Basal transcription of the human TBX3 gene, a key developmental regulator which is overexpressed in several cancers, requires functional NF-Y and Sp1 sites James Smith a , Shaheen Mowla b , Sharon Prince a, ⁎ a Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Observatory, 7925, Cape Town, South Africa b Department of Clinical Laboratory Sciences (Haematology), Faculty of Health Sciences, University of Cape Town, Observatory, 7925, Cape Town, South Africa abstract article info Article history: Accepted 9 July 2011 Available online 20 July 2011 Received by Richard Cordaux Keywords: Cancer Development Promoter T-box factors TBX3 is a member of the T-box family of genes that encode developmentally important transcription factors. Mutations resulting in decreased levels of functional TBX3 lead to Ulnar-Mammary Syndrome and increased levels of TBX3 have been linked to several cancers. To understand the mechanisms regulating TBX3 expression we have previously cloned the 5′-flanking region of the human TBX3 gene and here we describe cis-elements required for its basal transcription. Using site-directed mutagenesis, luciferase reporter assays and in vitro and in vivo DNA binding experiments we identify a Sp1 element and two CCAAT boxes to be essential for basal TBX3 promoter activity. Our results are consistent with reports that these sites are necessary for efficient basal transcription in genes which lack TATA boxes or an Initiator which we show to be the case for TBX3. © 2011 Elsevier B.V. All rights reserved. 1. Introduction TBX3 belongs to the T-box family of transcription factors which all share a conserved DNA binding domain called the T-box (Bamshad et al., 1997). During embryogenesis, TBX3 plays critical roles in the development of several organs such as the heart, limb and mammary gland (Davenport et al., 2003; Hoogaars et al., 2007). Its importance in these processes is emphasized by haploinsufficiency of TBX3 resulting in Ulnar-Mammary Syndrome (UMS), a disorder characterized by several defects including the development of the limbs, mammary glands, and in extreme cases, heart (Bamshad et al., 1997; Klopocki et al., 2006; Meneghini et al., 2006). The degree of deformities which UMS patients suffer is dependent on the impact of the mutation on functional TBX3 levels. Increased levels of TBX3, on the other hand, have been linked with cancer development. It is overexpressed in a number of cancers including melanoma, breast, liver, bladder and pancreatic cancers (Fan et al., 2004; Ito et al., 2005; Lomnytska et al., 2006; Lyng et al., 2006; Renard et al., 2007; Rowley et al., 2004). Furthermore we have recently shown that silencing TBX3 in melanomas and breast cancer cells that overexpress this protein, reverses key features of tumourigenesis (Peres et al., 2010). Taken together, TBX3 therefore appears to function in a dose dependent manner with deregulated levels of this protein having catastrophic consequences for development and disease. This would suggest that its protein levels have to be tightly regulated and it is therefore clearly important to understand the mechanisms which regulate its expression. While several signaling pathways including PKC/AP-1 (Mowla et al., 2010), Wnt/β-catenin (Renard et al., 2007) and BMP (Yamada et al., 2000) have been shown to regulate TBX3 expression, their detailed mechanisms remain largely unknown. Furthermore, no reports have identified the cis-acting elements in the core and proximal TBX3 promoters responsible for basal TBX3 expression. The core promoter co-ordinates the assembly of RNA plolymerase II and its general transcription factors on the gene promoter and determines the site of transcription initiation (Butler and Kandonaga, 2002). Typical eukaryotic core promoters often, but not always, contain cis-regulatory elements such as a TATA box, initiator (Inr) and/or a downstream promoter element (DPE) (Breathnach and Chambon, 1981; Corden et al., 1980; Smale and Kadonaga, 2005). These sites are found at specific locations between -40 and +40 bp relative to the transcription start site. In addition to the elements found in the core promoter, DNA motifs for sequence specific transcription factors such as Sp1 and NF-Y are also found in the proximal promoter within 200 bp upstream of the transcription start site. These sites are necessary for efficient basal transcription and become particularly important in genes which lack TATA boxes or an Inr. We report here that the TBX3 gene lacks a TATA box and Inr and using a series of 5′-deletion and site directed mutant TBX3 promoters, we show that TBX3 basal transcription requires a Sp1 site and two CCAAT boxes located in the proximal promoter. Furthermore, using in vitro and in vivo DNA binding experiments we demonstrate that Sp1 and NF-Y bind these sites. Gene 486 (2011) 41–46 Abbreviations: UMS, Ulnar-Mammary Syndrome; ChIP, Chromatin Immuno- Precipitation; DAI, DNA affinity immunoblotting; DPE, downstream promoter element; Inr, Initiator; PCR, polymerase chain reaction. ⁎ Corresponding author. Tel.: + 27 21 406 6240; fax: + 27 21 448 7226. E-mail address: sharon.prince@uct.ac.za (S. Prince). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.07.013 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene