Promoter binding of NF-κB p65 subunit in <i>C. parvum</i>-induced transactivation of miRNA genes in H69 cells.

Expression of pri-miRNAs corresponding to C. parvum-upregulated mature miRNAs was quantified by real-time PCR. These showed significant upregulation in cells following C. parvum infection were presented as “+” and those without significant increase were presented as “−”. Effects of NF-κB inhibitor SC-514 on C. parvum-induced upregulation of pri-miRNAs were also assessed by real-time PCR and presented as “+” (if the inhibitory effect was significant) and “−” (if not significant). Putative promoter element for each miRNA gene was either based on previous studies as referred in Table 1 or identified by RACE-PCR in this study (indicated by asterisks). Potential NF-κB binding site(s) in the promoter region was identified by the TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) and MOTIF (http://motif.genome.jp/). Positive promoter binding of p65 subunit to the predicted NF-κB binding site was confirmed by ChIP analysis and marked as “+”; otherwise marked as “−” if no p65 binding was detected. C. parvum-induced transactivation of miRNA gene by p65 was further confirmed by using luciferase reporter gene constructs that spanned the promoter region of each individual gene. If C. parvum infection increased luciferase activity in cells transfected with the luciferase constructs containing the binding site for p65 and this induction was blocked by SC-514, it was presented as “+”; otherwise presented as “−”. NS = not selected for further ChIP analysis or luciferase reporter assay in this study. In such cases, these genes are most likely not regulated by p65 promoter binding because their transactivation was not induced by C. parvum infection or the induced transactivation was not inhibited by SC-514 as assessed by real-time PCR. Refer to Figures S4, S5, S6 and S7 for details.