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. 2012 Nov 29;109(52):E3678–E3686. doi: 10.1073/pnas.1214572109

Fig. 3.

Fig. 3.

DNA PEs regulate silencing and mutually exclusive expression of var genes through specific promoter–promoter interaction. The deletion of a PE or insertion of an extra PE results in the activation of a var gene. In the schematic of the construct, the TG-rich element is shown as a red circle, the PbDT 3′ UTR is boxed, and the hrp2 3′ UTR is shown as a bold black line. (A) (Upper) Schematic of the pVΔ102LhIDb in which the last 102 bp of the var promoter containing the PE were deleted. (Lower) Schematic of the pVLhPEIDh episome, which is identical to pVLhIDb except that an additional PE is inserted upstream of the var intron. These modifications resulted in activation of the var promoter, which no longer is recognized by the mechanism that controls mutually exclusive expression. (B) Results of pVΔ102LhIDb (Upper) and pVLhPEIDb (Lower) in the DC-J parasite line. Quantification of the ratio between var promoter and intron by qRT-PCR (Left), steady-state mRNA levels of each individual var gene measured by qRT-PCR presented as relative copy number to the housekeeping genes arginyl-tRNA synthetase (PFL0900c) (Center), and the levels of luciferase expression (Right). The D10 parasite line (22) constitutively expressing luciferase from an endogenous var promoter was used as a positive control. (C) The interaction between the two DNA elements is specific to var genes. Schematic of the pHLhIDb (Upper) and pHPELhIDb (Lower) constructs made by replacing the var promoter from pVLhIDb with the hrp2 promoter with or without the last 102 bp of the var promoter that contains the PE. The promoters of both plasmids were constitutively active simultaneously with the endogenous var gene in the DC-J parasite line. (D) Results of pHLhIDb (Upper) and pHPELhIDb (Lower) in the DC-J parasite line. (Left) Quantification of the ratio between var promoter and intron by qRT-PCR. Steady-state mRNA levels of each individual var gene measured by qRT-PCR are presented as relative copy number to the housekeeping genes arginyl-tRNA synthetase (PFL0900c) (Center) and the levels of luciferase expression (Right). The D10 parasite line (22) constitutively expressing luciferase from an endogenous var promoter was used as a positive control. All values presented are the average of at least two biological replicates. Error bars represent SEs.