Abstract

Transcription factor fusion proteins can transform cells by inducing global changes of the transcriptome often creating a state of oncogene addiction. Here, we investigate the role of epigenetic mechanisms in this process, focusing on Ewing sarcoma cells that are dependent on the EWS-FLI1 fusion protein. We established reference epigenome maps comprising DNA methylation, seven histone marks, open chromatin states, and RNA levels, and we analyzed the epigenome dynamics upon downregulation of the driving oncogene. Reduced EWS-FLI1 expression led to widespread epigenetic changes in promoters, enhancers, and super-enhancers, and we identified histone H3K27 acetylation as the most strongly affected mark. Clustering of epigenetic promoter signatures defined classes of EWS-FLI1- regulated genes that responded differently to low-dose treatment with histone deacetylase inhibitors. Furthermore, we observed strong and opposing enrichment patterns for E2F and AP-1 among EWS-FLI1-correlated and anticorrelated genes. Our data describe extensive genome-wide rewiring of epigenetic cell states driven by an oncogenic fusion protein.

Data

DNA Methylation (WGBS & RRBS) Histone Marks (ChIP-seq) Open Chromatin (ATAC-seq) Gene Expression (RNA-seq) EWS-FLI1 (ChIP-seq)
Bilke S et al., 2013 doi:10.1101/gr.151340.112
Sequencing Data DNA methylation RRBS aligned reads
(zipped BAM archive, 5.4 GB)
DNA methylation WGBS aligned reads
(zipped BAM archive, 47 GB) 		ChIP-seq aligned reads
(zipped BAM archive, 68 GB) 		ATAC-seq aligned reads
(zipped BAM archive, 14 GB) 		RNA-seq aligned reads
(zipped BAM archive, 25 GB) 		ChIP-seq aligned reads
(zipped BAM archive, 5.8 GB)
Processed Results DNA methylation values
(zipped BED files, 1.1 GB) 		ChIP-seq histone peaks
(zipped BED files, 71 MB) 		ATAC-seq peaks and dips
(zipped BED files, 7.2 MB) 		RNA-seq gene expression values
(zipped TSV archive, 41 MB) 		ChIP-seq EWS-FLI1 peaks
(zipped BED files, 144 kB)
Genome Browser Tracks DNA methylation (track hub) Histone marks (track hub) ATAC marks (track hub) Gene expression (track hub) EWS-FLI1 marks (track hub)

Integrative Analysis

Analysis Analysis Description Downloads and Resources
Transcript assembly and annotation De novo transcript assembly, pre-filtering, and post-filtering (by requiring H3K4me3 peaks near TSSs). Filtered results are annotated with histone marks, clustering, and DNA methylation. ▸ TSS models GTF file (16 MB zip file)
▸ TSS annotation matrix (26,750 filtered TSSs)
Gene Clustering Genes chosen for expert classification. ▸ EWS-FLI1 correlated genes
▸ EWS-FLI1 anticorrelated genes
Enrichments Results of a custom location-based enrichment analysis tool testing for enrichment of TSSs or enhancers to ENCODE, DNase Hypersensitive Sites, and Cistrome databases. TSS results include enrichment of gene names to MSigDB. ▸ Interactive Enrichment Results Browser (TSSs)
▸ Interactive Enrichment Results Browser (Enhancers)
Differential Histone Marks Differential histone peaks for all marks, as called by diffBind based on the MACS2 peak calls. ▸ Differential Histone Peaks
Super-enhancers Super-enhancers as defined by ROSE, based on our H3K27ac data, in both EWS-FLI1 high and EWS-FLI1 low conditions. ▸ Super-enhancers in EWS-FLI1 high
▸ Super-enhancers in EWS-FLI1 low
DNA Methylation RnBeads analysis of DNA methylation data. These results comprise lists of differentially methylated regions and several other types of analysis. ▸ RnBeads Report
Drug Response FPKM (fragments per kilobase per million) values for RNA-seq after treatment with 3 HDAC inhibitors (Vorinistat, Entinostat, and Romedepsin), and DMSO control, plus original untreated RNA-seq data. 2 replicates per treatment group. ▸ Drug Response (6 MB zip file)

Genome Browser Links

Manuscript location Description Genomic Location Genome Browser Link
Figure 1B BCL11B locus (EWS-FLI1 regulated gene) chr14: 99,610,000 - 99,770,000 ▸ UCSC
Figure 3F LOX locus (cluster 1 gene) chr5: 121,391,624 - 121,436,625 ▸ UCSC
Figure 3G PTX3 locus (cluster 2 gene) chr3: 157,149,000 - 157,168,000 ▸ UCSC
Figure 3H POSTN locus (cluster 3 gene) chr13: 38,130,000 - 38,199,000 ▸ UCSC
Figure 3I DKK1 locus (cluster 4 gene) chr10: 54,068,131 - 54,083,327 ▸ UCSC
Figure 6E Typical enhancer chr2: 226,540,001 - 226,780,000 ▸ UCSC
Figure 6E Super-enhancer chr1: 163,521,167 - 163,583,365 ▸ UCSC
Figure 7C CCND1 locus chr11: 69,410,616 - 69,463,477 ▸ UCSC
Figure 7D MYB locus chr6: 135,461,500 - 135,576,000 ▸ UCSC
Figure 7E FOSL1 locus chr11: 65,659,000 - 65,673,000 ▸ UCSC
Figure S1A LINC00277 locus (long non-coding RNA) chr15: 69,358,216 - 69,403,137 ▸ UCSC
Figure S1B Predicted non-coding transcript chr1: 41,880,000 - 41,940,000 ▸ UCSC
Figure S1C Novel transcript chr13: 31,568,823 - 31,600,640 ▸ UCSC
Figure S1D HOOK1 locus (alternative TSS) chr1: 60,249,774 - 60,372,809 ▸ UCSC

Citation

Eleni M. Tomazou*, Nathan C. Sheffield*, Christian Schmidl, Michael Schuster, Andreas Schönegger, Paul Datlinger, Stefan Kubicek, Christoph Bock#, Heinrich Kovar#.
Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer reprogramming by the oncogenic fusion protein EWS-FLI1.
Cell Reports, Volume 10, 24 February 2015 doi:10.1016/j.celrep.2015.01.042