upstream regulatory region of L1 gene allowed to discriminate between CIN3 and CIN2 or lower grade lesions [41]. To our knowledge, the current study is the first one to show that methylation profile of non viral promoter gene can be assessed by direct BP on LBC samples. With this technique, we showed that UTF1 hypermethylation status might be a useful biomarker to discriminate normal Ecto and LSIL from HSIL and SCC and to improve cervical cancer diagnosis. The specificity of stem cell (SC) markers such as UTF1, Oct4A, Sox2 or KLF4 is well established for embryonic or induced pluripotent SC [42]. In the tumor context, cancer SC (CSC) have been proposed to be involved in resistance to therapy and metastasis formation [5,43]. However the involvement of SC markers and their epigenetic regulation during epithelial carcino- genesis are still poorly defined. This is in part due to the rarity of CSC and to the phenotypic cell heterogeneity within tumor mass [5,44]. Here, we demonstrated the presence of UTF1 in normal and tumor squamous epithelial tissues, as well as in cancer cell lines. The UTF1 expression pattern that we observed did not fit the definition of CSC which account for a very small proportion of cells within tumor mass [6,44]. This hypothesis is supported by the work of Ji et al. showing that Sox2 expression increases during cervical carcinogenesis which is consistent with the expression profile of UTF1 [16]. This also in agreement with a very recent work proposing that UTF1 expression in adult tissue may have different roles than those observed stem cell regulation at the earliest stages of embryogenesis [24]. In addition, we did not detect any expression of Oct4A in cell lines, which is in agreement with other studies conducted in breast and in cerebral tumors [31,45]. It has been also previously described that UTF1 depletion decreases the tumorigenicity of teratoma subcutaneously injected in nude mice [46]. In some LSIL and SCC samples, a cytoplasmic staining of UTF1 was also observed, which is in agreement with a recent work investigating skin keratinocytes [24]. One possible explanation could be related to a role of UTF1 in cell division as it was proposed for the B lymphoma Mo-MLV insertion region 1 homolog (BMI-1) gene in skin cancer but the mechanism behind these observations is still unknown [47]. Therefore, we can postulate that UTF1 upregulation by itself might participate to the global gene expression alterations leading to cancer development by locking tumor-specific gene expression pattern through its fixation to chromatin [18,48]. This mechanism could be mediated by estrogens, which were shown to increase DNA methyltransferase activity or cervical carcinogenesis [49,50]. This is supported by the fact that, in endometriosis, an estrogen-dependent disease characterized by the presence of endometrium outside the uterine cavity, higher levels of UTF1 mRNA were found in comparison with normal endometrium [51]. Consequently, in response to microenvironment or intracellular signaling, UTF1 might be overexpressed, participate in epigenetic cellular reprogramming and confer preneoplastic cells a proliferating advantage. This would ultimately lead to cancer cell engraftment and growth, in agreement with its known role as an enhancer of colony formation of induced pluripotent SC [42]. The treatment with 5-aza induced a global decrease of 21% and 26% of UTF1 promoter in A431 and HeLa cells, respectively. This range of variation in response to 5-aza has been previously described for other genes such as somatostatin in gastric cancer cell lines, GADD45A in osteosarcoma cell lines or LCR and L1 of HPV- 16 in cervical cancer cell line [52–54].The correlation between UTF1 expression and promoter methylation was also unexpected. However, similar results have already been reported. For example, in healthy tissue, the profiling of DNA methylation on chromo- UTF1 and Cervical Carcinogenesis somes 6, 20 and 22 showed that, for 63% of the genes tested, DNA methylation was not related to inhibition of expression [55]. In endometrial cancer, survivin promoter hypermethylation of CpG within transcriptional repressor binding sites was correlated with its upregulation [56]. In the same way, in breast cancer samples, the promoter of the KLHDC7B gene was hypermethylated and associated with its overexpression in comparison with normal mammary tissue [57]. In vitro, the methylation of the human glycoprotein hormone a-subunit (GPHa) gene was found to be hypermethylated in cell lines producing high amount of this protein, whereas low level were detected in cell lines with hypomethylated GPHa gene sequence [58]. Our results suggest that, in the absence of Oct4A and Sox2, UTF1 promoter hypermethylation might also participate in gene expression. This is supported by the methylation status of UTF1 promoter in NCCIT cells differentiated or not with retinoic acid which appear totally hypomethylated in both conditions (Figure S6). In NCCIT UN, UTF1 expression is stimulated by Oct4a and Sox2 (Fig. 5B and [21,22]), whereas in NCCIT differentiated with RA, UTF1, Oct4a and Sox2 are not expressed (Figure 5B and [18,22]). On the contrary, in untreated epithelial cancer cell lines, UTF1 promoter appears to be heavily methylated and expressed in spite of the absence of Oct4a and Sox2. This strongly suggests that UTF1 promoter methylation is involved in the control of its expression in the absence of its two well described regulators. This is also corroborated by the positive correlation between UTF1 methylation and expression in 2 cell lines (A431 and HeLa) of the 4 that were treated with 5-aza, with a third cell line (CaSki) being near significance. However the molecular mechanism involved need to be elucidated and might be cell-specific as showed by the lack of effect of 5-aza in SiHa cells. The p53 or Sp1 transcription factors might participate in this pathway. Indeed p53 protein is able to inhibit survivin expression when its promoter is demethyl- ated [56]. This is supported by the fact that HeLa cells express higher level of p53 than SiHa cells [59]. In osteoblastic cells the binding of Sp1 and its transcriptional activity were demonstrated to be enhanced when podoplanin promoter was hypermethylated [60]. In conclusion, we demonstrated that UTF1 promoter hyper- methylation is associated with its overexpression during cervical carcinogenesis, suggesting that UTF1 promoter methylation profile might be a useful diagnosis biomarker. We also showed that, in vitro, UTF1 expression is not restricted to SC and that it is localized in the nucleus of somatic tumor cells independently of Oct4A and Sox2 expression. Supporting Information Figure S1 Validation of UTF1 mRNA expression and UTF1 antibody specificity. A) RT-PCR of UTF1 performed with previously described UTF1 primers (see ref [21]). B) Western Blot of A431 and SiHa cell lines transiently transfected with either empty plasmid (PCDNA.3) or plasmid containing UTF1 cDNA (UTF1). NCCIT UN and NCCIT RA are positive and negative controls for UTF1 expression, respectively. (TIF) Figure S2 Structure of UTF1 gene. Red triangles, CpG analysed by microarray; black box, DNA sequence analysed by direct bisulfite pyrosequencing; blue and green circles, binding sites for Sox2 and Oct4A. (TIF) Figure S3 Validation of UTF1 promoter methylation by direct bisulfite pyrosequencing in samples used for PLoS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e42704