Hypermethylation of genomic 3.3-kb repeats is frequent event in HPV-positive cervical cancer

Abstract

Background: Large-scale screening methods are widely used to reveal cancer-specific DNA methylation markers. We previously identified non-satellite 3.3-kb repeats associated with facioscapulohumeral muscular dystrophy (FSHD) as hypermethylated in cervical cancer in genome-wide screening. To determine whether hypermethylation of 3.3-kb repeats is a tumor-specific event and to evaluate frequency of this event in tumors, we investigated the 3.3-kb repeat methylation status in human papilloma virus (HPV)-positive cervical tumors, cancer cell lines, and normal cervical tissues. Open reading frames encoding DUX family proteins are contained within some 3.3-kb repeat units. The DUX mRNA expression profile was also studied in these tissues.

Methods: The methylation status of 3.3-kb repeats was evaluated by Southern blot hybridization and bisulfite genomic sequencing. The expression of DUX mRNA was analyzed by RT-PCR and specificity of PCR products was confirmed by sequencing analysis.

Results: Hypermethylation of 3.3-kb repeats relative to normal tissues was revealed for the first time in more than 50% (18/34) of cervical tumors and in 4 HPV-positive cervical cancer cell lines. Hypermethylation of 3.3-kb repeats was observed in tumors concurrently with or independently of hypomethylation of classical satellite 2 sequences (Sat2) that were hypomethylated in 75% (15/20) of cervical tumors. We have revealed the presence of transcripts highly homologous to DUX4 and DUX10 genes in normal tissues and down-regulation of transcripts in 68% of tumors with and without 3.3-kb repeats hypermethylation.

Conclusion: Our results demonstrate that hypermethylation rather than hypomethylation of 3.3-kb repeats is the predominant event in HPV-associated cervical cancer and provide new insight into the epigenetic changes of repetitive DNA elements in carcinogenesis.

Figure 1

Diagram of a 3.3-kb monomer. Two squares denote double homeobox region. Sq-1, Sq-2, and Sq-3 mark the regions selected for sequencing analysis. The hybridization probes and RT-PCR primers are indicated by horizontal lines and arrows, respectively. The broken arrow indicates the position of DUX4 transcription start site. Vertical lines indicate NaeI sites.

Figure 2

Methylation status of 3.3-kb repeats by sodium bisulfite sequencing in cervical tissues, tumors, and cell lines. Sq-1, Sq-2, and Sq-3 mark three regions of 3.3-kb monomer selected for sequencing. Each row of circles represents a cloned DNA molecule; closed circle, methylated CpG; open circle, unmethylated CpG; dot, CpG site lost from the consensus sequence due to mutations; cross, CpG site status was not determined. The percentage of methylated CpGs among all CpGs analyzed is given below each sample. Nc, normal cervix from non-cervical-cancer patients; T, tumor; N, tumor – tumor-adjacent normal tissue. Numbers of patients are indicated as in Figure 3C. Only P values below 0.05 are given (χ-square test).

Figure 3

Analisis of methylation status of 3.3-kb and Sat2 repeats in HPV-positive cervical tumors by Southern blot hybridization. Nc, normal cervix from non-cervical-cancer patients; T, tumor; N, tumor-adjacent normal tissue. Lines to the left of the panel are molecular mass markers (from top to bottom, 24.3, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.5 kb). (A) Hybridization with the 3.3-kb repeat-specific probe, NaeI digests of DNAs. Numbers under the panels indicate the percentage of the high-molecular-weight hybridization signal (>4.4 kb) to the total hybridization signal for each track. (B) Hybridization with the Sat2-specfic oligonucleotide, BstBI digests of DNAs. (C) Hypermethylation of 3.3-kb repeats in tumors. (D) Hypomethylation of Sat2 in tumors. The asterisk and triangle indicate samples with hypermethylation and hypomethylation of 3.3-kb repeats, respectively; the circle – unchanged methylation status of 3.3-kb repeats.

Figure 4

Analysis of DUX gene expression in cervical tumors and cell lines by RT-PCR. Top row: Southern blot hybridization of PCR products with the DUX4-specific [32] P-labeled oligonucleotide. Middle and bottom rows, electrophoregrams after ethidium bromide staining. GAPDH, a control for RNA stability and concentration. RT, reverse transcriptase; Nc, normal cervix from non-cervical-cancer patients; T, tumor; N, tumor-adjacent normal tissue. Patient numbers are as in figure 3.