Fig 2: RARα association to transcribed regions in AF9-or BRD4-independent genes.The response of TTNPB-inducible genes (FC>2 after 4 hours) in P19wt was compared to that in P19Af9(−) or P19Brd4(−) in similar conditions. Genes losing their responsiveness to TTNPB (FC<1.2) in either the P19Brd4(−) background (cluster B), the P19Af9(−)background (cluster C) or both (cluster D) were identified by microarray data analysis. Genes maintaining an inducibility similar to that observed in P19wt in either the P19Af9(−) or the P19Brd4(−) background were grouped in Cluster A. Genes in each cluster were searched for the occurrence of RAR binding sites on the basis of RAR ChIP-Seq data carried out in mouse ES cells [42]. Three representative genes were selected from each cluster and their inducibility was validated by RT-QPCR in each condition (n = 3, left inset). RARα and RNApol II association to an upstream region (UR), RAR binding site (RAR BS), transcriptional start site region (TSS) and an exon (Exon) was assessed in independent, duplicate ChIP-PCR assays after a 4-hour challenge of P19wt with TTNPB. Input lanes showed an equal loading but were omitted for space purposes.

Fig 3: Antibody Epitopes Compared to MLL-FP Sequences in KOPN-8 (MLLENL) and THP-1 (MLL-AF9) Cells, Related to Figure 4(A) Fusion sequence between MLL ex8 (yellow) and ENL ex7 (green) in KOPN-8 cells. The breakpoint was determined by sequencing cDNA from KOPN-8 cells.(B) Wild-type ENL protein sequence, sequence in KOPN-8 MLL-ENL fusion (green) and sequence recognized by Bethyl antibody A302-268A (Bold, underline).(C) Fusion sequence between MLL ex8 (yellow) and AF9 ex5 (green) in THP-1 cells, sequence taken from Odero et al. (2000).(D) Wild-type AF9 protein sequence, sequence in THP-1 MLL-AF9 fusion (green) and sequence recognized by Bethyl antibody A300-595A (Bold, underline).

Fig 4: MLL-AF4 Activates the RUNX1 Gene and the RUNX1 Protein Interacts with the AF4-MLL Complex and Activates Gene Targets(A) RUNX1 can interact with either coactivators or corepressors to cause gene activation or repression. In t(4;11) cells, RUNX1 can also interact with the AF4-MLL complex.(B) In t(4;11) leukemias, MLL-AF4 is expressed from one translocated chromosome, and the MLL-AF4 protein binds to and activates the RUNX1 gene by stabilizing AF9 and ENL binding. AF4-MLL is expressed from the other translocated chromosome, and the RUNX1 protein interacts with the AF4-MLL complex and binds to target genes.

Fig 5: In t(4;11) Cells, RUNX1 Is Highly Expressed and Has High Levels of ENL and AF9 Bound to the Locus(A) Real-time PCR quantification (see gene expression analysis in Extended Experimental Procedures) of HOXA9 (top), HOXA10 (middle), and RUNX1 (bottom) gene expression in patient cell lines. The cell lines analyzed are: RS4;11 (t-4;11), SEM (t-4;11), MV4-11 (t-4;11), THP-1 (MLL-AF9), NOMO-1 (MLL-AF9), MONO-MAC1 (MLL-AF9), KOPN-8 (MLL-ENL), ML-2 (MLL-AF6 and an MLL deletion), SHI-1 (MLL-AF6), RCH-ACV (normal MLL), CCRF-CEM (normal MLL), JURKAT (normal MLL), and K562 (normal MLL). Error bars represent the ±SD of two independent experiments. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; N.D., not detected.(B) Western blot of RUNX1 in the cell lines as described in (A) with a short exposure (top panel) and a long exposure (middle panel).(C) Western blot of nuclear extracts in the cell lines indicated and probed with the antibodies as indicated.(D) MLL-N, AF4-C, ENL, AF9, and Cyclin T1 ChIP in RS4;11 (dark red bars), SEM (spotted red bars), MV4-11 (bright red bars), THP-1 (black bars), KOPN-8 (blue bars), and CCRF-CEM (gray bars) patient cell lines. The control primer set is from a random gene-poor region on human chromosome 8; otherwise, primer sets are as indicated in Figures 1B, 1C, and 2A. Error bars represent the ±SD of two independent experiments.See also Figure S3.