Subtype-Associated Epigenomic Landscape and 3D Genome

Iyyanki et al. Genome Biology (2021) 22:105 https://doi.org/10.1186/s13059-021-02325-y RESEARCH Open Access Subtype-associated epigenomic landscape and 3D genome structure in bladder cancer Tejaswi Iyyanki1,2†, Baozhen Zhang2,3†,QixuanWang2†,YeHou2†,QiushiJin2, Jie Xu1,2, Hongbo Yang2, Tingting Liu2, Xiaotao Wang2, Fan Song1,2,YuLuan2, Hironobu Yamashita4,5,RubyChien6,HuijueLyu2, Lijun Zhang7,LuWang2, Joshua Warrick4,5, Jay D. Raman5, Joshua J. Meeks8, David J. DeGraff4,5* and Feng Yue2,9* * Correspondence: ddegraff@ pennstatehealth.psu.edu; yue@ Abstract: Muscle-invasive bladder cancers are characterized by their distinct northwestern.edu expression of luminal and basal genes, which could be used to predict key clinical † Tejaswi Iyyanki, Baozhen Zhang, features such as disease progression and overall survival. Transcriptionally, FOXA1, Qixuan Wang and Ye Hou contributed equally to this work. GATA3, and PPARG are shown to be essential for luminal subtype-specific gene 4Department of Pathology and regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are Laboratory Medicine, The critical for regulation of basal subtype-specific genes. Despite these advances, the Pennsylvania State University, College of Medicine, Hershey, PA, underlying epigenetic mechanisms and 3D chromatin architecture responsible for USA subtype-specific regulation in bladder cancer remain unknown. 2Department of Biochemistry and Molecular Genetics, Feinberg School Result: We determine the genome-wide transcriptome, enhancer landscape, and of Medicine Northwestern transcription factor binding profiles of FOXA1 and GATA3 in luminal and basal University, Chicago, IL, USA subtypes of bladder cancer. Furthermore, we report the first-ever mapping of Full list of author information is available at the end of the article genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct transcription factor binding at distal enhancers of luminal and basal bladder cancers. Finally, we identify a novel clinically relevant transcription factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration. Conclusion: In summary, our work identifies unique epigenomic signatures and 3D genome structures in luminal and basal urinary bladder cancers and suggests a novel link between the circadian transcription factor NPAS2 and a clinical bladder cancer subtype. Introduction Urinary bladder cancers (BLCA) are the second most commonly diagnosed urologic malignancy in the USA, with over 81,400 total new cases diagnosed in 2019 [1, 2]. As BLCA is a morbid disease that is costly to treat, increased molecular understanding is required [3]. Expression of luminal (FOXA1, GATA3, PPARG, etc.) and basal (KRT1, KRT5, KRT6A, etc.) [4, 5] genes have been used to molecularly characterize muscle © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Iyyanki et al. Genome Biology (2021) 22:105 Page 2 of 20 invasive BLCA. In particular, the presence of basal BLCA, which is often enriched for squamous differentiation, is associated with significant morbidity, disease progression, and lower survival [6, 7]. Recent studies have identified both luminal (FOXA1, GATA3, and PPARG [8]) and basal (TP63 [9–12], STAT3 [4, 13–15], TFAP2A, and TFAP2C [16]) -specific transcription factors (TFs) with functional roles in BLCA. For instance, we previously reported that FOXA1 and GATA3 cooperate with PPARG activation to drive trans- differentiation of basal BLCA cells to luminal subtype [8]. This observation is in agree- ment with the role of these factors in maintaining urothelial cell differentiation [17, 18] and supports a role for inactivation of FOXA1, GATA3, and/or PPARG during BLCA progression to a basal subtype. Subtype-specific TFs also appear to drive programs in oncogenesis and tumor progression. For example, basal factors STAT3, TP63, and TFAP2A/C have been shown to promote tumor cell invasion and/or metastasis [12, 13, 16]. Similarly loss of FOXA1 [19] and GATA3 [20] have been shown to promote cell migration and invasion. Therefore, specific TFs play a key role in subtype specification. In addition to directly regulating transcription, studies show that TFs regulate gene expression through epigenetic histone modifications and open chromatin accessibility in breast cancers [21–24]. However, the degree to which the specific repertoire of TFs, epigenetic open chromatin TF accessibility, histone modifications, and 3D genome architecture cooperate for subtype expression is unknown. Therefore, we performed the most comprehensive set of genome-wide experiments to systematically map the epigenome, transcriptome, TF binding, and 3D chromatin loops. To our knowledge, this is the first report identifying 3D genome architecture in bladder cancer. Our work highlights the rele- vance of epigenetic modifications, open chromatin accessibility, and TF repertoire and identifies a novel identified basic helix loop helix (bHLH) TF NPAS2, all of which cooperate in the coordination of subtype-specific gene expression in bladder cancer. Results Comprehensive epigenomic profiling in both BLCA lines and primary tumors In this project, we performed RNA-Seq, ChIP-Seq for Histone 3 lysine 27 acetylation (H3K27ac), Assay for Transposase-Accessible Chromatin using sequencing (ATAC- Seq), and genome-wide chromatin confirmation capture experiments (Hi-C) on 4 bladder cancer cell lines (Fig. 1a), two of which (RT4 and SW780) were previously anno- tated as luminal and the two others (SCABER and HT1376) that were characterized as basal [8, 25]. Based on the RNA-Seq data generated in this study, we used a previously reported molecular subtyping approach [26] to confirm assignment to luminal and basal states. Our results confirmed RT4 and SW780 as belonging to the Luminal- papillary subtype, while SCABER and HT1376 belong to the Basal/squamous subtype (Additional file 1: Table S1). Each experiment in bladder cancer cell lines has at least two biological replicates (Additional file 2: Table S2) and we observed a high correl- ation between the two replicates (Additional file 3: Table S3). More importantly, we performed the same set of experiments on four patient muscle-invasive bladder tumors as well. By using the same molecular subtyping method, we determined their subtypes as the following: T1 is Luminal-papillary, T3 is Stroma-rich, and T4 and T5 are basal/ squamous. Iyyanki et al. Genome Biology (2021) 22:105 Page 3 of 20 Fig. 1 Luminal and basal transcriptional BLCA subtypes are associated with distinct promoter and distal enhancers’ activity at the epigenetic level. a Overall design of the study. b Differential expression gene (DEG) analysis of luminal cell lines (RT4 and SW780) and basal cell lines (SCABER and HT1376) shows 427 basal-specific upregulated genes and 524 luminal-specific upregulated genes. c Heatmap of differential H3K27ac ChIP-Seq at promoters (left). Signal H3K27ac intensity profiles for each cluster of BLCA cells (right). d Genome browser signal tracks for a panel of luminal and basal genes. Shown here are the tracks of H3K27ac ChIP-Seq, ATAC-Seq, and RNA-Seq data in RT4, SW780, SCABER, and HT1376 cells. e Promoter H3K27ac and its associated RNA-Seq signals for selected luminal and basal genes shows remarkable similarity. f Integrated H3K27ac peaks at distal enhancers and RNA-Seq gene expression association model identifies putative enhancers and gene regulation. Top 10,000 most variable enhancers (left heatmap) are plotted along with their corresponding gene expression (right heatmap). g Correlations of genome-wide H3K27ac signals between the bladder cancer cell lines and tumor samples demonstrate similarity of enhancer landscape Luminal and basal transcriptional BLCA subtypes are associated with distinct promoter and distal enhancers activity at the epigenetic level Enrichment of H3K27ac signals has been used to predict both active promoters and distal enhancers [27, 28]. Therefore, we first performed ChIP-Seq for H3K27ac in all four cell types and four patient samples. We observed that biologic replicates following H3K27ac ChIP-seq always clustered together, indicating our results are highly reprodu- cible (Additional file 4: Figure S1A). Further, we found that two luminal

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