cervical cancer, SC markers SOX2 and SMOOTHENED were recently shown to be overexpressed by nearly all the cells within tumour mass [16,17]. Another specific agent regulating SC functions is the Undiffer- entiated cell Transcription Factor 1 (UTF1). Human UTF1 gene codes for only one isoform, and up to now, no pseudogene has been found [18]. Its expression was described to be restricted to SC, teratocarcinoma cell lines and testicular germ cell tumors [18– 21]. Recent studies indicated that UTF1 is a stably chromatin- associated transcriptional repressor protein involved in the initiation of SC differentiation, but not in the SC self-renewal [19,20,22]. During development, in adult somatic tissues, its expression is downregulated and hypermethylation of the Oct4/ Sox2 enhancer, upstream the coding region of the gene, was proposed as a mechanism participating to its inhibition [22]. In a cancer context, UTF1 was shown to be highly expressed in germ cells tumors [19,21]. However, in somatic tumors, UTF1 expression was suggested to be of potential interest to differentiate between grade I–III and grade IV neuroblastoma tumors whereas it was shown to be expressed at the same level in healthy epidermis and in skin SCC [23,24]. In this study, we were interested to identify SC gene methylation signatures during cervical carcinogenesis. We demonstrated that UTF1 gene promoter is hypermethylated and associated with UTF1 overexpression in cervical SCC in comparison with normal ectocervix. In vitro experiments allowed to detect UTF1 in the nucleus of epithelial somatic cancer cell lines even in the absence of its main known regulators, Sox2 and Oct4A. Our results suggest that UTF1 promoter methylation profile could be used as a biomarker for cervical cancer diagnosis and raise the questions about the mechanisms involved in the regulation of its expression in epithelial tumors. Materials and Methods Ethics statements The study involving human biopsy samples was conducted in accordance with the Declaration of Helsinki and approved by the local ethics committee of the University Hospital of Liege (reference: B70720109045). Patients gave written informed con- sent for the sample collection. Tissue collection For frozen tissues, 4 samples of Ecto originating from total hysterectomy for non cervical pathologies and 5 samples of SCC were used. For Tissue microarray (TMA) construction, 66 paraffin-embedded tissue blocks of conisation or hysterectomy specimens representing 48 cases including various (pre)neoplastic cervical squamous intraepithelial lesions (11 LSIL, 18 HSIL and 19 SCC) and 18 tumor-free ectocervix samples (Ecto) from hysterectomy for noncervical pathologies were collected [25]. Liquid-based cytology samples (LBC) were composed of 17 Ecto, 10 LSIL, 11 HSIL and 9 SCC. Laser Capture Microdissection and DNA isolation Serial frozen sections (10 mm thick) of 5 SCC and 4 Ecto were obtained using a Microm HM 500 M cryostat (Microm Interna- tional, Francheville, France) and mounted on glass slides covered with a thin membrane (Carl Zeiss Microscopy, Munich, Germany). Sections were then stained with Gill III hematoxylin (RNase free, Merck, Darmstadt, Germany) for 1 minute, washed in distilled water and in ethanol, and dried at RT. Microdissection was performed using a P.A.L.M. microdissector (Carl Zeiss UTF1 and Cervical Carcinogenesis Microscopy) and was supervised by a histopathologist to ensure .95% specificity of captured cells. DNA isolation and bisulfite conversion DNA from microdissected tissue was extracted using QIAamp DNA micro kit (Qiagen, Valencia, CA) according to manufactur- er’s instructions. DNA from LBC samples was extracted using NucleospinH Tissue kit (Macherey-Nagel, Du¨ ren, Germany) according to manufacturer’s instruction. DNA concentration and quality was assessed with a ND-1000 spectrophotometer (Nano- Drop, Wilmington, DE). Bisulfite conversion of DNA was performed with EZ DNA Methylation kit (Zymo Research, Irvine, CA) for methylation microarray (500 ng DNA/sample) and methyl-specific PCR (1 mg DNA/sample) or with Epitect bisulfite kit (Qiagen) for BP analyses (250 ng DNA/sample) according to manufacturer’s instructions. Methylation microarray and bioinformatic analysis Microarrays were performed by the GIGA-Genotranscriptomic platform (GIGA, Lie`ge University, Belgium). The HumanMethy- lation27 BeadChip (Illumina, San Diego, CA) has been described previously [26]. After bisulfite conversion, DNA was whole- genome amplified, enzymatically fragmented and 200 ng of DNA was used for microarray. After extension, the array was fluorescently stained, scanned and the intensities of the non- methylated and methylated bead types were measured. DNA methylation values, described as b-values, were recorded for each locus of each sample and processed with BeadStudio (Illumina). All the beads of one probeID from all samples in each group (Ecto and SCC) were aggregated to calculate the mean values, standard deviations, detection p-values and b-values of the given probeID. The detection p-value is defined as the minimum of the 2 separate p-values of the 2 variables (green A/B or red A/B), where each p- value is the result of testing (Mann-Whitney-U-test) against the negative beads of each channel. The Benjamini-Hochberg correction was applied before choosing the minimum p-value [27]. To compare methylation between Ecto and SCC, the difference of b-values of the two groups was tested using Mann- Whitney-U-test. Clustering was performed using Cluster 3.0 [28]. The MIAME-compliant microarray data are available in GEO (GEO accession number GSE36637). Direct bisulfite pyrosequencing Bisulfite converted DNA was eluted in 20 mL of elution buffer and was subjected to PCR amplification of the specific region by use of a primer set designed to amplify both methylated and unmethylated sequences of the UTF1 gene promoter. The primers were designed using PSQ assay design software (Qiagen). One of the primers was biotin labelled. The primers sequences (in 59- -39 orientation) are, for the first step, forward: AGGGGTTT- TAGTTTTTTTAGTAGAGGTGTT-Btn and reverse: AACCCCTAACCCAATAACAAACT and for sequencing: GGGGGAGGATGTTAAG. Hot start Taq DNA polymerase High fidelity (Qiagen) was used to perform the PCR reaction. The PCR product was checked by 1.5 % agarose gel electrophoresis to confirm the quality, the size of the product and rule out the formation of primer dimers. The specific PCR products were then subjected to quantitative pyrosequencing analysis using a Biotage PyroMark Q24 system (Qiagen) according to manufacturer’s instruction. The results were analyzed by Pyro Q-CpG 1.0.9 software (Qiagen). Based on control normal samples and internal quality controls provided in the software analysis, a cut-off value was set at 20%, meaning that CpG methylation above this limit PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42704