Figure 7.

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.