Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by methyl-CpG binding proteins and histone modifications

Abstract

Background: The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs) and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation.

Methods: In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies.

Results: Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3) at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR.

Conclusions: This study is one of the first to reveal the histone code and MBD profile at the promoters of CD44, Cyclin D2, GLIPR1 and PTEN in different tumour cells and associated changes after stimulation with methylation inhibitor 5-aza-CdR.

Figure 1

mRNA expression of CD44, Cyclin D2, GLIPR1 and PTEN in DU145, LNCaP and MCF-7 cells. The data assessed by RT-PCR (gel electrophoretically separated on a representative agarose gel, left panel) and quantitative real-time PCR (bar charts, right panels) show the relative levels and changes in the mRNA expression of the tumour-associated genes in unstimulated basal (b), 5-aza-CdR- (A), TSA- (T) and 5-aza-CdR+TSA- (A+T) stimulated DU145, LNCaP and MCF-7 cells. The housekeeping gene β-Actin was selected as internal control. * Statistical significance of p < 0.05 according to the Fisher's exact test in respect of changes in stimulated samples compared to the basal status.

Figure 2

Luciferase activities of the co-transfected reporter plasmids containing the promoters of CD44, Cyclin D2, GLIPR1 or PTEN in MCF-7, DU145 and LNCaP cells. MCF-7, DU145 and LNCaP cells were transiently transfected with pGL2-Luciferase reporter plasmids containing promoter fragments of CD44 (A), Cyclin D2 (B), GLIPR1 (C) or PTEN (D) immediately downstream of five Gal4 binding motifs (left diagrams), or with methylated reporter plasmids containing the same promoters without the Gal4 motifs (right diagrams). The co-transfected expression plasmids encoded either for Gal4-fused TRDs of MBD1, MBD2, or MeCP2 (left diagrams) or for full length proteins of MBD1v1, MBD1v3, MBD2a, MBD2b or MeCP2 proteins (right diagrams). The activities derived from the reference Renilla Luciferase were used for normalization of the data. The relative luciferase activities of the reporter constructs co-transfected with the empty expression plasmids (-) were arbitrarily set to 100%. * Statistical significance of p < 0.05 according to the Fisher's exact test in respect of repression of promoters by MBDs compared to the basal level activity (-).

Figure 3

Protein expression of MBD1, MBD2 and MeCP2 in basal and stimulated MCF-7 cells and in MBD1^-/- mouse embryonic fibroblasts. The MBD protein levels in basal, 5-aza-CdR-, TSA- and 5-aza-CdR&TSA- stimulated MCF-7 cells were evaluated by Western Blot analysis using antibodies specific for MBD1 (60 kDa), MBD2 (49 kDa), MeCP2 (70 kDa) and HSC70 (70 kDa, loading control) (A). The MBD1 protein level in MBD1^-/- mouse embryonic fibroblasts was evaluated in comparison to basal MCF-7 cells with antibodies specific for MBD1 (60 kDa) and HSC70 (70 kDa, loading control) (B).

Figure 4

Chromatin immunoprecipitation using antibodies specific for MBD1, MBD2 and MeCP2. Quantitative real-time PCR analysis of the immunoprecipitated DNA (IP) derived from unstimulated (basal) and 5-aza-CdR-stimulated DU145, LNCaP and MCF-7 cells was performed using primer pairs specific for the promoter fragments of RPLP0 (A), CD44 (B), Cyclin D2 (C), GLIPR1 (D), and PTEN (E). All values obtained were normalized and referred to 100% of the input DNA. * Statistical significance of p < 0.05 according to the Fisher's exact test. ** Statistical significance of p < 0.05 according to the analysis of variance (ANOVA).

Figure 5

Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones. Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.