Yan-Yi Jiang1, Yuan Jiang2, Chun-Quan Li3, Ying Zhang4, Pushkar Dakle4, Harvinder Kaur4, Jian-Wen Deng4, Ruby Yu-Tong Lin4, Lin Han4, Jian-Jun Xie5, Yiwu Yan6, Ngan Doan7, Yueyuan Zheng8, Anand Mayakonda4, Masaharu Hazawa9, Liang Xu4, YanYu Li3, Luay Aswad10, Maya Jeitany4, Deepika Kanojia4, Xin-Yuan Guan11, Jonathan W Said7, Wei Yang6, Melissa J Fullwood12, De-Chen Lin13, H Phillip Koeffler14. 1. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California; Cancer Science Institute of Singapore, National University of Singapore, Singapore. 2. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California; Cancer Science Institute of Singapore, National University of Singapore, Singapore. Electronic address: jiangyuanjy2016@gmail.com. 3. School of Medical Informatics, Daqing Campus, Harbin Medical University, Daqing, China. 4. Cancer Science Institute of Singapore, National University of Singapore, Singapore. 5. Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, China. 6. Cedars-Sinai Medical Center, Departments of Surgery and Biomedical Sciences, Los Angeles, California. 7. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California. 8. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California. 9. Cell-Bionomics Research Unit, Innovative Integrated Bio-Research Core, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Ishikawa, Japan. 10. Cancer Science Institute of Singapore, National University of Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore. 11. Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China. 12. Cancer Science Institute of Singapore, National University of Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore. Electronic address: mfullwood@ntu.edu.sg. 13. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California. Electronic address: dchlin11@gmail.com. 14. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California; Cancer Science Institute of Singapore, National University of Singapore, Singapore; National University Cancer Institute, National University Hospital Singapore, Singapore.
Abstract
BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.
BACKGROUND & AIMS: We investigated the transcriptome of esophageal squamous cell carcinoma (ESCC) cells, activity of gene regulatory (enhancer and promoter regions), and the effects of blocking epigenetic regulatory proteins. METHODS: We performed chromatin immunoprecipitation sequencing with antibodies against H3K4me1, H3K4me3, and H3K27ac and an assay for transposase-accessible chromatin to map the enhancer regions and accessible chromatin in 8 ESCC cell lines. We used the CRC_Mapper algorithm to identify core regulatory circuitry transcription factors in ESCC cell lines, and determined genome occupancy profiles for 3 of these factors. In ESCC cell lines, expression of transcription factors was knocked down with small hairpin RNAs, promoter and enhancer regions were disrupted by CRISPR/Cas9 genome editing, or bromodomains and extraterminal (BET) family proteins and histone deacetylases (HDACs) were inhibited with ARV-771 and romidepsin, respectively. ESCC cell lines were then analyzed by whole-transcriptome sequencing, immunoprecipitation, immunoblots, immunohistochemistry, and viability assays. Interactions between distal enhancers and promoters were identified and verified with circular chromosome conformation capture sequencing. NOD-SCID mice were given injections of modified ESCC cells, some mice where given injections of HDAC or BET inhibitors, and growth of xenograft tumors was measured. RESULTS: We identified super-enhancer-regulated circuits and transcription factors TP63, SOX2, and KLF5 as core regulatory factors in ESCC cells. Super-enhancer regulation of ALDH3A1 mediated by core regulatory factors was required for ESCC viability. We observed direct interactions between the promoter region of TP63 and functional enhancers, mediated by the core regulatory circuitry transcription factors. Deletion of enhancer regions from ESCC cells decreased expression of the core regulatory circuitry transcription factors and reduced cell viability; these same results were observed with knockdown of each core regulatory circuitry transcription factor. Incubation of ESCC cells with BET and HDAC disrupted the core regulatory circuitry program and the epigenetic modifications observed in these cells; mice given injections of HDAC or BET inhibitors developed smaller xenograft tumors from the ESCC cell lines. Xenograft tumors grew more slowly in mice given the combination of ARV-771 and romidepsin than mice given either agent alone. CONCLUSIONS: In epigenetic and transcriptional analyses of ESCC cell lines, we found the transcription factors TP63, SOX2, and KLF5 to be part of a core regulatory network that determines chromatin accessibility, epigenetic modifications, and gene expression patterns in these cells. A combination of epigenetic inhibitors slowed growth of xenograft tumors derived from ESCC cells in mice.
Authors: Ying Lyu; Yinglu Guan; Lisa Deliu; Ericka Humphrey; Joanna K Frontera; Youn Joo Yang; Daniel Zamler; Kun Hee Kim; Vakul Mohanty; Kevin Jin; Vakul Mohanty; Virginia Liu; Jinzhuang Dou; Lucas J Veillon; Shwetha V Kumar; Philip L Lorenzi; Yang Chen; Kathleen M McAndrews; Sergei Grivennikov; Xingzhi Song; Jianhua Zhang; Yuanxin Xi; Jing Wang; Ken Chen; Priyadharsini Nagarajan; Yejing Ge Journal: Genes Dev Date: 2022-08-25 Impact factor: 12.890
Authors: Zhong Wu; Jin Zhou; Xiaoyang Zhang; Zhouwei Zhang; Yingtian Xie; Jie Bin Liu; Zandra V Ho; Arpit Panda; Xintao Qiu; Paloma Cejas; Israel Cañadas; Fahire Goknur Akarca; James M McFarland; Ankur K Nagaraja; Louisa B Goss; Nikolas Kesten; Longlong Si; Klothilda Lim; Yanli Liu; Yanxi Zhang; Ji Yeon Baek; Yang Liu; Deepa T Patil; Jonathan P Katz; Josephine Hai; Chunyang Bao; Matthew Stachler; Jun Qi; Jeffrey J Ishizuka; Hiroshi Nakagawa; Anil K Rustgi; Kwok-Kin Wong; Matthew Meyerson; David A Barbie; Myles Brown; Henry Long; Adam J Bass Journal: Nat Genet Date: 2021-05-10 Impact factor: 41.307
Authors: Sai Ma; Bo Zhou; Qian Yang; Yunzhi Pan; Wei Yang; Stephen J Freedland; Ling-Wen Ding; Michael R Freeman; Joshua J Breunig; Neil A Bhowmick; Jian Pan; H Phillip Koeffler; De-Chen Lin Journal: Cancer Res Date: 2021-01-05 Impact factor: 13.312