EXPERIMENT_TITLE: GSM7438626: CTL_293T_HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In situ Hi-C experiments were conducted following the method described by Rao and colleagues with slight modifications (Rao et al., Cell. 2014). Briefly, HindIII enzyme was used to digest cells overnight at 37°C with 500U, followed by biotin filling and proximity ligation for 4 hours at 18°C with 2000U T4 DNA Ligase. After reverse-crosslinking, DNA was purified and sheared to ~300 bp fragments using Covaris S220 sonicator. The biotin-containing fragments were then immobilized on MyOne Streptavidin T1 beads. The NEXTflex adaptors were ligated, and the fragments were PCR amplified for 8 cycles using the KAPA HiFi Library Amplification Kit. Double-size selection was performed using AMPure XP beads to isolate fragments between 300 and 800bp
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The p65/RelA factor of NF-?B plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA governs gene transcription and alternative splicing (AS) through promoter enrichment and genomic exon occupancy, respectively. However, the mechanisms underlying the coordination of these processes remain elusive. In this study, we employed the HTLV-1 Tax viral oncoprotein, a potent activator of NF-?B, to investigate the integrative relationship between 3D chromatin architecture and NF-?B-regulated AS. Our analysis revealed that Tax induces a significant reorganization of the 3D genome, resulting in the formation of multigene complexes that comprise genes co-regulated in transcription and in AS. Notably, we found that the Tax-induced gene-gene contact between the two master genes NFKBIA and RELA is associated with their differential regulation in gene expression and alternative splicing, respectively. Through dCas9-mediated approaches, we demonstrated that NFKBIA-RELA interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on RELA. Via this mechanism, Tax promotes the expression of the splice isoform RELA?E6 that exhibits specific gene transactivation capacity at the protein level. Our findings shed light on new regulatory mechanisms upon HTLV-1 Tax and underscore the crucial role of p65/RelA in coordinated regulation of NF-?B-responsive genes at both transcriptional and alternative splicing levels in the context of the 3D genome. Overall design: Hi-C experiments on 293 HEK cells expressing or not the HTLV-1 TAX protein
SAMPLE_TITLE: CTL_293T_HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "embryonic kidney"}, {"TAG": "tissue", "VALUE": "embryonic kidney"}, {"TAG": "cell line", "VALUE": "293T HEK"}, {"TAG": "cell type", "VALUE": "fibroblast"}, {"TAG": "genotype", "VALUE": "WT"}, {"TAG": "treatment", "VALUE": "psg5M-empty"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: CTL_293T_HiC
RUN_SET_RUN_Pool_Member_@attributes_sample_title: CTL_293T_HiC
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