Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by methyl-CpG binding proteins and histone modifications.
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.
Müller
Imke
I
Department of Tumour Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Wischnewski
Frank
F
Pantel
Klaus
K
Schwarzenbach
Heidi
H
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D002843:Chromatin / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D056742:Cyclin D2 / Q000235:genetics*; D019175:DNA Methylation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006657:Histones / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins; D051783:Methyl-CpG-Binding Protein 2 / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D051059:PTEN Phosphohydrolase / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
C080336:CCND2 protein, human; C497483:CD44 protein, human; D002843:Chromatin; D056742:Cyclin D2; D004791:Enzyme Inhibitors; C094538:GLIPR1 protein, human; D006657:Histones; D018960:Hyaluronan Receptors; D008565:Membrane Proteins; D051783:Methyl-CpG-Binding Protein 2; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human