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

Coimmunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays were performed as previously described (22, 29) . For Co-IP, HepG2 or HepG2.2.15 cells grown in a 100-mm petri dish were harvested into 1 ml of immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1 mM dithiothreitol, 20 mM ␤-glycerophosphate, 1 mM sodium vanadate and 1 mM phenylmethylsulfonyl fluoride). Flag-HBx, CRTC1-V5 and endogenous CREB proteins were immunoprecipitated from the cleared lysate by incubation at 4 • C for 2 h. For ChIP, HepG2 cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Cells were washed and then lysed in the presence of protease inhibitor cocktail. The DNA-protein Nucleic Acids Research, 2014, Vol. 42, No. 20 12457 complex was immunoprecipitated. The cross-link was reversed by proteinase K. The DNA was purified by phenolchloroform extraction. Promoter sequence spanning the two CRE enhancers in the preS2 promoter was analyzed by quantitative PCR using primers 5 -CCCTGCTCCG AATATTGCCT C-3 and 5 -ACACACGGGT GATCCC-CCTA G-3 . For cccDNA ChIP, quantitative PCR was performed with primers 5 -CTCCCCGTCT GTGCCTTCT-3 and 5 -GCCCCAAAGC CACCCAAG-3 that selectively amplify cccDNA as previously described (7, 12, 29) .