Sequence-specific binding between let-7i miRNA and the TATA-box motif facilitates IL-2 PIC assembly and transcription initiation. (A, top) Mutations were introduced into the predicted let-7i–binding site in IL-2 core promoter region (in red). (Bottom) Wild-type and mutated IL-2 promoter–driven reporter constructs were transfected into HEK293T cells with let-7i mimic or negative control. Cells were harvested at 36 h post transfection and the promoter activities were determined by dual-luciferase assay. Up-regulation folds of the promoter activities relative to the control by let-7i were shown. n = 5 biological replicates. (B) A series of mutations were generated in the 5′ seed region, the sequence complementary to IL-2 TATA box, or 3′ region of let-7i miRNA, respectively (mutated nucleotide in red). The wild-type let-7i mimic or these mutated mimics were transfected to HEK293T cells, respectively, with the IL-2 promoter–driven luciferase construct. The IL-2 promoter activities were examined as described above. n = 5 biological replicates. (C) A mutated let-7i mimic was co-transfected with an IL-2 promoter–driven reporter which harbored mutations base-pairing complementary to the mutated let-7i mimic, and promoter activities were measured as described above. n = 5 biological replicates. (D) ChIP assay with antibody against TBP, TFIIA, or Pol II in Jurkat cells expressing GFP only (plko-gfp) or let-7i precursor (plko-let-7i) to examine the IL-2 promoter DNA associated with the general transcription factors. The enrichment of the promoter DNA by the transcription factors was normalized to IgG. These data represent three independent experiments. (E) Nuclear run-on assay of IL-2 promoter activity. The IL-2 promoter–driven luciferase construct was co-transfected into HEK293T cells with let-7i precursor or the empty vector. GAPDH was selected as an input control and mRFP as negative control. This figure represents three independent experiments. (F) Effects of inhibiting Ago1, Ago2, or both genes on the activation of IL-2 promoter by let-7i miRNA mimics. Ago proteins were knockdown with specific siRNAs in HEK293T cells, and the promoter activities of IL-2 in the presence of let-7i or control mimic were measured as described above. n = 3 biological replicates. (G) ChIP experiments were performed with antibody against AGO1 or AGO2 in Jurkat cells expressing GFP (plko-gfp) or let-7i precursor (plko-let-7i) to examine their association with the IL-2 promoter. The plko-gfp cells and plko-let-7i cells were generated as described above. The primers covering -146 to -8 relative to the TSS of IL-2 promoter were used to detect the associated promoter DNA. The enrichments of the promoter DNA by Ago proteins were normalized to IgG. This figure represents three independent experiments. P-values were calculated using the two-tailed unpaired Student's t-test with equal variances. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.
