Role for tissue-dependent methylation differences in the expression of FOXE1 in nontumoral thyroid glands

Abstract

Background: Discordance of monozygotic twins for thyroid dysgenesis suggests that epigenetic mechanisms may underlie defects in thyroid gland development. This prompted us to evaluate whether differentially methylated regions (DMRs) can be found between human thyroids (either eutopic or ectopic) and matched leukocytes.

Methods: To compare the genome-wide methylation profile of thyroids and leukocytes, immunoprecipitated methylated DNA was interrogated on human promoter plus CpG island tiling arrays. In addition, the methylation profile of the human FOXE1, PAX8, and NKX2.1 promoter was examined using bisulfite sequencing. Finally, the functional impact of CpG methylation of the promoter on FOXE1 expression was assessed with luciferase assays.

Results: Genome-wide methylation profiling and bisulfite sequencing of CpG islands of PAX8 and NKX2.1 promoters revealed no DMR between thyroid and leukocytes. However, bisulfite sequencing revealed that the methylation level of two consecutive CpG dinucleotides (CpG14 and CpG15, which were not covered by the genome-wide array) in one CpG island of the FOXE1 promoter (-1600 to -1140 from the transcription start site) is significantly higher in leukocytes than in eutopic or ectopic thyroid tissues, suggesting that methylation of this region may decrease FOXE1 gene expression. Indeed, luciferase activities were decreased when FOXE1 promoter constructs were methylated in vitro. Moreover, derepression of luciferase activity was observed when the methylation of CpG14 and CpG15 was prevented by mutations.

Conclusion: We report a tissue-dependent DMR in the FOXE1 promoter. This DMR contains two consecutive CpG dinucleotides, which are epigenetic modifiers of FOXE1 expression in nontumoral tissues.

Figure 1

CpG methylation profile of the human FOXE1 promoter. A, CpG methylation profile was determined using European Molecular Biology Open Software Suite (EMBOSS) CpGblot (http://www.ebi.ac.uk/tools/emboss/). B, Schematic representation of the FOXE1 promoter, numbered from the TSS (+1), with the CpG island 1 (−1600 to −1140 from TSS). C, Nucleotide sequence of CpG island 1, with CpG dinucleotides numbered; CpG₁₄ and CpG₁₅ are highlighted in a box.

Figure 2

DNA methylation status of CpG island 1 (−1600 to −1140 from the TSS) in the 5′-untranslated region of the FOXE1 gene using bisulfite sequencing. A and B, CpG island of FOXE1 (−1600 to −1140 from TSS, +1) is globally more methylated in leukocytes (10%) compared with the thyroids (3%, P < .001). In addition, two CpG dinucleotides (ie, CpG₁₄ and CpG₁₅, located −1417 and −1412 relative to TSS) show higher methylation in leukocytes (51% for CpG₁₄ and 43% for CpG₁₅) when compared with thyroids (5%; P < .001). C, Genomic DNA was extracted from four different human cell lines and subjected to sodium bisulfite sequencing to analyze the methylation profile of CpG island 1 of the FOXE1 promoter. Seven (REH cells) to 14 (other cell lines) different clones were sequenced. All leukemia cell lines showed significantly higher CpG island 1 methylation (Jurkat, 51%; K562, 15%; and REH, 86%) when compared with the thyroid cell line WRO (4.4% methylation; P < .001 when compared with each leukemia cell line); this methylation difference is even more pronounced for CpG₁₄ and CpG₁₅ (Jurkat, 100%; K562 39%; REH 100% compared with 0% in WRO). Each line represent the sequencing results of distinct tissues or cell lines. Circles represent the 33 CpG dinucleotides of CpG island 1, which are labeled as follows: black, methylation greater than 75%; dark gray, methylation of 50%–75%; gray, methylation of 10%–49%; white, methylation less than 10%. Numbers of clones analyzed for each tissue or cell line are listed on the right of the figure.

Figure 3

Confirmation of DNA methylation status by bisulfite pyrosequencing. The methylation profile of the first 17 CpG dinucleotides in CpG island 1 was analyzed using sodium bisulfite pyrosequencing. The figure represents the means ± SEM percentage of DNA methylation of the 17 CpG dinucleotides in leukocytes (in black) vs thyroid tissues (in gray). *, P < .05, two-tailed paired t test, corrected for multiple comparisons with the Holm-Sidak method.

Figure 4

Semiquantitative RT-PCR analysis of FOXE1 expression. FOXE1 RT-PCR in normal leukocytes and thyroid tissues (A), in human leukemia cell lines (Jurkat, K562, and REH), and in thyroid cancer WRO cells (B). The numbers denote the ratio of FOXE1: γ-actin values, normalized to that of ectopic thyroid tissue number 3 (set as 1.0).

Figure 5

Effect of global CpG methylation on FOXE1 promoter activity. FOXE1 promoter activity was tested in PCCL3 cells using either M.SssI methylated constructs (black bars) or mock methylated constructs (gray bars). Values are expressed as fold of the basic empty vector. Data represent means ± SEM of three independent experiments, each in triplicate. *, P <.05; **, P < .01 (Student’s t test). All methylated constructs showed significant decreased luciferase activity. Methylated constructs with CpG₁₄-CpG₁₅ mutations and with CpG island 1 deletion showed a significant derepression when compared with the methylated WT construct.

Figure 6

Effect of regional methylation of the DMR on FOXE1 promoter activity. FOXE1 promoter activity was tested in Nthy-ori 3–1 cells using either M.SssI regionally methylated constructs (black bars) or mock methylated constructs (gray bars). Values are expressed as fold of the basic empty vector. Data represent means ± SEM of two independent experiments, each in triplicate. *, P < .05; **, P < .01 (n.s., nonsignificant; Student’s t test). Regional methylation of CpG island 1 encompassing the wild-type CpG₁₄ and CpG₁₅ (at −1417 and −1412) leads to a significant decrease by reducing porter gene activity by 33% when compared with mock methylated control (48% if corrected for the background obtained for transfections with empty basic vector). Regional methylation of the CpG island 1 construct with mutated CpG₁₄ and CpG₁₅ induced no difference between methylated and mock methylated constructs.