Genome-wide ChIP-seq analysis of EZH2-mediated H3K27me3 target gene profile highlights differences between low- and high-grade astrocytic tumors

Abstract

Enhancer of zeste homolog-2(EZH2) is a key epigenetic regulator that functions as oncogene and also known for inducing altered trimethylation of histone at lysine-27 (H3K27me3) mark in various tumors. However, H3K27me3 targets and their precise relationship with gene expression are largely unknown in astrocytic tumors. In this study, we checked EZH2 messenger RNA and protein expression in 90 astrocytic tumors of different grades using quantitative PCR and immunohistochemistry, respectively. Further, genome-wide ChIP-seq analysis for H3K27me3 modification was also performed on 11 glioblastomas (GBMs) and 2 diffuse astrocytoma (DA) samples. Our results showed EZH2 to be highly overexpressed in astrocytic tumors with a significant positive correlation with grade. Interestingly, ChIP-seq mapping revealed distinct differences in genes and pathways targeted by these H3K27me3 modifications between GBM versus DA. Neuroactive ligand receptor pathway was found most enriched in GBM (P = 9.4 × 10-25), whereas DA were found to be enriched in metabolic pathways. Also, GBM showed a higher enrichment of H3K27me3 targets reported in embryonic stem cells and glioma stem cells as compared with DAs. Our results show majority of these H3K27me3 target genes were downregulated, not only due to H3K27me3 modification but also due to concomitant DNA methylation. Further, H3K27me3 modification-associated gene silencing was not restricted to promoter but also present in gene body and transcription start site regions. To the best of our knowledge, this is the first high-resolution genome-wide mapping of H3K27me3 modification in adult astrocytic primary tissue samples of human, highlighting the differences between grades. Interestingly, we identified SLC25A23 as important target of H3K27me3 modification, which was downregulated in GBM and its low expression was associated with poor prognosis in GBMs.

Polycomb complex mediated epigenetic reprogramming alters TGF‐β signaling via a novel EZH2/miR‐490/TGIF2 axis thereby inducing migration and EMT potential in glioblastomas

“…Our in‐house data for ChIPseq analysis revealed H3k27me3 modified targets across GBM patients genome that were encoded with a large set of genes and ncRNAs in gliomas . Although a large set of altered ncRNAs were identified in our study, none of these was explored in detail.…”

“…Our in-house data for ChIPseq analysis revealed H3k27me3 modified targets across GBM patients genome that were encoded with a large set of genes and ncRNAs in gliomas. 10 Although a large set of altered ncRNAs were identified in our study, none of these was explored in detail. Thus, in the present study, we specifically analyzed H3K27me3 modified target miRNAs enriched in our study as their role is comparatively better explored in gliomas.…”

“…For ChIP studies, 11 GBM tissue samples data were analyzed from our previous studies . Briefly, Low Cell ChIP Kit (M/s Diagenode, Belgium) reagents and protocol were followed with some modifications.…”

“…For validation of ChIP, qPCR reactions were performed using SYBR green chemistry to look for HOXA9 (positive control) and GAPDH (negative control) promoter enrichment in ChIP precipitated DNA. Sequencing, bioinformatics pipeline and data analysis details are mentioned in Sharma et al . miRNA targets identified in each patient with p ‐value < 0.001 cut off were taken for downstream analysis.…”

“…For ChIP studies, 11 GBM tissue samples data were analyzed from our previous studies. 10 Briefly, Low Cell ChIP Kit (M/s Diagenode, Belgium) reagents and protocol were followed with some modifications. Fragmented chromatin was immunoprecipitated using ChIP-grade antibodies: H3K27me3 (M/s Abcam, Cambridge, MA ), Histone H3 (M/s Abcam) and IgG What's new?…”