DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells

Abstract

Background: The neural transcription factor SOX11 is present at specific stages during embryo development with a very restricted expression in adult tissue, indicating precise regulation of transcription. SOX11 is strongly up-regulated in some malignancies and have a functional role in tumorgenesis. With the aim to explore differences in epigenetic regulation of SOX11 expression in normal versus neoplastic cells, we investigated methylation and histone modifications related to the SOX11 promoter and the possibility to induce re-expression using histone deacetylase (HDAC) or EZH2 inhibitors.

Methods: The epigenetic regulation of SOX11 was investigated in distinct non-malignant cell populations (n = 7) and neoplastic cell-lines (n = 42) of different cellular origins. DNA methylation was assessed using bisulfite sequencing, methylation-specific melting curve analysis, MethyLight and pyrosequencing. The presence of H3K27me3 was assessed using ChIP-qPCR. The HDAC inhibitors Vorinostat and trichostatin A were used to induce SOX11 in cell lines with no endogenous expression.

Results: The SOX11 promoter shows a low degree of methylation and strong enrichment of H3K27me3 in non-malignant differentiated cells, independent of cellular origin. Cancers of the B-cell lineage are strongly marked by de novo methylation at the SOX11 promoter in SOX11 non-expressing cells, while solid cancer entities display a more varying degree of SOX11 promoter methylation. The silencing mark H3K27me3 was generally present at the SOX11 promoter in non-expressing cells, and an increased enrichment was observed in cancer cells with a low degree of SOX11 methylation compared to cells with dense methylation. Finally, we demonstrate that the HDAC inhibitors (vorinostat and trichostatin A) induce SOX11 expression in cancer cells with low levels of SOX11 methylation.

Conclusions: We show that SOX11 is strongly marked by repressive histone marks in non-malignant cells. In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications. The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation. In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.

Figure 1

Epigenetic profiling of SOX11 in normal cells. (A) The SOX11 promoter 2000 bp upstream of transcription start site contains four CpG islands with analyzed CpG sites marked. (B) Mean SOX11 promoter methylation within 28 CpG sites close to the transcription start site. (C) Enrichment of repressive H3K27me3, determined by ChIP-qPCR, within the SOX11 promoter in naïve, GC and memory B-cells. (D) Methylation status of five CpG sites within the SOX11 promoter, measured with Illumina 450 K methylation array, for several types of non-malignant mammary cell-types. (E) Enrichment of active (H3K4me3) and repressive (H3K27me3) histone marks in primary human mammary epithelial cells. ChIP-seq data was extracted from the ENCODE project.

Figure 2

SOX11 promoter methylation in tumor cell-lines. SOX11 promoter methylation status assessed with MethyLight and MS-MCA. The methylation levels analyzed by MethyLight are presented as percent methylated reference (PMR). PMR < 1 was considered as unmethylated promoter. SOX11 promoter methylation was investigated in lymphoma cell lines with (A) MethyLight and (B) MS-MCA. SOX11 promoter methylation was investigated in solid tumor cell lines with (C) MethyLight and (D) MS-MCA.

Figure 3

Correlation between SOX11 promoter methylation and expression. The correlation between DNA methylation and gene expression was analyzed with RT-qPCR and western blot. In RT-qPCR, CT values >35 were considered below detection limit and corresponding SOX11 levels were set to zero. (A) MethyLight (x-axis) and melt curve analysis (see filled, grey or open diamonds) showed an inverse correlation between SOX11 mRNA and promoter methylation in lymphoma cell lines. (B) Likewise, inverse correlation between SOX11 mRNA and promoter methylation was seen for solid cancer cell lines. For clarity, SOX11 positive (western blot) cell line names are shaded. (C) Western blot analysis of SOX11 and GAPDH in Burkitt’s lymphoma, follicular lymphoma, diffuse large B-cell lymphoma and mantle cell lymphoma cell-lines. (D) Western blot analysis of SOX11 and GAPDH in breast cancer, ovarian cancer, lung cancer, brain cancer and neuroblastoma cell lines.

Figure 4

Correlation between SOX11 promoter methylation and ER positive breast cancer. A)SOX11 promoter methylation is significantly (p < 0.01) enriched in ER positive breast cancer (n = 460) compared to ER negative breast cancer (n = 139). B)SOX11 gene expression is significantly (p < 0.01) enriched in ER negative breast cancer compared to ER positive breast cancer.

Figure 5

Enrichment of H3K27me3 within the SOX11 promoter. Histone methylation of lysine 27 on histone 3 (H3K27me3) was assessed using chromatin immunoprecipitation and RT-qPCR for GAPDH (negative control), TSH2B (positive control) and SOX11. (A)Enrichment of H3K27me3 in unmethylated cell lines lacking SOX11. (B) Enrichment of H3K27me3 in methylated cell lines lacking SOX11.

Figure 6

TSA and Vorinostat induce SOX11 expression and EZH2 down-regulation in unmethylated cancer cell-lines. Cells were treated with 0, 0.5 and 5 μM TSA or vorinostat for 24 hours followed by detection of SOX11 and EZH2 with western blot. (A) Unmethylated cancer cell lines JIMT-1, KCN69n and SK-BR-3 expressed SOX11 after treatment while DMS-114 and JVM-2 remained SOX11 negative. (B) JIMT-1 and KCN69n also showed a clear down-regulation of EZH2 upon treatment with HDAC inhibitors while the other cell lines did not show any change in EZH2 levels (data not shown).