Systematic Identification of cis-Regulatory Sequences Active in Mouse and Human Embryonic Stem Cells (original) (raw)
Figure 4
The Predicted Regulatory Motifs Are Sequence-Specific, Active in Both Mouse ES and EG Cells, and Act as Functional Enhancers of the Endogenous Eed Promoter
(A) Mutations in the predicted regulatory motifs. Sequences containing the predicted regulatory motifs (depicted in red) flanked by endogenous sequences are shown on the left. Sequences containing point mutations (depicted in blue) are shown on the right.
(B) Regulatory activity of the predicted motifs and their mutated counterparts in mouse ES cells. Data were collected and analyzed as described in Figure 3B. Representative results from five independent experiments are shown. Bars represent averages of triplicates performed in each single experiment. Error bars depict standard deviation. Wild-type sequences, red bars; mutated sequences, blue bars.
(C) Regulatory activity of predicted motifs and their mutated counterparts in mouse EG cells. Data were collected and analyzed as described in Figure 3B. Representative results from two independent experiments are shown. Bars represent averages of triplicates performed in each single experiment, error bars depict standard deviation. Wild-type sequences, red bars; mutated sequences, blue bars.
(D) Activity of regulatory motifs 2 and 6 present in Eed upstream genomic sequence. A 1.7-kb fragment of Eed upstream genomic sequence (bp −1,605 to +109 relative to the transcription start site) was cloned and fused to the Firefly luciferase reporter gene (Eed). Mouse ES cells were transfected, and the activity of the Eed construct was compared to the activities of the construct containing luciferase reporter gene alone (ctrl), a TK-bearing construct containing the Oct4 DE (Oct4), an Eed construct containing four point mutations in motif 2 (Eed 2M), and an Eed construct containing four point mutations in motif 6 (Eed 6M). Data were collected and analyzed as described in Figure 3B. Results from two independent experiments are shown. Bars represent averages of triplicates performed in each single experiment; error bars depict standard deviation. Wild-type sequences, red bars; mutated sequences, blue bars. The same mutations also significantly reduce activity of the Eed promoter in EG cells (unpublished data).