Human miR-181a-1 promoter is regulated by C/EBPα. (A) C/EBPα binds specifically to a site within the human miR-181a-1 proximal promoter. Nuclear extracts from HEK-293T cells transiently transfected with pcDNA3-FLAG (vect.), pcDNA3-C/EBPα-p30-FLAG (α-p30), or pcDNA3-C/EBPα-p42-FLAG (α-p42) were used in electrophoretic mobility shift assay (EMSA). The radiolabeled probes contained either wild-type predicted C/EBP-binding site, or mutant (mut.) sequences (shown below). Lane labeled “probe” contains binding reaction in the absence of nuclear extract. Where indicated by “+” above the lanes, C/EBPα specific antibody was added to the binding reactions. Solid arrowhead shows protein/DNA complex, whereas open arrowhead indicates binding complex super-shifted with the antibody. Unbound probe is shown on the bottom of the gel (free probe). (B) Relative expression of C/EBPα proteins in nuclear extracts used in EMSA in panel A is demonstrated in Western blot stained with C/EBPα-specific antibody. The p42 and p30 C/EBPα polypeptides are indicated to the right. (C) C/EBPα-dependent transactivation of the miR-181a-1 promoter. Human miR-181a-1 192 bp promoter fragment containing wild-type C/EBP-binding site (boxed sequence) or mutated sequence (indicated below the box) were linked to firefly luciferase gene (black box; diagrammed on top) and transiently transfected to HEK-293T cells with either empty expression vector pcDNA3-FLAG (−), or pcDNA3-C/EBPα-FLAG vectors (p42 or p30). Cell lysates were analyzed for luciferase activity and normalized to cotransfected Renilla luciferase activity. For control, C/EBPα expression vectors were also tested with promoter-less luciferase vector, pGL4-11 (vector). Each bar represents average of 3 transfection experiments and SEM bars are shown. On the right, representative aliquots of cell lysates used for luciferase assay were also analyzed by Western blot to demonstrate comparable levels of C/EBPα protein.