Figure 3

Nanog, Ets1 and Gata4 interact with the functional domain of the Ens LTR promoter in cES cells. (A) Binding sites for Gata, Nanog, Ets and Churchill transcription factors in the functional domain of the Ens-1 LTR are represented in bold and upper characters. The mutations used in Figure 1 are indicated by underline and italic characters. (B) Real time PCR analysis performed on cDNA from cES induced to differentiate 48 h with retinoic acid. Expression levels of the indicated transcripts are represented as percentages of the levels obtained in undifferentiated cES. The numbers that are underlined identify the member of the Gata or of the Ets transcription factors family indicated below. Means are from three experiments +/- s.d. For Gata3 s.d. is +/- 1097. T test is relative to the values obtained in undifferentiated cells: *p < 0.05, **p > 0.05. (C) Chromatin-immunoprecipitation of the Ens-1 LTR promoter with anti-flag antibodies in cES transfected with expression vectors encoding for Nanog, Gata4 or Ets1 proteins in fusion with a flag tag. cES transfected with untagged transcription factors were used as negative controls. Results obtained with the irrelevant transcription factor YY1 in fusion with a flag tag are also represented. Results represent the fold enrichment compared to cells transfected with empty vector. The non-specific binding of the tagged transcription factors on upstream and on downstream regions is represented as well as the non specific interaction of the untagged transcription factors with the binding domain. Means of at least two experiments +/- s.d. are shown. T test is relative to the values obtained with the tagged YY1 transcription factor, *p < 0.05, **p > 0.05.