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Figure 1 | Retrovirology

Figure 1

From: The endogenous retrovirus ENS-1 provides active binding sites for transcription factors in embryonic stem cells that specify extra embryonic tissue

Figure 1

Four domains cooperate to fully control the p455 promoter activity in cES cells. (A) Sequence of the active domain in the p455 luciferase-reporter construct. Deletions performed between positions -179 and -123 of the p455 promoter relatively to the transcription start site are indicated in reduced characters, underlined and called a, b, c, d, e, S1, S2, S3, S4. The sequence of the p40 oligonucleotide used as a probe in EMSA experiments, is bounded above. (B) Luciferase activity from p455 constructs carrying the mutations presented in (A). Mutations inhibiting the activity are designed S1 to S4 while the others are called a to e. All luciferase activities were normalized by co-transfection of cES cells with a CMV-renilla luciferase reporter and results are the means of three independent experiments +/- s.d. Statistics are the results of a t test relatively to the values obtained with the non mutated p455 construct. (C) EMSA were performed with p40 or with the mutated p40 labelled probes (MutS1: GAGCG in place of AGATA; MutS3: AAA in place of GGG) using nuclear extracts from cES cells. (D) EMSA with p40 probe using nuclear extracts from cES cells or from cES cells induced to differentiate for 4 days with retinoic acid (p40 diff). Mut S2 probe (GCA in place of ATG) was used with cES extracts. The position of the DNA-protein complexes x, y and z or the probe used alone are indicated with arrows. For each probe competition, experiments were performed with a 100-fold molar excess of the same unlabelled nucleotide for specific specific binding (s lanes) or an unrelated nucleotide for nonspecific binding (ns lanes). Results are representative of two others.

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