Fig 1: 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.
Fig 2: MC-HBV–based screen identifies cohesin complex as a novel host cccDNA binding factor. (A) MC-HBV was biotinylated and detected by dot blot using horseradish-peroxidase–streptavidin (upper panel); the input, unbound, and streptavidin-beads pull-down DNA was analyzed by dot blot to detect pull-down efficiency (lower panel). (B) Biotin-MC-HBV and MC-HBV were incubated with nuclear protein of HepG2 cells, and the interaction of H3, H3K27Ac, and H3K122me3 with cccDNA was identified by pull-down assay. (C) Schematic diagram of cccDNA-interacting factors screen with biotin-MC-HBV/pull-down/MS/bioinformatic analysis, biotin-linear-MC-HBV (linearized by EcoRI), and unlabeled MC-HBV taken as control. (D) Venn diagram of MS-identified proteins among 3 groups. A total of 306 candidates specifically were identified in the biotin-MC-HBV group and (E) analyzed for GO enrichment, or (F, left panel) were analyzed for enriched ontology clusters and a visualized network, (F, right panel) together with the 134 candidates classified into the DNA conformation regulation set. A circle node represented an enriched cluster, and nodes of the same color belong to the same cluster. (G) The protein–protein interaction network of these 134 proteins was mapped onto 6 pathways by MCODE (left panel), among which the chromosome segregation pathway was highlighted with red. Right: Cohesin composition and the information of 6 cohesin subunits and 2 regulators identified by MS. (H) A total of 1 μg biotin-MC-HBV, biotin-linear MC-HBV, and unlabeled MC-HBV were incubated with 400 μg HepG2 cells nuclear protein for 6 hours, then the interaction of SMC3 and SMC1A with cccDNA was identified by pull-down assay (upper panel); and 1 μg biotin-MC-HBV was incubated with 400 μg HepG2 cells nuclear protein with/without the presence of 1–3 μg unlabeled MC-HBV, then SMC3 were identified by pull-down assay (lower panel). (I) MC-HBV was transfected into Huh7 cells, or HBV-infected HLCZ01 cells and PHH cells at 200 genome equivalents (Geq), the enrichment of endogenous SMC3 and SMC1A on cccDNA was measured by ChIP assay (n = 3). (J) The green fluorescent protein (GFP)-SMC1A and GFP-SMC3 proteins were incubated with increasing concentrations of MC-HBV, the specific interactions were quantified by MST and plotted with the dissociation constant (Kd) equation, GFP protein served as control (n = 3). Data are presented as means ± SD. ∗∗P < .01.
Fig 3: CTCF mediates cohesin loading onto HBV cccDNA. (A) HLCZ01 cells were infected with HBV (200 genome equivalents [Geq]) for 72 hours, the interaction of CTCF with cccDNA was analyzed by ChIP (n = 3). (B) The green fluorescent protein (GFP)-CTCF protein was incubated with increasing concentrations of MC-HBV, specific interactions were quantified by MST and plotted with the dissociation constant (Kd) equation, GFP protein served as control (n = 3). (C) Huh7 cells were transfected with Flag-CTCF and MC-HBV for 72 hours, binding of CTCF with cccDNA was first estimated with ChIP using anti-Flag antibody, after competitively eluting with Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DDDDK) peptide, reChIP assay was performed using an anti-SMC3 antibody to detect the occupancy of SMC3 with CTCF on cccDNA (n = 3). (D) siCTCF was cotransfected into Huh7 with MC-HBV for 72 hours. ChIP assay was performed with an anti-SMC3 antibody to analyze the enrichment of SMC3 on cccDNA (n = 3). (E) Co-IP was performed with an anti-CTCF antibody or IgG to analyze the interaction of CTCF with SMC1A and SMC3. (F) siSMC3 was transfected into Huh7 for 72 hours, and Co-IP was performed with an anti-CTCF antibody to analyze the interaction of CTCF and SMC1A. Relative quantification was performed by analyzing the relative ratio of band density using ImageJ software (National Institutes of Health, Bethesda, MD) as follows: relative SMC1A level interacting with CTCF = band density ratio (immunoprecipitated SMC1A/immunoprecipitated CTCF)/band density ratio (input SMC1A/input CTCF), scramble group set as 1. Data are presented as means ± SD. ∗∗P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Fig 4: The loading of cohesin onto cccDNA is necessary for its antiviral role. (A) MC-HBV was cotransfected with siWAPL, siNIPBL, or siMAU2 into Huh7 cells for 72 hours, (B) HBV-infected (200 genome equivalents [Geq]) HLCZ01 cells for 24 hours, then siWAPL, siNIPBL, and siMAU2 were transfected for another 48 hours. RT-qPCR detected the knockdown effect and the enrichment of SMC3 on cccDNA was measured by ChIP (n = 3). (C) MC-HBV was cotransfected with Flag-SMC3 and siNIPBL or siMAU2 into Huh7 cells for 72 hours, and HBsAg, HBeAg, and pgRNA levels were measured (n = 3). (D) MC-HBV was cotransfected with WT SMC3 or SMC3-K38A mutant into Huh7 cells for 72 hours, and HBsAg, HBeAg, HBc, and pgRNA levels were measured (n = 3). SMC1A binding on cccDNA was detected by ChIP (n = 3). Data are presented as means ± SD. ∗P < .05, ∗∗P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA.
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