SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis
Yuchen Xie, Merve Sahin, Toru Wakamatsu, Akane Inoue-Yamauchi, Wanming Zhao, Song Han, Amrita M. Nargund, Shaoyuan Yang, Yang Lyu, James J. Hsieh, Christina S. Leslie, Emily H. Cheng
Yuchen Xie, Merve Sahin, Toru Wakamatsu, Akane Inoue-Yamauchi, Wanming Zhao, Song Han, Amrita M. Nargund, Shaoyuan Yang, Yang Lyu, James J. Hsieh, Christina S. Leslie, Emily H. Cheng
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Research Article Oncology

SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis

Abstract

SETD2, a H3K36 trimethyltransferase, is the most frequently mutated epigenetic modifier in lung adenocarcinoma, with a mutation frequency of approximately 9%. However, how SETD2 loss of function promotes tumorigenesis remains unclear. Using conditional Setd2-KO mice, we demonstrated that Setd2 deficiency accelerated the initiation of KrasG12D-driven lung tumorigenesis, increased tumor burden, and significantly reduced mouse survival. An integrated chromatin accessibility and transcriptome analysis revealed a potentially novel tumor suppressor model of SETD2 in which SETD2 loss activates intronic enhancers to drive oncogenic transcriptional output, including the KRAS transcriptional signature and PRC2-repressed targets, through regulation of chromatin accessibility and histone chaperone recruitment. Importantly, SETD2 loss sensitized KRAS-mutant lung cancer to inhibition of histone chaperones, the FACT complex, or transcriptional elongation both in vitro and in vivo. Overall, our studies not only provide insight into how SETD2 loss shapes the epigenetic and transcriptional landscape to promote tumorigenesis, but they also identify potential therapeutic strategies for SETD2 mutant cancers.

Authors

Yuchen Xie, Merve Sahin, Toru Wakamatsu, Akane Inoue-Yamauchi, Wanming Zhao, Song Han, Amrita M. Nargund, Shaoyuan Yang, Yang Lyu, James J. Hsieh, Christina S. Leslie, Emily H. Cheng

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Figure 4

SETD2 loss induces human ETV1 expression through activation of an intronic enhancer.

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SETD2 loss induces human ETV1 expression through activation of an intron...
(A) Sequence homology between ATAC-Seq peak regions at the intron 4 of mouse Etv1 and the intron 5 of human ETV1. (B) Whole cell lysates (WCL) and histone fractions from JHRCC12 cells infected with control retrovirus or retrovirus expressing SETD2ΔN or from 786-O cells were analyzed by immunoblots. The mRNA levels of ETV1 were assessed in the indicated JHRCC12 cells by qPCR and normalized against β-actin (mean ± SD, n = 3). (C) JHRCC12 cells infected with control retrovirus or retrovirus expressing SETD2ΔN were assessed by ChIP-qPCR using the indicated antibodies for the promoter and intron 5 of ETV1 and the promoter of GAPDH. Data shown are the percent input (mean ± SD, n = 3). (D) A schematic of the strategy used to delete the conserved region (1,193 bp) at the intron 5 (9,157 bp) of ETV1 in JHRCC12 cells using CRISPR/Cas9-mediated genome editing. The positions of primers (P1 and P2) used for PCR-based validation of genome editing are indicated. PCR-based genotyping using the P1 and P2 primers was performed on JHRCC12 cells ± CRISPR/Cas9-mediated deletion of the intron 5 of ETV1. (E) The mRNA levels of ETV1 in JHRCC12 cells infected with lentivirus expressing the indicated sgRNAs were assessed by qPCR and normalized against β-actin (mean ± SD, n = 3). (F) A549 cells were transiently transfected with pGL2-pro vector or pGL2-pro containing the putative intron 5 enhancer of ETV1 without or with deletion of the FOS binding motif together with the pRL-SV40 plasmid (Promega) as a normalization control. The firefly and Renilla luciferase activities were assessed and normalized (mean ± SD, n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test.