PMC

Epigenetic signature of MLL target genes. (a) Distribution of expressed genes (RPKM>0.5), silent genes, MLL-AF9 and MLL WT target genes. (b) Overview of AF9, MLL, H3K79me2, H3K27ac and H3K4me3 binding and transcriptional activity at the ZEB2 locus in THP-1 cells. (c) Average signal of H3K27ac, H3K4me3 and H3K79me2 at MLL-AF9 (top), and MLL WT (middle) target genes, as compared with a random set of expressed genes (bottom) (d) Expression levels of MLL-AF9 and MLL WT target genes. ***P<0.001 (Welch’s t-test). (e) Motif families enriched over background in MLL-AF9 target gene promoters (top left), MLL WT target gene promoters (top right) and motif families in MLL-AF9 target gene promoters enriched over MLL WT target gene promoters (bottom).

Comparison of MLL-AF4 and MLL-AF9 target genes. (a) Overview of AF4, MLL, H3K79me2, H3K27ac and H3K4me3 binding and transcriptional activity at the ZEB2 locus in MV4-11 cells. (b) Genomic distribution of MLL-AF4 and MLL WT binding events in the ‘broad’ and ‘sharp’ modes. (c) Expression levels of MLL-AF4 and MLL WT target genes. ***P<0.001 (Welch’s t-test) (d) Distribution of MLL-AF9-specific and MLL-AF9 and MLL-AF4 common target genes (top). Distribution of MLL WT target genes in THP-1 and MV4-11 (bottom). (e) Average signal of H3K4me3, H3K27ac and H3K79me2 on MLL-AF4 and MLL-AF9 common and specific target genes. (f) Expression level of MLL-AF9 target genes shared (+) or not shared (−) by MLL-AF4 in THP-1 and MV4-11 cells (left). Expression level of MLL-AF4 target genes shared (+) or not shared (−) by MLL-AF9 in THP-1 and MV4-11 cells (right). ***P<0.001 (Welch’s t-test).

Characterization of MLL-AF9-bound distal regulatory elements. (a) Average signal of RUNX1 and CTCF on MLL-AF9 and MLL WT target genes. (b) Rate of co-occupancy of MLL-AF9 and MLL WT target genes by RUNX1 and CTCF (left). Expression level of MLL-AF9 target genes grouped by RUNX1 co-occupancy. ***P<0.001 (Welch’s t-test) (right). (c) Average signal on MLL-AF9 (left) and MLL WT (right) bound enhancers for H3K4me3 and H3K27ac (top), MLL and AF9 (middle) and RUNX1, CTCF and H3K79me2 (bottom). (d) Genomic distribution of MLL-AF9 and MLL WT enhancers (top left). Co-occupancy of MLL-AF9 and MLL WT bound enhancers by CTCF, H3K79me2 and RUNX1 z(top right). Expression levels of MLL-AF9 and MLL WT intergenic enhancers (bottom left). Expression level of MLL-AF9-bound enhancers grouped by H3K79me2 co-occupancy. *P<0.05 (Welch’s t-test). (e) Motif family enrichment for MLL-AF9-bound enhancers. (f) Overview of HOXA locus in THP-1 cells showing alignment of AF9, MLL, H3K79me2, H3K4me3, H3K27ac, RUNX1 and RNA expression signal with the HOXA TAD boundary (gray box). (g) Pathway enrichments of active genes nearest to an MLL-AF9-bound enhancer within the same TAD. (h) Long range interactions from the BCL2 and PHLPP1 promoters as measured by 4C-seq in THP-1 cells (black bars, q<0.01) aligned with MLL, AF9 and H3K27ac ChIP-seq patterns on MLL-AF9-bound enhancers (gray boxes). Arrows highlight examples of interactions of the baited promoters with MLL-AF9-bound enhancers.

Gene expression levels of MLL-AF9 patient blasts as compared with CD34⁺ cells, monocytes and AML blasts. (a) Distance-based clustering on the expression of all hg19 refSeq genes. (b) PC analysis on the expression of MLL-AF9 target genes. (c) Pathway enrichments for MLL-AF9 target genes in PC1 (top left) and PC2 (top right). Expression levels of MLL-AF9 target genes in PC1 (bottom left) and PC2 (bottom right). (d) Distribution of differentially expressed AP2, C2H2-Zf, ETS, NR, POU and T-box TFs. Green dots: Benjamini–Hochberg-adjusted P-value<0.01 and fold change >4; orange dots: Benjamini–Hochberg-adjusted P-value<0.05; red dots: fold change >1. (e) Euclidean distance clustering of 74 TFs expressed significantly different in MLL-AF9 cells versus monocytes, CD34⁺ and MLL-AF4 cells. (f and g) Mean RPKM of MLL-AF9 AML high (f) and low (g) expressed TFs in CD34⁺ cells (n=3), THP-1 (n=2), MV4-11 (n=1), monocytes (n=3), MLL-AF9 blasts (n=5), AML-ETO blasts (CGA, n=7), CBFß-MYH11 blasts (CGA, n=11), MLLr blasts (CGA, n=11), PML-RAR blasts (CGA, n=16) and other AMLs (CGA, n=134).

Genome-wide binding patterns of MLL and AF9 in THP-1 cells. (a) Schematic representation of MLL, AF9 and MLL-AF9. Antibody binding locations are indicated with dotted lines, primer regions used in panel (b) with a filled line. (b) Reverse transcriptase–qPCR experiments (n=5) in THP-1 cells with primers against the C and N termini of MLL and AF9 normalized to GAPDH. The N-terminus of AF9 is not expressed, indicating that there is no WT expression of AF9 in this cell line. ***P<0.001 (Welch’s t-test). (c) ChIP-qPCR experiments using two anti-MLL-1 and two anti-AF9 antibodies in THP-1 cells and primers for HOXA7, 9, 10 and MEIS1. (d) ChIP-seq overview of MLL and AF9 binding at the HOXA, ZEB2 and CDKN2C loci in THP-1 cells. (e) Classification of MLL and AF9 binding events in ‘broad’ and ‘sharp’ modes. Left: boxplot showing dispersion of peak lengths. Right: barplot showing genomic distributions. (f) Classification of MLL-AF9 and MLL WT binding events. Average profiles showing ChIP-seq signal intensities for MLL-AF9 and MLL WT binding events in THP-1 cells. (g) Left: Distribution of MLL-AF9 and MLL WT binding events in the ‘broad’ and ‘sharp’ modes. Right: Genomic distribution of MLL-AF9 and MLL WT binding events in the ‘broad’ and ‘sharp’ modes. (h) Venn diagram illustrating the overlap between our human (THP-1) MLL-AF9 AML targets, MLL-AF9 targets in a mouse LSC model (Bernt et al.) and human MLL-AF4 ALL targets (Guenther et al.).