Fig 5. Genetic depletion of bromodomain proteins phenocopies treatment with I-BET151.

(A) Left, Chromatin immunoprecipitation and sequencing (ChIP-seq) of four endogenously HA-tagged trypanosome bromodomain proteins showing localization to sites of transcription initiation for three of them. The region shown is a 184 kb region from chromosome 7. Arrows represent direction of transcription for PTUs. Areas where the arrows diverge are sites of transcription initiation. (B) Left, the percent of all MACS-derived peaks lying within 100 bp of a divergent strand switch region (Transcription Start Site, TSS), convergent strand switch region (Transcription Termination Site, TTS), both (a peak that is within a 100 bp of both a TSS and a TTS) or neither, a peak that is >100 bp away from an TTS or TSS. Right, the percent of all transcription start sites within 100 bp of a peak are shown for Bdf1,3,4. No Bdf2 MACS-derived peaks were within the FDR < 0.1 cutoff and are thus not included in this analysis. The total number of peaks called for Bdf1, 3 and 4 are 240, 307, and 175 respectively. Note that “head to tail” sites were not included in this analysis. (C) Isothermal titration calorimetry measuring binding of Bdf2 and Bdf3 to I-BET151. Conserved tyrosine and asparagine residues within the AcK binding pocket are mutated to alanine in the mutant constructs. (D) Volcano plot showing the most significant up-regulated and down-regulated genes in the trypanosome genome from an RNA-seq experiment comparing Dox-treated Bdf3^kd (top) or Bdf2^KO (bottom) to untreated control cells. DESeq was used to normalize counts, compute log₂(fold change) of treated cells over untreated cells, and calculate p-adjusted values. Dots in red indicate genes with fold changes of > 2 and a p-adjusted value < 0.05. (E) Flow cytometry measuring surface EP1 expression in two-d Dox-treated Bdf2^KO (top) or Bdf3^kd cells (middle), or I-BET151-treated cells (bottom) following washout of IBET-151 or Dox, and subsequent exposure to a 24 h treatment with one of two different differentiation triggers: incubation at 27°C (middle) or treatment with cis-aconitate (CA) at 37°C (right) compared to a control without any trigger (left, No stimulus). (F) Flow cytometry measuring the amount of primary anti-VSG antibody remaining on the surface following a 5-min incubation at the indicated temperatures, washout, fixation, and staining with secondary antibody in Dox-treated Bdf2^KO (top panels) and Bdf3^kd cells (bottom panels) compared to controls. Numerical data for Fig 5 is in S5 Data, except wiggle plots shown in Fig 5A, which are in S6 Data.