Requirement of CRTC1 coactivator for hepatitis B virus transcription

Tang, Hei-Man Vincent; Gao, Wei-Wei; Chan, Chi-Ping; Cheng, Yun; Chaudhary, Vidyanath; Deng, Jian-Jun; Yuen, Kit-San; Wong, Chun-Ming; Ng, Irene Oi-Lin; Kok, Kin-Hang; Zhou, Jie; Jin, Dong-Yan

We next employed RNAi to silence endogenous CRTC1 expression in HepG2 cells. Two independent siRNAs directed against CRTC1 (siCRTC1-a and siCRTC1-b) efficiently depleted its protein expression ( Figure 4D , lanes 2 and 3 compared to 1 in the blot). The silencing effect was highly specific since only CRTC1 mRNA was affected, whereas the levels of CRTC2 transcript remained unaltered ( Figure 4D , groups 3 and 5 compared to 1, and groups 4 and HepG2 cells were transfected with preS2-Luc (100 ng) and pcDNA3.1-V5/His-CRTC1/2/3 (100 ng). Dual luciferase assay was performed. Results represent means from three independent experiments and error bars indicate SD. The difference between groups 1 and 3 is statistically significant by Student's t test (P = 0.000024, highlighted with ***). (C) Effect on X promoter activity. The difference between groups 1 and 2 is statistically not significant (n.s.) by Student's t test (P = 0.49). (D) CRTC recruitment to CREs in the preS2 promoter. HepG2 cells were transfected with the indicated plasmids. ChIP was performed to precipitate V5-CRTC1/2/3-preS2 promoter complex using mouse anti-V5 (Invitrogen) and the CRE sequence in the preS2 promoter was analyzed by quantitative PCR. Results represent relative recruitment measured in arbitrary units (AU). The difference between groups 2 and 5 is statistically significant by Student's t test (P = 0.0032, highlighted with **); N.D.: not detected. (E) Impact of CRTC coactivators on CREB recruitment to the preS2 promoter. ChIP was performed to precipitate CREB-preS2 promoter complex using rabbit anti-CREB (Cell Signaling). The difference between groups 2 and 5 is statistically significant by Student's t test (P = 0.029, highlighted with *). (F) CRTC recruitment to cccDNA. ChIP was performed to obtain V5-CRTC1/2/3-DNA complex using mouse anti-V5 and the cccDNA-specific sequence was analyzed by quantitative PCR. The difference between groups 2 and 3 is statistically significant by Student's t test (P = 0.031, highlighted with *). 6 compared to 2 in the bar chart). In keeping with this, the recruitment of endogenous CREB to preS2-CRE was prevented in CRTC1-depleted cells ( Figure 4E , groups 3 and 4 compared to 2), indicating the physiological importance of CRTC1 in mobilizing CREB to the preS2 promoter to activate transcription. Furthermore, the levels of pgRNA, nuclear cccDNA and secreted HBsAg were declined when CRTC1 was compromised ( Figure 4F-H, groups 3 and 4 compared to 2). Collectively, our results suggest that CRTC1 is required for HBV transcription and replication.