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Evolution of a New Function by Degenerative Mutation in Cephalochordate Steroid Receptors

Figure 5

BfER repression of BfSR by competition for EREs.

A) BfER's DNA-binding domain recognizes an estrogen response element (ERE). A fusion of the BfER-DBD with a constitutive activation domain activates transcription of an ERE-driven luciferase reporter gene. Negative control (pCMV-AD vector only) and positive control (HsER, human ERα DBD) are shown for comparison. Replacement C205A in the BfER-DBD abolishes recognition of an ERE. B) Neither the BfER-DBD nor the BfSR-DBD recognize steroid response elements (SRE). Negative control (pCMV-AD vector only) and positive control (HsGR, human GR DBD) are shown for comparison. C) BfER and BfSR bind directly to EREs. An electromobility shift assay shows that each receptor binds to a biotin-labeled ERE and migrates as expected (BfER, 521 amino acids, ∼58 kD; BfSR, 616 amino acids, ∼68 kD). When the two receptors are co-transfected, ERE-binding by the BfER homodimer is so strong that it is unclear whether BfER and BfSR heterodimerize. D) Mutation C205A, which compromises DNA binding, abrogates BfER's capacity to repress BfSR-mediated transcription of an ERE-driven reporter. Full-length BfSR, wild-type BfER (BfER-WT), and the BfER C205A mutant were cotransfected. Cells were treated with no hormone (white bars) or 1 µM estradiol (black bars).

Figure 5