Figure 4

TR binds to the putative TREs in Dot1L gene in vitro. (A) Schematics diagram of the putative TREs in Xenopus tropicalis Dot1L gene. The TREs are shown as white boxes with arrows indicating the orientation of TREs. The black box shows exon. (B) The sequences of wild type and mutant Dot1L TRE1 and TRE2 used in the gel shift assay in comparison to the consensus TRE and the TRE of Xenopus laevis TRβA gene. Bold letters show conserved nucleotides and the mutated nucleotides are underlined. (C) Both Dot1L TRE1 and TRE2 compete against the TRE of Xenopus laevis TRβA for binding to TR/RXR heterodimers. The labeled Xenopus laevis TRβA promoter TRE was mixed with in vitro translated TR/RXR heterodimers in the presence or absence of 4×, 20×, or 100× unlabeled wild type or mutant Dot1L TRE1 or TRE2 as indicated. The reaction mixture was analyzed by gel retardation assay. Arrowhead indicates the TR/RXR-TRE complex. Note that both wild-type Dot1L TREs competed while the mutations reduced or abolished their ability to compete for binding to TR/RXR. (D) Dot1L TRE2 has higher affinity than TRE1 for TR/RXR heterodimer in vitro. Gel mobility shift assay was done as in (C) except with 20× unlabeled Xenopus laevis TRβA TRE, Dot1L TRE1, and Dot1L TRE2. Note that Xenopus laevis TRβA TRE competed most effectively, followed by Dot1L TRE2, while Dot1L TRE1 was least effective.