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. 2020 Mar 16;215(1):89–101. doi: 10.1534/genetics.120.303144

Figure 6.

Figure 6

Promoter-proximal BEAF facilitates local and long-range interactions between Sry-δ and promoters, and directly activates a housekeeping promoter. (A) Schematic of constructs used to drive firefly luciferase expression in transfected S2 cells. All transfections also had a plasmid with an Act5C promoter driving Renilla luciferase expression to normalize for transfection efficiency, with or without a plasmid with an Act5C promoter driving expression of Sry-δ or Sry-δ-VP16 (endogenous Sry-δ is expressed in S2 cells). Transfections with each set of plasmids were further normalized to firefly luciferase expression when only the BEAF-binding site was present. Error bars indicate the SD of three biological replicates. Test plasmids had either a minimal y (developmental) or RpS12 (housekeeping) promoter. Also shown is a model of Sry-δ interacting with BEAF to facilitate long-range activation of the promoter. The y promoter was tested (B) without an Sry-δ-expressing plasmid; (C) with an Sry-δ plasmid; and (D) with an Sry-δ-VP16 plasmid. Average luminometer readings for BF.y were 102,245 compared to a mock transfection background of 99. The RpS12 promoter was tested (E) without an Sry-δ-expressing plasmid; (F) with an Sry-δ plasmid; and (G) with an Sry-δ-VP16 plasmid. Average luminometer readings for BF.R were 1,145,492 compared to a mock transfection background of 102. (H) Testing the aurA promoter with and without mutations in the BEAF-binding site. Average luminometer readings for aurA were 924,577 compared to a mock transfection background of 122. In (B–G), comparison of the BF, ppTF.mBF, and ppTF.BF values show local interactions between Sry-δ and BEAF cooperatively activate the reporter gene, while comparison of the BF, pdTF.mBF, and pdTF.BF values indicate long-range interactions between Sry-δ and BEAF activate the reporter gene. In (B–D), comparisons of mBF and BF show that BEAF does not activate the y promoter. In (E) through (H), comparisons of mBF and BF show that BEAF activates the RpS12 and aurA promoters. BEAF, Boundary Element-Associated Factor; BF, promoter-proximal wild-type BEAF-binding site; mBF, promoter-proximal mutant BEAF-binding site; pdTF, promoter-distal four tandem Sry-δ TF-binding sites; ppTF, promoter-proximal four tandem Sry-δ TF-binding sites; prom., promoter; Sry-δ, Serendipity δ; TF, transcription factor.