PfSir2 Paralogues Directly Regulate var Gene Promoters and Contribute to Spreading of Transcriptional Silencing into Neighbouring Regions
(A) Plasmids bearing two expression cassettes were introduced into 3D7, ΔPfSir2A, and ΔPfSir2B parasites. The bsd cassette is driven by the housekeeping hsp86 promoter, which provides a selection mechanism for parasites stably maintaining episomes. UpsB and UpsC var promoters were placed in front of the selectable marker hdhfr and therefore var promoter activity can be monitored. All values plotted were normalised against the var promoter in its activated state.
(B) PfSir2A and B paralogues differentially affect the silencing of isolated var promoters. hdhfr transcript level was measured in 3D7 parasites with silenced and activated episomally located UpsB and UpsC promoters as well as the level of hdhfr transcription in the absence of PfSir2 paralogues. Both PfSir2 paralogues can directly affect the activity of a var promoter strongly suggesting that PfSir2 paralogues do not function independently of one another on different var promoters but rather cooperate to regulate expression. In both subtelomeric and internally derived var promoters isolated from their natural context PfSir2B has a larger effect on the suppression of var promoter activity.
(C) PfSir2A and PfSir2B control the spreading of silencing from var promoters. Upon activation of the downstream var promoter activity of bsd expression cassette increases suggesting that factors that normally silence var gene promoters can spread in cis to modulate expression of neighbouring regions. In the absence of both PfSir2A and PfSir2B spreading of silencing is reduced suggesting that both paralogues mediate the spreading of silencing from both internal and subtelomerically derived var gene promoters. In both cases PfSir2B has a greater influence on spreading of silencing to within the bsd cassette. qPCR was performed on three biological replicates harvested from ring-stage parasites. 95% confidence error bars derived from a calculated standard error are also displayed.