Gene Expression of the Tumour Suppressor LKB1 Is Mediated by Sp1, NF-Y and FOXO Transcription Factors Nicolas Lu¨ tzner*, Johanna De-Castro Arce, Frank Ro¨ sl Research Program Infections and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Abstract The serine/threonine kinase LKB1 is a tumour suppressor that regulates multiple biological pathways, including cell cycle control, cell polarity and energy metabolism by direct phosphorylation of 14 different AMP-activated protein kinase (AMPK) family members. Although many downstream targets have been described, the regulation of LKB1 gene expression is still poorly understood. In this study, we performed a functional analysis of the human LKB1 upstream regulatory region. We used 200 base pair deletion constructs of the 59-flanking region fused to a luciferase reporter to identify the core promoter. It encompasses nucleotides 2345 to +52 relative to the transcription start site and coincides with a DNase I hypersensitive site. Based on extensive deletion and substitution mutant analysis of the LKB1 promoter, we identified four cis-acting elements which are critical for transcriptional activation. Using electrophoretic mobility shift assays as well as chromatin immunoprecipitations, we demonstrate that the transcription factors Sp1, NF-Y and two forkhead box O (FOXO) family members FOXO3 and FOXO4 bind to these elements. Overexpression of these factors significantly increased the LKB1 promoter activity. Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1’s main substrate AMPK in three different cell lines. Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription. Citation: Lu¨ tzner N, De-Castro Arce J, Ro¨ sl F (2012) Gene Expression of the Tumour Suppressor LKB1 Is Mediated by Sp1, NF-Y and FOXO Transcription Factors. PLoS ONE 7(3): e32590. doi:10.1371/journal.pone.0032590 Editor: Raya Khanin, Memorial Sloan Kettering Cancer Center, United States of America Received October 26, 2011; Accepted January 30, 2012; Published March 6, 2012 Copyright: 2012 Lu¨ tzner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum (DKFZ) and Israel’s Ministry of Science and Technology (MOST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: n.luetzner@dkfz.de Introduction Liver Kinase B1 (LKB1, also called STK11) was initially identified as the tumour suppressor gene mutated in the inherited Peutz-Jeghers cancer syndrome, an autosomal dominant genetic disorder [1,2,3,4]. It encodes a ubiquitously expressed and evolutionary well conserved protein kinase that is also inactivated in a large percentage of sporadic lung and cervical carcinomas [5,6,7,8]. Indeed, examining the HPV18-positive cervical carci- noma cell line HeLa, LKB1 is not transcribed. Transcription, however, can be reconstituted by trans complementation [9] after somatic cell hybridization with normal human fibroblasts, leading to cellular hybrids with a non-tumourigenic phenotype. Tumouri- genic segregants derived from the same hybrids again have lost LKB1 expression, suggesting that LKB1 down-regulation may favor progression towards malignancy [6]. Furthermore, LKB1 (+/2) mice develop hepatocellular carcino- mas after loss of heterozygosity and loss of LKB1 also correlates with increased metastasis in a well-studied mouse model of lung carcinogenesis [10]. In this model, LKB1-deficient tumours showed even more frequent metastasis than tumours lacking the tumour suppressor p53 [11]. Known mechanisms that explain how LKB1 operates as a tumour suppressor, mainly depend on direct phosphorylation of different AMP-activated protein kinase (AMPK) family members [12,13,14,15]. AMPK is a multi-component enzyme complex that acts as metabolic stress-sensor. Once activated, AMPK switches off many ATP-utilizing processes in order to sustain energy homeostasis. AMP binding allosterically activates AMPK, facilitating the binding of upstream kinases that enhance its activity [16]. Although various downstream targets such as the mammalian target-of-rapamycin (mTOR) pathway have been studied in detail [14], the regulation of LKB1 gene expression is still poorly understood. Hence, analysis of the transcriptional regulation of LKB1 should not only be helpful to identify important trans-acting regulatory proteins that can alter gene expression, but also to define critical cis-regulatory regions indispensable for its transcrip- tional control. Such regions could be affected in a variety of cases in which LKB1 was inactivated without having mutations within the coding sequence [17,18]. Here, gene silencing via de novo DNA- methylation of CpG-rich stretches could be such a scenario [19]. Therefore, identification and characterization of the LKB1 promoter and transcriptional regulators is not only important to unravel the complexity of LKB1 gene silencing, but also to understand how upstream regulatory proteins mediate metabolic sensoring of nutritional depletion and in turn cell cycle control. In this study we performed a functional analysis of the LKB1 promoter and identified distinct cis-regulatory elements, including three CCAAT boxes and a non-canonical GC-box that critically affected LKB1 gene expression. These elements bind NF-Y and Sp1, representing two ubiquitous transcription factors involved in the regulation of various genes [20,21]. Furthermore, two forkhead box O (FOXO) transcription factors that bind the PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32590