Demethylation of a nonpromoter cytosine-phosphate-guanine island in the aromatase gene may cause the aberrant up-regulation in endometriotic tissues  Masao Izawa, Ph.D., Fuminori Taniguchi, M.D., Takashi Uegaki, M.D., Eri Takai, M.D., Tomio Iwabe, M.D., Naoki Terakawa, M.D., Tasuku Harada, M.D.  Fertility and Sterility  Volume 95, Issue 1, Pages 33-39 (January 2011) DOI: 10.1016/j.fertnstert.2010.06.024 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Aromatase mRNA expression in endometriotic and endometrial cells. (A) The reverse transcriptase–polymerase chain reaction (RT-PCR) analysis. Stromal cells were collected from endometriotic and endometrial tissues. Single-stranded cDNA was prepared from total cellular RNA and subjected to PCR. A sequence corresponding to the total open reading frame from Exon II to Exon X (1,518 bp) was amplified and subjected to a 1.5% agarose gel electrophoresis. As an internal control, β-tubulin mRNA was assayed in parallel. The amplified signal was visualized using ethidium bromide staining under ultraviolet light. A representative result is shown. (B) Promoter usage of aromatase gene expression. Stromal cells were randomly picked up, and unique Exon I (PII, I.1, I.3, I.4, I.5, and I.6) and Exon II primers were used for the PCR in the promoter assay as described previously (3). (C) Effect of 5-Aza-dC treatment on aromatase mRNA expression in endometrial cells. Stromal cells collected from endometrial tissues (proliferative phase) were treated with 5-Aza-dC at a concentration of 5 μM for 96 hours, and total cellular RNA and the single-stranded cDNA were prepared. A sequence corresponding to the total open reading frame (1,518 bp) was amplified and subjected to agarose gel electrophoresis as described in A. As an internal control, β-tubulin mRNA was assayed in parallel. A representative result is shown. (D) Promoter usage of aromatase gene expression in 5-Aza-dC-treated endometrial cells collected from two endometrial tissues (proliferative phase) as described in C. Fertility and Sterility 2011 95, 33-39DOI: (10.1016/j.fertnstert.2010.06.024) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Methylation status of a CpG island predicted in a nonpromoter region of the aromatase gene. (A) Schematic representation of genomic organization of the human CYP19 gene, and a CpG island predicted by the CpG Island Searcher (http://cpgislands.usc.edu/). The predicted CpG island is depicted as a shaded box (49139456–49138748) between the most upstream (49210015) and the end of Exon X (49080810) according to NM 031226 (chromosome 15). The sequence location from the most upstream (49210015) is shown as base pairs in parenthesis. The sequence of 733 bp in length was divided into two sequences, S1 (1–376) and S2 (352–733), for further analysis. Horizontal arrows associated with S1, S2, Bs1, and Bs2 lines indicate the locations of PCR primers used. (B) Survey for methylated CpG sequence. According to the protocol developed by Yamada et al. (13), cellular DNA (0.5 μg) prepared from stromal cells from endometrium or chocolate cysts was digested with HhaI or McrBC for 18 hours at 37°C, and the digested DNA was used for PCR to amplify S1 and S2 sequences depicted as solid lines in A. At the end of PCR, amplified sequences were separated by electrophoresis on a 1.5% agarose gel electrophoresis. The amplified signal was visualized using ethidium bromide staining under ultraviolet light. Two representative results are shown for stromal cells from endometrium (patient 1, secretory phase and patient 2, proliferative phase) and chocolate cysts (patients 11 and 12, luteal phase), respectively, along with a result of 5-Aza-dC-treated cells from endometrium (patient 2). (C) Bisulfite sequencing analysis. To survey the methylated CpGs, the bisulfite reaction was performed in DNA samples from stromal cells from six endometrium (patients 2, 3, 5, and 6, proliferative phase, and patients 1 and 4, secretory phase) and five chocolate cysts of endometriosis (patients 11, 12, 14, and 15, luteal phase, and patient 13, follicular phase) according to the manufacturer's protocol (EpiTect Whole Bisulfitome kit). The PCR primers were selected to cover the HhaI sites of S1 and S2 sequences, which are depicted as dotted lines Bs1(269–454) and Bs2(455–716) in A. Direct sequencing analysis was carried out using ABI 310. Results from the Bs1 and Bs2 sequences are shown. The open circle indicates unmethylated CpG and the closed circle indicates methylated CpG. A half-closed circle means the mixture of methylated and unmethylated CpG, indicating a heterogeneous cell population. (D) Methylated CpGs in Bs1 sequence. Relative number of methylated CpGs (fully and partially methylated) was expressed as the percentage of seven CpGs within Bs1 sequence. Statistical analysis from the results depicted in C. Values are expressed as the mean (%) ± SE. P<.001 between chocolat cyst and endometrium. Fertility and Sterility 2011 95, 33-39DOI: (10.1016/j.fertnstert.2010.06.024) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Methylation status of a CpG island predicted in a nonpromoter region of the aromatase gene. (A) Schematic representation of genomic organization of the human CYP19 gene, and a CpG island predicted by the CpG Island Searcher (http://cpgislands.usc.edu/). The predicted CpG island is depicted as a shaded box (49139456–49138748) between the most upstream (49210015) and the end of Exon X (49080810) according to NM 031226 (chromosome 15). The sequence location from the most upstream (49210015) is shown as base pairs in parenthesis. The sequence of 733 bp in length was divided into two sequences, S1 (1–376) and S2 (352–733), for further analysis. Horizontal arrows associated with S1, S2, Bs1, and Bs2 lines indicate the locations of PCR primers used. (B) Survey for methylated CpG sequence. According to the protocol developed by Yamada et al. (13), cellular DNA (0.5 μg) prepared from stromal cells from endometrium or chocolate cysts was digested with HhaI or McrBC for 18 hours at 37°C, and the digested DNA was used for PCR to amplify S1 and S2 sequences depicted as solid lines in A. At the end of PCR, amplified sequences were separated by electrophoresis on a 1.5% agarose gel electrophoresis. The amplified signal was visualized using ethidium bromide staining under ultraviolet light. Two representative results are shown for stromal cells from endometrium (patient 1, secretory phase and patient 2, proliferative phase) and chocolate cysts (patients 11 and 12, luteal phase), respectively, along with a result of 5-Aza-dC-treated cells from endometrium (patient 2). (C) Bisulfite sequencing analysis. To survey the methylated CpGs, the bisulfite reaction was performed in DNA samples from stromal cells from six endometrium (patients 2, 3, 5, and 6, proliferative phase, and patients 1 and 4, secretory phase) and five chocolate cysts of endometriosis (patients 11, 12, 14, and 15, luteal phase, and patient 13, follicular phase) according to the manufacturer's protocol (EpiTect Whole Bisulfitome kit). The PCR primers were selected to cover the HhaI sites of S1 and S2 sequences, which are depicted as dotted lines Bs1(269–454) and Bs2(455–716) in A. Direct sequencing analysis was carried out using ABI 310. Results from the Bs1 and Bs2 sequences are shown. The open circle indicates unmethylated CpG and the closed circle indicates methylated CpG. A half-closed circle means the mixture of methylated and unmethylated CpG, indicating a heterogeneous cell population. (D) Methylated CpGs in Bs1 sequence. Relative number of methylated CpGs (fully and partially methylated) was expressed as the percentage of seven CpGs within Bs1 sequence. Statistical analysis from the results depicted in C. Values are expressed as the mean (%) ± SE. P<.001 between chocolat cyst and endometrium. Fertility and Sterility 2011 95, 33-39DOI: (10.1016/j.fertnstert.2010.06.024) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Association of methyl-binding proteins with methylated CpG sequence within a predicted CpG island: chromatin immunoprecipitation (ChIP) assay. The ChIP assay using chromatin samples from stromal cells from endometrium (proliferative and secretory phases) and chocolate cysts (luteal phase) were performed according to the manufacturer's protocol (SimpleChIP). In brief, cells were cross-linked in 1% formaldehyde, and fragmented chromatin was prepared using a digestion with micrococcal nuclease. After ChIP and DNA purification, relative concentration of S1 (224–376) and S2 (524–733) sequence (see Fig. 2A) was assessed by PCR. Antibodies used are anti-methyl-CpG-binding domain protein 2 (MeCP2) and anti-methyl-CpG-binding domain protein 1 (MBD1). As positive and negative controls, anti-histone H3 and normal rabbit IgG were used, respectively. The promoter region of ribosomal protein L30 gene was assayed in parallel as unmethylated and positive controls. Fertility and Sterility 2011 95, 33-39DOI: (10.1016/j.fertnstert.2010.06.024) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions