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. Author manuscript; available in PMC: 2022 Mar 18.
Published in final edited form as: Mol Cell. 2021 Aug 5;81(17):3604–3622.e10. doi: 10.1016/j.molcel.2021.07.018

Figure 5. Genome-wide binding profiles reveal the co-occupancy of ZMYND8 and BRD4 in active enhancer regions.

Figure 5.

(A) Meta-profile (top) and density plot (bottom) of CUT&RUN peaks at 13,125 ZMYND8-occupied regions in MOLM-13 cells. Peaks are ranked by ZMYND8 CUT&RUN tag counts.

(B) Pie chart annotating the distribution of 13,125 ZMYND8 peaks in MOLM-13 cells. TTS, transcription termination site. Other, UTR and non-coding RNA regions.

(C) ZMYND8 CUT&RUN-derived de novo motif analysis in MOLM-13 cells using HOMER. Statistical analysis (p value) was calculated using the binomial test.

(D) Venn diagram displaying CUT&RUN peak overlap between ZMYND8, BRD4, and H3K27ac occupancy in MOLM-13 cells.

(E) Gene tracks of H3K27ac, H3K14ac, BRD4, and ZMYND8 enrichment with 4C-seq analysis at leukemic MYC enhancer locus (ME1-ME5, gray box) in MOLM-13 cells. 4C-seq was performed using MYC promoter as the “viewpoint.”

(F) Top: schematic of dCas9-KRAB-mediated epigenomic silencing. Locations of different sgRNAs targeting H3K27ac-enriched regions +23–86 kb from the IRF8 TSS are shown by red lines. Bottom: gene tracks of H3K27ac, H3K14ac, BRD4, and ZMYND8 enrichment in addition to 4C-seq analysis at the IRF8 locus in MOLM-13 cells. Putative IRF8 enhancer is labeled in a gray box. IE, IRF8 enhancer.

(G) RT-qPCR analysis of mRNA expression of IRF8 in dCas9_KRAB+ MOLM-13 cells transduced with indicated sgRNAs in Figure 5F and harvested after 5 days post-infection. sgIRF8_TSS (purple) targets the IRF8 TSS region. Effective sgRNAs that induce >2-fold downregulation of IRF8 are labeled in green. Relative mRNA levels were normalized to GAPDH levels. sgNeg, negative control; TSS, transcription start site. Plotted are the mean ± SEM (n = 3).

(H) Competition-based proliferation assays performed in dCas9_KRAB+ MOLM-13 cell lines. Cells were transduced with sgNeg (n = 4) or sgIRF8_TSS, sgIR-F8_enh-2, −3, −5, or −9 (n = 2).

(I) Leukemic MYC enhancer (left, ME1-ME5, gray box) or IRF8 enhancer (right, IE, gray box) region in indicated cell lines. H3K27ac ChIP-seq data in THP-1, HEL, and HUH7 cells were extracted from GSE109493, GSE123872, or GSE89212.

Error bars represent mean ± SEM. See also Figure S5.