 2000). A general mechanism of gene regulation relies on the - 220 interactions of transcription factors with co-activators - 112 that contain histone acetyltransferase (HAT) activity and co-repressors that contain histone deacetylase - 54 (HDAC) activity (Berger, 2002). HDAC inhibitors, such as trichostatin A (TSA; Sigma, Tres Cantos, Madrid, pGL3-B Spain), induce many genes coding for proteins involved in cell cycle control, including GADD45a and GADD45b 0 20 40 60 80100 120 (Della-Ragione et al., 2001; Chen et al., 2002; Hirose Relative luciferase activity et al., 2003), and stop tumor growth by eliciting either cell cycle arrest or apoptosis in tumor cells (Johnstone, Figure 1 Deletion analysis of the murine GADD45g promoter. Several 50-deleted promoter fragments were cloned into pGL3- 2002). Basic (pGL3-B). The resulting plasmids (125 ng) were transiently Since GADD45g expression is reduced in tumors and transfected into NIH3T3 cells and luciferase activities were its forced expression has tumor-suppressive properties, analysed, normalized to protein concentration and b-galactosidase a detailed knowledge of the molecular mechanisms activity, and referred to the normalized luciferase activity of the À818 promoter reporter (relative luciferase activity). Data are regulating its expression might help to develop new shown as means (bars, s.e.) of six independent experiments. antitumor therapeutic strategies. We therefore analysed the mechanisms of GADD45g transcriptional regula- tion. Our results indicate that functional Oct and NF-Y elements are essential for the promoter activity, that -818 -482 -220 -112 -54 +15 GADD45g promoter can be regulated through histone acetylation/deacetylation, and that GADD45g expres- sion can be readily induced in tumor cells by HDAC Oct1 NF-Y TATA inhibitors possibly through the NF-Y and Oct elements of its promoter. -818 Results Octm NFYm Deletion and point mutation analysis of the murine GADD45g promoter OctmNFYm To gain insight into the molecular mechanisms regulat- ing GADD45g expression, we first identified the motifs pGL3-B required for basal activity of the murine GADD45g promoter. To this end, a set of 50 serially deleted promoter fragments, spanning from nucleotides À818 to 0 20406080100 120 þ 15 according to the transcription start site (Balliet Relative luciferase activity et al., 2003), were cloned upstream from the luciferase Figure 2 Mutational analysis of the GADD45g promoter. A gene in pGL3-Basic. The resulting reporter constructs schematic representation of the GADD45g promoter showing the were transiently transfected in NIH3T3 cells and the approximate positions of the Oct, NF-Y and TATA box elements luciferase activity was assayed for each construct. As is shown. Reporter plasmids (125 ng) containing either the wild- shown in Figure 1, although the promoter activity type promoter (nucleotides À818 to þ 15) or promoters devoid of either the Oct site (Octm), the NF-Y site (NFYm) or both sites decreased when the region À818 to À112 was deleted, an (OctmNFYm) were transiently transfected into NIH3T3 cells and almost complete ablation of its activity was achieved luciferase activities were analysed. Relative luciferase activities only upon deletion of the À112 to À54 region. These were calculated as in Figure 1. Data are shown as means (bars, s.e.) results suggest that although elements within the À818 of ten independent experiments. to À112 can contribute to this promoter regulation, the region between À112 and À54 is essential for its activity. A search for putative transcription factor binding sites using MatInspector (Cartharius et al., 2005) revealed the presence in the À112 to À54 region of or combined. As shown in Figure 2, mutation of the potential Oct (ATTTGCAT), and NF-Y (CCAAT) individual sites or their combination decreased the binding sites. Site-directed mutagenesis was used to activity to 35–50% of the wild-type promoter activity, generate mutated versions of the À818 construct devoid suggesting that these elements significantly contribute to of the Oct (Octm) and NF-Y (NFYm) sites, individually promoter activation. Oncogene Induction of GADD45c by HDAC inhibitor MR Campanero et al 1265 Identification of proteins