Obesity Risk Gene TMEM18 Encodes a Sequence-Specific DNA-Binding Protein Jaana M. Jurvansuu*, Adrian Goldman Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland Abstract Transmembrane protein 18 (TMEM18) has previously been connected to cell migration and obesity. However, the molecular function of the protein has not yet been described. Here we show that TMEM18 localises to the nuclear membrane and binds to DNA in a sequence-specific manner. The protein binds DNA with its positively charged C-terminus that contains also a nuclear localisation signal. Increase in the amount of TMEM18 in cells suppresses expression from a reporter vector with the TMEM18 target sequence. TMEM18 is a small protein of 140 residues and is predicted to be mostly alpha-helical with three transmembrane parts. As a consequence the DNA binding by TMEM18 would bring the chromatin very near to nuclear membrane. We speculate that this closed perinuclear localisation of TMEM18-bound DNA might repress transcription from it. Citation: Jurvansuu JM, Goldman A (2011) Obesity Risk Gene TMEM18 Encodes a Sequence-Specific DNA-Binding Protein. PLoS ONE 6(9): e25317. doi:10.1371/ journal.pone.0025317 Editor: Brian P. Chadwick, Florida State University, United States of America Received June 28, 2011; Accepted August 31, 2011; Published September 28, 2011 Copyright: ß 2011 Jurvansuu, Goldman. 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 Academy of Finland (1128207 to J.M.J., 1114752 to A.G., www.aka.fi); and the Sigrid Juse´lius Foundation (www. sigridjuselius.fi). 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: [email protected] Introduction TMEM18 is a small protein of 140 amino acids. It has a functional nuclear localisation signal and it is predicted to have According to the NCBI’s Entrez Gene search, humans have three transmembrane helices [2]. Although, the studies related to over 200 proteins named merely transmembrane protein obesity highlight the expression of TMEM18 in brain, it is (TMEM). The first publication mentioning TMEM18 appeared expressed robustly in most of the tissues studied in human, mouse, in 2008 when TMEM18 was identified as a terminal oligo- rat, and fruit fly [6,16,17][18]. This abundant expression pattern pyrimidine track gene [1]. Terminal oligo-pyrimidine track is an would imply that the protein has some general function in cells. mRNA cis-regulatory sequence that inhibits translation from the Moreover, TMEM18 is well conserved during evolution from mRNA, for example, in growth arrested cells. The same year plants to animals. Yeast and roundworm C. elegans are two known TMEM18 emerged from a screen designed to identify proteins, exceptions of the sequenced eukaryotes to lack an obvious which enhance neural stem cell migration towards gliomas [2]. In homologue to TMEM18 [16]. Thus, TMEM18 seems to be the article it was shown that TMEM18 regulates neuronal stem clearly beneficial yet not indispensable. cell mobility in vivo as well as in vitro. Abdullah et al. found What does TMEM18 do in the cell? Here we provide evidence TMEM18 mRNA to be among the transcripts that correlated with and hypothesise that TMEM18 might be involved in gene tumorigenicity of human tumour-derived cell lines [3]. silencing. From the beginning of 2009 there has been a spate of articles linking TMEM18 to obesity [4–12]. The first two articles were Results meta-analyses of genome wide association studies of single- nucleotide polymorphism (SNP) and body mass index (BMI) Recombinant TMEM18 localises to nuclear membrane [5,6]. In these studies tens of thousands of individuals were We used fluorescence microscopy and cell fractionation to analysed, which gave exceptional power to detect genes with small determine the localisation of recombinant TMEM18. Cells were effects on BMI. The strongest impact on BMI was with Fat mass transfected either with a vector expressing GFP or TMEM18-GFP. and obesity associated (FTO), a gene already identified as an Nuclei were stained with DAPI before detection by fluorescence obesity risk [13]. The second best association to BMI was with microscopy. GFP alone localises throughout the cell, whereas TMEM18. The SNP variant linked to TMEM18 added 0.26 kg/ TMEM18-GFP showed a clear ring-like structure around the m2 to BMI, which for 170 cm tall adult corresponds to 750 g [6]. nucleus (Figure 1A). This suggests that the TMEM18-GFP is in the For FTO the change in BMI per allele was 0.33 kg/m2. nuclear envelope. TMEM18-GFP localised also to cytoplasmic Subsequent articles confirmed the role of TMEM18 as an obesity structures, most probably to the endoplasmic reticulum (ER). risk in adult Europeans, extended the linkage to childhood and Next we did a cell fractionation to verify the nuclear localisation adolescent obesity, and the obesity of Japanese [8–10,14][15]. In of TMEM18. HA-tagged TMEM18 was transfected into cells, and the articles it was speculated that the detected TMEM18 the next day cytosol and nuclei were separated by hypotonic expression in brain and particularly in hypothalamus would treatment and centrifugation. Nuclear proteins were further translate into feeding behaviour. separated into detergent soluble and insoluble fractions. The PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e25317 TMEM18 Encodes a DNA-Binding Protein Figure 1. TMEM18 localises to nuclear membrane and cytoplasm. (A) The localisation of TMEM18-GFP in cells was studied by fluorescence microscopy. The cells in the first row express GFP alone and in the second TMEM18-GFP. The first column shows GFP expression, the second DAPI stained nuclei, and the third the superimposition of the two pictures. (B) Western blot analysis of cell fractions from TMEM18-HA overexpressing cells. TMEM18 is mainly in cytosolic and nuclear detergent soluble fractions. A small amount of TMEM18 can be seen also in detergent insoluble nuclear fraction containing proteins bound to DNA. Laminin A/C was used as the control for the purity of the cell fractionation. doi:10.1371/journal.pone.0025317.g001 detergent insoluble pellet was treated with acid to release DNA- modelling or by an ab initio protocol for proteins, like TMEM18, bound proteins. Laminin A/C is a soluble nuclear protein and was without Protein Data Base homologues [19]. The Robetta used as a positive control for the purity of nuclear preparation. predicted TMEM18 to consist mainly of alpha helixes According to the Western blot (Figure 1B) most of the TMEM18- (Figure 2A). The C-terminal part of TMEM18 has a nuclear HA was in the cytosol and detergent soluble nuclear fractions. The localisation signal and thus should be inside the nucleus. The C- cytosolic fraction contains soluble proteins as well as proteins terminus has an array of large hydrophilic amino acids bound to endoplasmic reticulum (ER). The nuclear detergent (ERRKEKKRRRKED) and must thereby extend outwards from soluble fraction contains soluble nuclear proteins and proteins the membrane (Figure 2A). This protruding part with several bound to nuclear envelope. A small amount of TMEM18 positive amino acids, i.e. lysines and arginines, is a good candidate remained in the insoluble nuclear pellet. Detergent insoluble to bind DNA. Because the ROSETTA software does not take into nuclear proteins pellet together with the chromatin. account the lipid membrane, the mainly non-structured N- Recombinant TMEM18 localised to cytoplasm and nuclear terminus shown to be inside the membrane in Figure 2A is membrane. This pattern of localisation resembles that reported for unlikely to be in that position. the natural protein [2]. Moreover, we saw that some TMEM18 We used chromosomal DNA linked to cellulose to study the was also found in nuclear detergent insoluble fraction, which binding of TMEM18 to DNA. All DNA binding experiments indicates that TMEM18 might be bound to chromatin. included the non-ionic detergent, dodecyl maltoside, in the buffers to ensure native conformation of TMEM18. Protein extracts from TMEM18 binds DNA with its C-terminus TMEM18-HA overexpressing cells were incubated with DNA- Robetta server uses ROSETTA software to model three- cellulose, washed, and protein bound to the DNA was eluted with dimensional protein domains either by fragment homology high salt concentration. As shown in Figure 2B, TMEM18 could PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25317 TMEM18 Encodes a DNA-Binding Protein Figure 2. The positively charged C-terminus of TMEM18 binds DNA. (A) Robetta server-predicted (robetta.bakerlab.org) structure model of TMEM18. Dashed lines depict nuclear membrane, the three transmembrane domains are in gray, the C-terminal DNA-binding domain, ERRKEKKRRRKED, is coloured black, and white C’s indicate the sites of cysteines. The structural model was edited with PyMOL. (B) TMEM18 binds to dsDNA and ssDNA-cellulose resin. Western blot of Flag-tagged TMEM18 shows the amount of TMEM18 in flow through (FT), wash, and elute. DNase I treatment of the DNA-cellulose resins erased the TMEM18 binding demonstrating that TMEM18 does not bind to the cellulose matrix. (C) TMEM18 lacking the last 13 C-terminal amino acids was unable to bind DNA-cellulose. Western blot results are shown for both dsDNA and ssDNA-cellulose binding assays. doi:10.1371/journal.pone.0025317.g002 be eluted from both single and double-stranded DNA-cellulose. TMEM18. To control for nonspecific binding, cells were Dnase I treated DNA-cellulose was used as a negative control to transfected with empty vector, TMEM18-HA, or TMEM18-Flag.