Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer
Manjie Xing, … , Bin Tean Teh, Patrick Tan
Manjie Xing, … , Bin Tean Teh, Patrick Tan
Published May 4, 2020
Citation Information: J Clin Invest. 2020;130(6):3005-3020. https://doi.org/10.1172/JCI126726.
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Research Article Gastroenterology Oncology

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer

Abstract

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA–binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.

Authors

Manjie Xing, Wen Fong Ooi, Jing Tan, Aditi Qamra, Po-Hsien Lee, Zhimei Li, Chang Xu, Nisha Padmanabhan, Jing Quan Lim, Yu Amanda Guo, Xiaosai Yao, Mandoli Amit, Ley Moy Ng, Taotao Sheng, Jing Wang, Kie Kyon Huang, Chukwuemeka George Anene-Nzelu, Shamaine Wei Ting Ho, Mohana Ray, Lijia Ma, Gregorio Fazzi, Kevin Junliang Lim, Giovani Claresta Wijaya, Shenli Zhang, Tannistha Nandi, Tingdong Yan, Mei Mei Chang, Kakoli Das, Zul Fazreen Adam Isa, Jeanie Wu, Polly Suk Yean Poon, Yue Ning Lam, Joyce Suling Lin, Su Ting Tay, Ming Hui Lee, Angie Lay Keng Tan, Xuewen Ong, Kevin White, Steven George Rozen, Michael Beer, Roger Sik Yin Foo, Heike Irmgard Grabsch, Anders Jacobsen Skanderup, Shang Li, Bin Tean Teh, Patrick Tan

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Figure 2

EBF1 is transcriptionally silenced in GC via repressive histone modifications and DNA methylation.

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EBF1 is transcriptionally silenced in GC via repressive histone modific...
(A) Correlation of mRNA expression levels between EBF1 and TFs at genomic regions flanking the EBF1 TSS. Correlation coefficients were computed using the normalized expression matrix from TCGA (n = 415 tumors, n = 35 normal samples). *Q < 0.05, **Q < 0.01, and ***Q < 0.001, by Pearson’s correlation test with FDR multiple testing correction. (B) H3K27me3, EZH2, and SUZ12 signals flanking the EBF1 TSS. Top: RNA-Seq tracks depict EBF1 expression in GC lines. Bottom left: H3K27me3, EZH2, and SUZ12 enrichment. Yellow bar indicates the EBF1 promoter (TSS ± 2.5 kb). Bottom right: DNA methylation levels of GC lines. Colored graph shows the average β value of 12 CpG probes in the 5′-UTR and TSS of EBF1. EZH2 and SUZ12 ChIP-Seq data are from the ENCODE database. Layered signals from multiple cell lines are shown for EZH2 (H1 human embryonic stem cells [H1-hESCs], human mammary epithelial cells [HMECs], human umbilical vein endothelial cells [HUVECs], normal human astrocytes [NHAs], normal human epidermal keratinocytes [NHEKs], and normal human lung fibroblasts [NHLFs]). The SUZ12 profile is from H1-hESCs. (C) EZH2 and EBF1 expression levels in SNU484 cells after drug treatment. (D) DNA methylation 450K array data from TCGA. Data were plotted for the top and bottom 25th percentiles of EBF1-expressing samples (n = 191). (E) In-house DNA methylation 450K array data for 18 T/N GC pairs. Heatmap shows GCs with or without loss of EBF1. (D and E) Data are from 12 CpG probes in the 5′-UTR and TSS region of EBF1. (F) Response patterns of EBF1 and TERT mRNA levels following drug treatments. (C and F) Control cells were treated with DMSO. *Q < 0.05, by 2-sided t test with FDR multiple testing correction. qPCR results are shown as the mean ± SD of technical triplicates. All data are representative of 3 independent experiments. (G) H3K27me3 enrichment at the EBF1 locus from GC cell lines with DMSO, 5-aza, and GSK126 treatment. Red arrows indicate a decrease in H3K27me3 enrichment.