Figure 3.

Wild-Type BRG1 Significantly Enhances TR Transcription in a Dose-Dependent Manner, whereas a Mutant BRG1 with a Mutation in the ATPase Domain Inhibits TR Transcription

The mRNA for TR (1.15 ng/oocyte) was injected into the cytoplasm of 20 oocytes together with RXR mRNA (1.15 ng/oocyte) and either the Flag-tagged BRG1 (F-BRG1) or its mutant (F-BRG1mut) mRNA (1.15 ng/oocyte or 3.5 ng/oocyte), as indicated. Subsequently, the TRE-Luc firefly luciferase reporter vector and phRG-Tk Renilla luciferase control vector was coinjected into the nucleus of the oocytes. The oocytes were incubated overnight with (lanes 7–11) or without (lanes 1–6) 50 nm T₃. The oocytes were lysed and subjected to dual-luciferase assays. The relative activity of the reporter vs. that of the control was plotted with the basal activity set to 100 (lane 1). The average from five oocytes was plotted together with the se. Note that although we were not able to detect F-BRG1 or F-BRG1mut by Western blot (not shown), their effects were mRNA dose dependent, consistent with the prediction that more proteins were synthesized when more mRNA was injected. Also, although overexpression of BRG1 or its mutant appeared to reduce the transcription further in the absence of T₃ (lanes 3–6), the effect was variable and not significant because the absolute luciferase activity was very low and thus relatively more variable in the presence of unliganded TR/RXR (see text and Fig. 7B).