Figure 2. p300 compensates for the loss of BRD4 to rescue transcription after BET inhibition.

A.-C. Tornado plots of BRD4 and p300 binding differences between BETi and DMSO conditions in KASUMI-1, SKNO1 and OCI-AML3 cells (24h treatment). Positive enrichment (in red) shows stronger binding upon BETi. Negative enrichment, colored in blue, shows loss of binding upon BETi. Shown are results from representative matched pairs (color-tri: red = positive enrichment upon BETi treatment; white = no change; petrol blue = negative enrichment upon BETi treatment). With the exception of BRD4 ChIPSeq in KASUMI-1, which was performed once per condition, all experiments were performed with 2 biological replicates per condition.

D. Average binding curve profiles for p300 at the top 5% rescued sites in the indicated cell lines. Upper panels show binding at the 5% best-scoring sites that were found in KASUMI1, middle panels show binding profiles at the 5% best-scoring sites that were found in SKNO1, lower panels apply the same method for 5% best-scoring sites that were found in OCI-AML3.

E. Examples of BRD4 and p300 binding profiles in DMSO and BETi-treated KASUMI-1, SKNO1 and OCI-AML3 cells, to demonstrate the BETi-triggered increase of p300 binding at/near promoters of the main RUNX1 isoforms.

F. Bar plots of BRD4 (blue bars) and p300 (red bars) log2 fold changes of binding between the BETi and DMSO conditions in KASUMI-1 and SKNO1 cells (24h treatment) at the indicated MYC and RUNX1 regulatory regions. Shown are log2 fold changes of ChIP-qPCR results from 3 biological replicates and SD.

G. Western blot for p300 and GAPDH in SKNO1 cells during a time lapse of 0 (DMSO-treated control), 2, 4, 12 and 24 hr of PROTAC-mediated p300 degradation via deg_p300.

H. Scheme of treatment with BETi for 24 hr and degradation of p300 within the last 4 hr of BETi.

I. Bar plots of p300 binding ratios to IgG in BETi and DMSO conditions with or without the addition of deg_p300 within the last 4 hr of treatment in SKNO1 cells. Shown are ChIP-qPCR results from 3 biological replicates at the RUNX1 promoter and one MYC enhancer and SD.

J. Analysis of qPCR expression of the indicated transcripts/genes in SKNO1 cells after treatment with either BETi (24h of treatment), deg_p300 (4h) or BETi (24h) with the addition of deg_p300 during the last 4h of treatment. Shown are log2 Fold Changes normalized to DMSO-treated controls and SD from 3 biological replicates.

K. Volcano plots showing SLAMSeq expression changes in SKNO1 cells after 24 hr of treatment with DMSO or BETi (same as Fig.S1E) and DMSO or BETi plus the addition of deg_p300 within the last 4h of treatment. Three biological replicates were acquired per condition/time point.

L. (Left panel) SLAMSeq intensity at the 933 rescued genes at 24 hr of BET inhibition with the addition of deg_p300 during the last 4 hr of treatment, to be compared with the values at 0 hr (DMSO-treated controls), 4 hr and 24 hr of BETi treatment in the background. Shown are log2 fold change ratios to 0 hr (DMSO-treated controls). (Right panel) SLAMSeq intensity at the indicated exemplar rescued genes at 24 hr of BET inhibition with the addition of deg_p300 during the last 4 hr of treatment, to be compared with the values at 0 hr (DMSO), 4 hr and 24 hr of BETi treatment in the background. Shown are log2 fold change ratios to 0 hr (DMSO). Three biological replicates were acquired per condition and time point.