Identification of DNA Methylation-Independent Epigenetic Events Underlying Clear Cell Renal Cell Carcinoma

Abstract

Alterations in chromatin accessibility independent of DNA methylation can affect cancer-related gene expression, but are often overlooked in conventional epigenomic profiling approaches. In this study, we describe a cost-effective and computationally simple assay called AcceSssIble to simultaneously interrogate DNA methylation and chromatin accessibility alterations in primary human clear cell renal cell carcinomas (ccRCC). Our study revealed significant perturbations to the ccRCC epigenome and identified gene expression changes that were specifically attributed to the chromatin accessibility status whether or not DNA methylation was involved. Compared with commonly mutated genes in ccRCC, such as the von Hippel-Lindau (VHL) tumor suppressor, the genes identified by AcceSssIble comprised distinct pathways and more frequently underwent epigenetic changes, suggesting that genetic and epigenetic alterations could be independent events in ccRCC. Specifically, we found unique DNA methylation-independent promoter accessibility alterations in pathways mimicking VHL deficiency. Overall, this study provides a novel approach for identifying new epigenetic-based therapeutic targets, previously undetectable by DNA methylation studies alone, that may complement current genetic-based treatment strategies. Cancer Res; 76(7); 1954-64. ©2016 AACR.

Figure 1. Diagram of AcceSssIble assay

Tissues were obtained and processed as described in Materials and Methods. Tissues were processed and nuclei were either left untreated or were treated with the M.SssI methyltransferase enzyme. Endogenously methylated loci are unable to be interrogated (as illustrated). Following bisulfite conversion and HM450 analyses, untreated (endogenous) DNA methylation values from each sample were subtracted from respective enzyme-treated (exogenous) DNA methylation values, revealing chromatin accessibility changes.

Figure 2. Overview of AcceSssIble data in ccRCC tumor/normal tissue pairs

A) Δβ-values of no-enzyme-treated (endogenous methylation) and M.SssI-treated (accessibility) samples were plotted to visualize DNA methylation and accessibility changes simultaneously. Bar plots quantify these changes and illustrate the distribution of CpG-islands and probe location. B) Six groups of epigenetic changes analyzed: Group a, loss of chromatin accessibility but gain in DNA methylation; Group b, no change in chromatin accessibility but gain in DNA methylation; Group c, no change in chromatin accessibility but loss of DNA methylation; Group d, gain in DNA accessibility but loss of DNA methylation; Group e, loss of chromatin accessibility but no change in DNA methylation; Group f, gain in chromatin accessibility but no change in DNA methylation.

Figure 3. ChIP-seq data in regions changing in accessibility

Average lineplots of ChIP-seq (active histone marks H3K4me3 and H3K27ac, and repressive mark H3K27me3) data Z-scores for regions with detected accessibility changes in Patient 4, separated by A & C) accessibility loss and B & D) accessibility gain. Gene promoter regions (A & B) and enhancers (C & D) were analyzed separately.

Figure 4. Accessibility in gene promoters intersected with DNA methylation and expression data from a larger cohort (TCGA)

Data from Groups a, d-f (filtered for those within 200bp upstream of transcription start sites (TSSs)) were intersected with a large sets of ccRCC HM450 and RNA-seq data from TCGA. CpG islands are depicted in dark green, with gray indicating non-CpG islands. See materials and methods for details in analysis and filtering.

Figure 5. Comparison of genetic and epigenetic changes in ccRCC

Epigenetically regulated genes in ccRCC (Figure 4) were compared to genes significantly mutated in ccRCC (TCGA). A) Overlap between epigenetically regulated genes and commonly mutated genes. B) Frequency of significant methylation, expression, and genetic changes in ccRCC. C & D) Pathway analysis of genes commonly mutated (C) and epigenetically regulated (D) in ccRCC.

Figure 6. Epigenetic changes affect genes regulated by HIF1A

A) Pathway analyses reveal HIF1A as the top upstream regulator of epigenetically regulated genes (Figure 4) in ccRCC samples. B) Diagram of HIF1A-signaling pathway, in which 4 genes are epigenetically upregulated in our data.