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. 2022 Aug 17;14(6):1177–1198. doi: 10.1016/j.jcmgh.2022.08.002

Figure 4.

Figure 4

Cohesin reshapes cccDNA conformation to impede RNAPII recruitment. (A) In MC-HBV–transfected Huh7 and HBV-infected HLCZ01 models, SMC3 was overexpressed or knocked down, and cccDNA levels were detected by qPCR (n = 3). (B) Flag-SMC3 or siSMC3 was cotransfected with MC-HBV into Huh7 cells for 72 hours, enrichment of RNAPII and CTD-S2-RNAPII on cccDNA was measured by ChIP (n = 3). (C) Interaction of RNAPII with cohesin complex was analyzed by detecting SMC1/SMC3 enrichment using Co-IP assay. (D) Flag-SMC3 or siSMC3 was cotransfected with MC-HBV into Huh7 cells for 72 hours, and H3K27ac, H3K4me3, and H3K9me3 enrichment on cccDNA was measured by ChIP (n = 3). (E) Cohesin complex was purified from HepG2 cells with Co-IP using SMC1A antibody and glycine-HCl elution. (F) MC-HBV was incubated with purified cohesin complex at room temperature for 30 minutes, mock treatment and IgG precipitation were taken as control. MC-HBV conformation was analyzed with AFM imaging, and arrows indicate the classic cccDNA structure, the box represents the amplified region. Data are presented as means ± SD. ∗P < .05, ∗∗P < .01.