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

The importance of CREB in HBV transcription prompted us to investigate whether CRTC1 activation of the preS2 promoter requires CREB function. As a first step, we gen- erated additional reporter constructs in which the CRE elements in the preS2 promoter were disrupted. Similar constructs were previously described (10) . Indeed, progressive removal of CRE elements effectively blunted CRTC1 activation of the preS2 promoter ( Figure 5A , groups 5 and 6 compared to 4). Next, we asked whether functional CREB is critical in supporting CRTC1 activity by overexpressing A-CREB, a dominant inactive form of CREB (34) . CRTC1 activation of the preS2 promoter was erased by A-CREB ( Figure 5B , groups 3 and 4 compared to 2). Reciprocally, CREB activation of the preS2 promoter was progressively suppressed when we increased M1-CRTC1 expression in HepG2 cells ( Figure 5C , groups 3 and 4 compared to 2). In line with this, the recruitment of CRTC1 to preS2-CRE was abrogated when A-CREB was overexpressed in pHBV1.3D-transfected HepG2 cells ( Figure 5D , group 4 compared to 3). Reciprocal immunoprecipitation and immunoblotting experiments confirmed the formation were used to deplete endogenous CRTC1 at a concentration of 100 nM. siNC was used as a negative control. Endogenous CRTC1 in siRNA-transfected HepG2 cells was analyzed by western blotting at 72 h post-transfection using rabbit anti-CRTC1. Concurrently, total RNA was extracted and cDNA was synthesized. Quantitative RT-PCR was performed to analyze the relative levels of CRTC1 and CRTC2 transcripts. The difference between bars 1 and 5 is statistically significant by Student's t test (P = 0.0018, highlighted with **). The difference between bars 2 and 6 is statistically not significant (n.s.) by Student's t test (P = 0.74). (E) CREB recruitment to pre-S2 promoter was diminished in CRTC1-compromised HepG2 cells. After 24 h of CRTC1 knockdown, pHBV1.3D was transfected into HepG2 cells for another 48 h. ChIP was performed as in Figure 2D . The differences between groups 2 and 3 (P = 0.00023, highlighted with ***) as well as between groups 2 and 4 (P = 0.013, highlighted with *) are statistically significant; AU: arbitrary unit. (F) Knockdown of CRTC1 repressed pgRNA expression. The differences between groups 2 and 3 (P = 0.00031, highlighted with ***) as well as between groups 2 and 4 (P = 0.022, highlighted with *) are statistically significant by Student's t test. (G) Knockdown of CRTC1 dampened cccDNA level. Nuclear cccDNA was analyzed as in Figure 3B . The differences between groups 2 and 3 (P = 0.00012, highlighted with ***) as well as between groups 2 and 4 (P = 0.0089, highlighted with **) are statistically significant by Student's t test. (H) CRTC1 is required for production of cell-free virus. After 24 h of CRTC1 knockdown, pHBV1.3D was transfected into HepG2 cells for another 48 h. Culture medium was centrifugated to remove debris and then detected for HBsAg using Monolisa TM ULTRA kit (BioRad). Relative HBsAg level in siNC-transfected cells was taken as 1. The differences between groups 2 and 3 (P = 0.00071, highlighted with ***) as well as between groups 2 and 4 (P = 0.0047, highlighted with **) are statistically significant by Student's t test. The difference between groups 4 and 5 is statistically significant by Student's t test (P = 0.0014, highlighted with **); vec: empty vector. (B) CRTC1 activation of preS2 promoter was blunted by dominant inactive CREB. HepG2 cells were transfected with