A Histone Methyltransferase Modulates Antigenic Variation in African Trypanosomes
Figure 1
Disruption of DOT1B Results in Derepression of Silent VSGs
(A) Schematic representation of PN221 genetic background, in which the actively transcribed BES1 is tagged with a PUR and the silent BES17 is tagged with NEO. VSG221 (MITat1.2) and VSG13 (MITat1.13) genes are located ∼50 kb downstream of the promoter (arrow) and ∼1 kb upstream of the telomere (circle).
(B) Transcript quantification by quantitative RT-PCR of WT PN221 and two ΔDOT1B PN221 bloodstream clones, grown in the presence of 1 μg/ml puromycin. VSG224 (MITat1.3), VSG800 (MITat1.18), VSG VO2 (MITat1.9), VSG13, and NEO are genes present in silent BESs, whereas VSG221 is actively transcribed in BES1. PAG3 is present in one procyclin locus, which is transcriptionally downregulated in bloodstream forms. β-Tubulin genes are present in a tandem array that is transcribed by RNA polymerase II. Actin was used as the reference gene. Error bars indicate the standard deviation of sextuplicates of the same cDNA sample.
(C) Relative enrichment of ΔDOT1B transcripts relative to the wild-type parental reference (average of four independent measurements). When column height is >1, ΔDOT1B transcript levels are higher than those in parental PN221.