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

Abstract

BackgroundThe 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.MethodsIn 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.ResultsComparison 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.ConclusionsThis 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.

Epigenetic control of the basal-like gene expression profile via Interleukin-6 in breast cancer cells

“…Considering the close relationship existing between mammospheres and CSCs [4,10-14], these findings support the very recent observation [35] that IL-6 driven epigenetic changes are associated with CSCs features in breast cancer cells. In line with this reasoning, both CD133 and CD44 have been previously reported to be regulated by promoter methylation [30,31,36]. Moreover, multiple epigenetic promoter modifications have been found to control IL-6 gene expression [32,37].…”

Targeting Hyaluronidase for Cancer Therapy: Antitumor Activity of Sulfated Hyaluronic Acid in Prostate Cancer Cells

“…We also did not observe CD44 expression (standard (CD44s) and variant (CD44v) isoform(s)) in these cells (Supplement Table 2). Two publications have reported CD44 expression in LNCaP and C4-2 cells (46,47), including the expression of a CD44 variant, CD44-v9 (46). However, no PCR product was amplified from LNCaP, LNAI, LAPC-4, C4-2 and C42B cells using the same PCR primer pair that was used to amplify CD44-v9 (46,48).…”

Variable Expression of GLIPR1 Correlates with Invasive Potential in Melanoma Cells

“…DNA methylation analysis was performed using BiQ Analyzer v2.00 (18). We amplified a 320 bp region using primers for amplification and sequencing of GLIPR1 from bisulfite-treated DNA previously published by Muller et al (19). This region extends from −104 to −424 of the GLIPR1 translation start site, and corresponds to the CpG’s referred to as “A–D” in (2).…”