Ren et al. J Hematol Oncol (2021) 14:120 https://doi.org/10.1186/s13045-021-01131-0 RESEARCH Open Access High-resolution Hi-C maps highlight multiscale 3D epigenome reprogramming during pancreatic cancer metastasis Bo Ren1†, Jinshou Yang1†, Chengcheng Wang1, Gang Yang1, Huanyu Wang1, Yuan Chen1, Ruiyuan Xu1, Xuning Fan2, Lei You1* , Taiping Zhang1* and Yupei Zhao1* Abstract Background: Pancreatic cancer’s poor prognosis is caused by distal metastasis, which is associated with epigenetic changes. However, the role of the 3D epigenome in pancreatic cancer biology, especially its metastasis, remains unclear. Methods: Here, we developed high-resolution 3D epigenomic maps of cells derived from normal pancreatic epithe- lium, primary and metastatic pancreatic cancer by in situ Hi-C, ChIP-seq, ATAC-seq, and RNA-seq to identify key genes involved in pancreatic cancer metastasis Results: We found that A/B compartments, contact domains, and chromatin loops changed signifcantly in meta- static pancreatic cancer cells, which are associated with epigenetic state alterations. Moreover, we found that upregu- lated genes, which were located in switched compartments, changed contact domains, and metastasis-specifc enhancer-promoter loops, were related to cancer metastasis and poor prognosis of patients with pancreatic cancer. We also found that transcription factors in specifc enhancer-promoter loop formation were also associated with metastasis. Finally we demonstrated that LIPC, looped to metastasis-specifc enhancers, could promote pancreatic cancer metastasis. Conclusions: These results highlight the multiscale 3D epigenome reprogramming during pancreatic cancer metas- tasis and expand our knowledge of mechanisms of gene regulation during pancreatic cancer metastasis. Keywords: Hi-C, Pancreatic cancer, Metastasis, Epigenetics, Multi-omics Background metastasis and difculty in early diagnosis. Currently, the Te incidence of pancreatic cancer has steadily risen 5-year survival rate of patients with distant metastasis is in recent years. Despite the signifcant progress in the only 3% [1], which is much smaller than that of patients treatment of most human cancers, pancreatic cancer with localized lesions. Terefore, the identifcation of continues to be a deadly malignancy due to its distant fundamental mechanisms involved in pancreatic cancer metastasis would provide valuable information for its *Correspondence: [email protected]; [email protected]; diagnosis and treatment. [email protected] It is well known that genetic mutations play pivotal †Bo Ren and Jinshou Yang contributed equally. 1 Department of General Surgery, State Key Laboratory of Complex roles in primary tumorigenesis; however, no “metasta- Severe and Rare Diseases, Peking Union Medical College Hospital, sis-specifc” genetic mutations have been identifed [2]. Chinese Academy of Medical Sciences, Peking Union Medical College, A recent whole-genome sequencing study showed that Beijing 100023, People’s Republic of China Full list of author information is available at the end of the article there are few heterogeneities of known driver mutations © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ren et al. J Hematol Oncol (2021) 14:120 Page 2 of 19 in primary and metastatic pancreatic cancer tissues [3], characterization of the 3D epigenomic features provided indicating that genetic mutations per se may have rela- more detailed epigenetic mechanisms of pancreatic can- tively less infuence on metastasis. Increasing knowledge cer metastasis. of epigenetics has shown that DNA methylation and his- tone modifcation are associated with pancreatic cancer Methods pathobiology and subtyping [4], and they change signif- Cell culture and transfection cantly during pancreatic cancer progression. However, Normal human pancreatic epithelial cells (HPNE, as an integral part of epigenetic information [5], the 3D ATCC®CRL-4023), primary pancreatic cancer cells organization of chromatin and its reprogramming during (PANC-1, ATCC®CRL-1469), and metastatic pancreatic tumor metastasis remain to be elucidated, and even less cancer cells (Capan-1, ATCC®HTB-79) were obtained is known about pancreatic cancer. from American Type Culture Collection (ATCC) (https:// Appropriate gene expression is determined by correct www. atcc. org/). All cell lines were cultured under recom- chromatin folding. Hi-C, a genome-wide chromosome mended conditions and were authenticated by high-reso- conformation capture assay, showed that the human lution small tandem repeats (STR) profling. Te siRNA genome is folded three-dimensionally in the nucleus and targeting LIPC and a scramble control siRNA were pur- divided into several active or inactive compartments, chased from Guangzhou RiboBio Co., LTD. (Guangzhou, named A or B [6]. Subsequent analyses showed that China). Sequences of siLIPC is 5′-GCA AAG GAA TTG compartments are partitioned into ~ 1 Mb in size, called CTA GTA A-3′. Te full length LIPC cDNA was sub- “topologically associating domains” [7]. With increased cloned into the GV658 empty vector (https:// www. genec sequencing depth, high resolution Hi-C data showed hem. com. cn/ index/ suppo rts/ zaiti_ info. html? id= 208). that there are contact domains located in megabase- Transient transfections were performed by lipofectamine sized chromatin domains [8] and allowed the detec- 3000 (Invitrogen) according to the corresponding tion of loops across the entire genome. Tese chromatin protocol. loops are usually mediated by CCCTC-binding factor (CTCF) [8], and often connect regulatory regions, such Lentivirus production and infection as enhancer-promoter loops. Previous studies demon- strated that aberrant chromatin interactions contribute Lentivirus for LIPC stably overexpression and shRNA to tumorigenesis [9, 10], but no detailed studies have targeting LIPC were purchased from GENECHEM as delineated a 3D epigenomic map of cancer metastasis, viral particles with frefy luciferase cassette and puro- especially for pancreatic cancer. mycin resistance gene. Infection of PANC-1 and Capan-1 To investigate the 3D epigenomic features of pancre- was performed according to the manufacturer’s protocol. atic cancer metastasis, we performed multi-omic analy- After infection, cells were selected by 2 µg/mL puromy- ses of normal pancreatic and pancreatic cancer cells cin and maintained by 1 µg/mL puromycin. derived from the primary site and liver metastasis. We frst analyzed chromatin interactions by high-resolution qRT‑PCR in situ Hi-C, which is useful for mapping compartments Total RNA was extracted by RNA-Quick Purifcation Kit [6], contact domains, and chromatin loops [8]. Moreo- (ES Science, RN001) and reverse transcription was per- ver, the epigenetic states of chromatin interacting regions formed using the Takara PrimeScript™ reagent Kit with were characterized by chromatin immunoprecipitation gDNA eraser (RR047A). Te PCR primers for LIPC are sequencing (ChIP-seq) of several histone marks and F: ATC AAG TGC CCT TGG ACA AAG, R: TGA CAG CCC CTCF to characterize contact domains and loops medi- TGA TTG GTT TCT. GAPDH primers as internal control ated by CTCF or cis-regulatory elements (e.g., enhancer, were purchased from Sangon Biotech. insulator) by combining the ChIP-seq and Hi-C data. In addition, we performed Assay for Transposase-Accessi- Western blot analysis ble Chromatin using sequencing (ATAC-seq) to identify Western blot analysis was performed as described previ- nucleosome-depleted regions (NDRs) and infer essen- ously [11]. Briefy, we performed SDS-PAGE to separate tial transcription factors involved in chromatin loops. protein lysates (20 µg). Ten we used primary and sec- Finally, transcription profles were determined by RNA- ondary antibodies to detect target proteins, and used an seq. Tese sequencing data and integrated analyses ena- enhanced chemiluminescence assay to visualize protein bled to (a) develop comprehensive 3D epigenomic maps, bands. LIPC antibody was purchased from ProteinTech (b) investigate the epigenetic state of the compartments (21133-1-AP, ProteinTech). β-actin and GAPDH anti- and contact domains, and (c) identify and character- bodies were purchased from LabLead (A0101-1, G0100, ize metastasis-specifc enhancer-promoter loops. Tis LabLead). E-cadherin, N-cadherin, Vimentin antibodies Ren et al. J Hematol Oncol (2021) 14:120 Page 3 of 19 were purchased from Cell Signaling Technology (3195 T, 4-cutter restriction enzyme MboI, and the DNA ends 13116 T, 3390S, CST). were