Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3

Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Atarashi, Ryuichiro; Nishida, Noriyuki

Oct-1 is responsible for IRF-3 promoter activity. We identified putative transcription factor binding sites in nt -119 to -1 with the software TFSEARCH ver.1.3 (http://www.cbrc.jp/research/db/ TFSEARCH.html) and found that this region contains a potential Oct-1 binding site (59-ATTTGCAT-39, nt -42 to -35) and an acute myeloid leukemia 1 protein (AML1) binding site (59-TGCGGT-39, nt -49 to -44). In addition, an E2F transcription factor 1 (E2F1) binding site (59-TTTCCCAC-39, nt -116 to -109) was also conserved in murine (Fig. 3a) . To determine whether the Oct-1 site is important for promoter activity, an original and two different mutated plasmids from the Oct-1 site (M1 and M2) were prepared (Fig. 3b ) and the transiently transfected into N2a and 3T3 cells. After 24 h, we evaluated their respective promoter activity by reporter assay. The activities of pGL3 -119/-1 (M1) were also significantly reduced compared with the original promoter activities in N2a and 3T3 cells ( Fig. 3c and 3d ). The activities of pGL3 -1000/-1 (M1) and pGL3 -523/-1 (M1) were also significantly reduced ( Supplementary Fig. S3 ). We obtained similar results in the second mutated plasmid pGL3 -119/-1 (M2) where activity was also significantly reduced in N2a and 3T3 cells ( Fig. 3e and 3f) . These results indicate that the Oct-1 site might play a crucial role for murine IRF-3 promoter activity. We next determined whether the E2F1 site was important for the promoter activity. The original plasmid pGL3 -119/-1 and the E2F1 site mutated plasmid (M3: Fig. 3g ) were transiently transfected into N2a and 3T3 cells. After 24 h, we evaluated their respective promoter activity by reporter assay. The activities of pGL3 -119/-1 (M3) were not significantly different compared to the original plasmid promoter activity in N2a and 3T3 cells ( Fig. 3h and 3i) , indicating the E2F1 is not an important determining factor for the regulation of murine IRF-3 promoter. Since two different mutations in the AML1 site (M4 and M5: shown in Supplementary Fig. S4a ) had little effect on the promoter activity in N2a58 cells (N2a cells overexpress wild-type prion protein), indicating the AML1 is not also important ( Supplementary Fig. S4b ).