Functional analysis of wild-type and mutant CCAAT boxes of the TERC promoter. (A) Gel shift and supershift assays. Gel shift assay was performed with HeLa nuclear extract with wild-type probe (wt, 5′-Bio/cttggccaatccgtgcggtcgg-3′), a mutant probe (mt1, 5′-Bio/cttgggcaatccgtgcggtcgg-3′), and additional mutant control probes (mt2, 5′-Bio/cttggagtctccgtgcggtcgg-3′; mt3, 5′-Bio/cttggccattccgtgcggtcgg-3′); bold and underlined letters indicate mutated nucleotides. Anti-NF-YA antibody was used for supershift assay. Arrows show bands that were shifted and supershifted with the wt probe but not with the mt1 probe (−58C>G) or additional mutant control probes (mt2 and mt3). (B) A 200-fold molar excess of unlabeled mutant competitor (mt1, mt2, or mt3) did not compete with wild-type binding, whereas wild-type competitor did (indicated with an arrow). Mutant promoter activity in HEK293T (C) or HeLa cells (D) was reduced compared with wild-type in reporter gene assays. Depicted on the x-axis is the 5′ position from the transcriptional start site of TERC for each TERC promoter-luciferase construct; each construct ended at nucleotide +69. Luciferase activity is reported as relative fold increase compared with the empty vector pGL4.18[luc2P/Neo] (given an arbitrary value of 1), normalized to protein concentration. Results shown are means of 3 (HEK293T) or 1 (HeLa) independent experiment performed in duplicate. Error bars indicate SEM. Detailed methods that include sequences of primers and probes used in the present study will be provided on request.