KLF15 knockdown also reduced the HBV DNA level in the serum (Fig. 7C). Similar to HBsAg profiles, this reduction effect was more prominent with 50 than with

30 μg of KLF15 RNAi construct. To further confirm the effect of KLF15 on HBV replication, we generated an HBV genome with the CPm2 mutations that abolished the stimulatory effect of KLF15 on the core promoter (Fig. 2D). The replication efficiency of this HBV mutant plasmid in mice was then compared with that of the wild-type plasmid by hydrodynamic Alisertib molecular weight injection. As shown in Fig. 8, mice injected with the mutant genome had significantly lower levels of viral DNA in the sera than those injected with the wild-type genome (Mann-Whitney U = 27.0, P = 0.030, two-tailed). These results demonstrated the importance of the KLF15 response element in the core promoter in HBV replication. In this study, we demonstrated that the transcription factor, KLF15, could activate HBV major surface and core promoters (Figs. 1 and 2). The overexpression of KLF15 in hepatoma cell lines increased, whereas the suppression of KLF15 expression

with RNAi reduced, the activities of HBV surface and core promoters (Fig. 4). Consistent with these results, EMSAs and ChIP assays showed that KLF15 could bind to core and surface promoters (Fig. 5). The role of KLF15 in HBV gene expression was also confirmed in vivo using a mouse model, as we demonstrated that RNAi knockdown of KLF15 expression in the mouse liver could lead to a significant reduction in the expression of HBV core protein and HBsAg (Figs. 6 and 7), as well as HBV DNA copy number in mouse sera (Fig. 8). Therefore, KLF15 is important for modulating HBV gene expression Ivacaftor order and viral load. By performing MCE mutagenesis

studies, we demonstrated that mutations in the two Sp1-binding sites in the surface promoter (i.e., the Z1/Z2 mutant) could reduce the transactivation effect of KLF15 on this promoter (Fig. 1C). This observation is consistent with our ChIP assay results, which showed that these mutations reduced the binding of KLF15 to the surface promoter (Fig. 5F). Because the mutations in the Sp1 sites reduced, but did not abolish, the binding of KLF15 to the surface promoter, it is likely that the KLF15 binding sites partially overlap with the Sp1 sites. The possibility that there are cryptic KLF15 sites elsewhere in the surface promoter cannot be ruled out, at present. Interestingly, however, results from the ChIP assays showed that mutating the CCAAT site did not affect KLF15 binding to the surface promoter (Fig. 5), but yet it abolished the effect of KLF15 on this promoter (Fig. 1C). It is conceivable that KLF15 needs to cooperatively interact with NF-Y, which binds to CCAAT,12 to exert its effect on the S promoter. Using similar approaches, we also found that mutations in the consensus KLF15 sequence in the core promoter could abolish the effects of KLF15 on the core promoter (Fig. 2C and D).