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The fundamental cause of transcription dysregulation in hepatocellular carcinoma (HCC) remains elusive. To investigate the underlying mechanisms, comprehensive 3D-epigenomic analyses are performed in cellular models of THLE2 (a normal hepatocytes cell line) and HepG2 (a hepatocellular carcinoma cell line) using integrative approaches for chromatin topology, genomic and epigenomic variation, and transcriptional output. Comparing the 3D-epigenomes in THLE2 and HepG2 reveal that most HCC-associated genes are organized in complex chromatin interactions mediated by RNA polymerase II (RNAPII). Incorporation of genome-wide association studies (GWAS) data enables the identification of non-coding genetic variants that are enriched in distal enhancers connecting to the promoters of HCC-associated genes via long-range chromatin interactions, highlighting their functional roles. Interestingly, CTCF binding and looping proximal to HCC-associated genes appear to form chromatin architectures that overarch RNAPII-mediated chromatin interactions. It is further demonstrated that epigenetic variants by DNA hypomethylation at a subset of CTCF motifs proximal to HCC-associated genes can modify chromatin topological configuration, which in turn alter RNAPII-mediated chromatin interactions and lead to dysregulation of transcription. Together, the 3D-epigenomic analyses provide novel insights of multifaceted interplays involving genetics, epigenetics, and chromatin topology in HCC cells., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Advanced Genetics published by Wiley Periodicals LLC.)

Chromatin Interaction Analysis Using Paired-End Tag Sequencing (ChIA-PET) is an established method to map protein-mediated chromatin interactions. A limitation, however, is that it requires a hundred million cells per experiment, which hampers its broad application in biomedical research, particularly in studies in which it is impractical to obtain a large number of cells from rare samples. To reduce the required input cell number while retaining high data quality, we developed an in situ ChIA-PET protocol, which requires as few as 1 million cells. Here, we describe detailed step-by-step procedures for performing in situ ChIA-PET from cultured cells, including both an experimental protocol for sample preparation and data generation and a computational protocol for data processing and visualization using the ChIA-PIPE pipeline. As the protocol significantly simplifies the experimental procedure, reduces ligation noise, and decreases the required input of cells compared to previous versions of ChIA-PET protocols, it can be applied to generate high-resolution chromatin contact maps mediated by various protein factors for a wide range of human and mouse primary cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and data generation Support Protocol: Bridge linker preparation Basic Protocol 2: Data processing and visualization., (© 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.)