PGE2 inducesinterleukin-8derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

Abstract

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.

Systemic analysis of osteoblast-specific DNA methylation marks reveals novel epigenetic basis of osteoblast differentiation

“…While methylation at individual CpG sites may possibly be linked to gene expression regulation (Venza et al, 2012, Chen, 1983), methylation levels at neighboring CpGs are often highly correlated and methylation-mediated regulatory elements often extend across genomic regions (Davies et al, 2012, Baylin and Jones, 2011, Song et al, 2009). Therefore, we further conducted differentially methylated region (DMR) analysis based on the computed DMC data.…”

DNA Methylation and Differentiation: Silencing, Upregulation and Modulation of Gene Expression

“…We then mapped DM sites to specific regions (−2 kb to TSS, first exon or intron, internal exons, internal introns, last exon, transcription termination site to 2 kb distal and other intergenic regions) using R scripts. Analysis of individual DM sites is a valuable tool in DNA regions where there is low coverage by RRBS or where there may be individual CpGs whose DNA methylation status might affect gene expression irrespective of methylation of neighboring sequences [56,57]. …”

Arsenic exposure causes epigenetic dysregulation of IL-8 expression leading to proneoplastic changes in kidney cells

“…The bisulfite converted DNA was amplified by using specific Bisulfite PCR (BSP) primers (Venza et al, 2012). Jump Start TM RED Taq 1 Ready Mix TM PCR Master Mix (Sigma, USA) was used for amplification of the bisulfite converted DNA using the manufacturer provided protocol and BSP primers mentioned in Table 2.…”