Fig. 3: MLL-AF4 binding recruits transcription elongation factors to enhancers.

a Mean distribution of ENL and MENIN at MLL-AF4-bound and non-MLL-AF4-bound intergenic (solid line) and intragenic (dashed line) enhancers in SEM cells. b Schematic of wild-type N-terminal MLL structure, showing the domains used for immunoprecipitation. c Colloidal Blue-stained gel of control HEK-293 nuclear extracts (1) or HEK-293 nuclear extracts expressing the FLAG-C-PHD construct (2), immunoprecipitated with anti-FLAG antibody. Gel lanes were sliced and subjected to mass spectrometry (see “Methods”). Regions where the proteins HCFC1 and FACT complex component SPT16 were identified are indicated by arrowheads. The red arrowhead indicates the position of the FLAG-C-PHD protein. Image represents a single replicate used for MS. Source data are provided as a Source Data file. d Immunoblots for HCFC1, CTR9 (PAF1C component) and SPT16 following anti-FLAG immunoprecipitation of HEK-293 cell lysates expressing the indicated FLAG-tagged MLL domains. Representative of three experiments. Source data are provided as a Source Data file. e Silver-stained gel after affinity purification of GST-tagged MLL RD1 and RD2 domains following incubation with purified SPT16 and SSRP1 (FACT). Lower panel shows immunoblot for SPT16. Representative of two experiments. Source data are provided as a Source Data file. f Mean distribution of PAF1 at MLL-AF4-bound and non-MLL-AF4-bound intragenic and intergenic enhancers in SEM cells under control (NT; solid line) and 96 h MLL-AF4 knockdown (dashed line) conditions. g Mean distribution of PAF1C component LEO1 and FACT component SSRP1 at MLL-AF4-bound and non-MLL-AF4-bound intergenic (solid line) and intragenic (dashed line) enhancers in SEM cells. h ChIP-seq, ATAC-seq and Capture-C at the FLT3 locus in SEM cells. Reference-normalized ChIP-seq for PAF1 in SEM cells under control (NT) and 96 h MLL-AF4 knockdown conditions. The Capture-C viewpoint is the FLT3 TSS. i TOPmentation and ATAC-seq at the FLT3 locus in chALL patient #3. j Schematic showing the principle behind the TetR recruitment system. k ChIP-qPCR for the indicated proteins (left) at the TetO array inserted into mESCs expressing the indicated TetR fusion proteins (top). Dashed line shows ChIP-qPCR in cells treated with doxycycline for 6 h. Data are represented as mean ± SEM, n = 4 independent experiments for TetR FS2 in TetR, TetR FS2 in TetR-CXXC, TetR FS2 in TetR-Paf1, Enl in TetR, Enl in TetR-ENL, Paf1 in TetR, Paf1 in TetR-PAF1; n = 3 independent experiments for TetR FS2 in TetR-ENL, TetR FS2 in TetR-DOT1L, Enl in TetR-CXXC, Enl in TetR-Paf1, Paf1 in TetR-CXXC, Paf1 in TetR-ENL, Ssrp1 in TetR, Ssrp1 in TetR-CXXC, Ssrp1 in TetR-ENL, Ssrp1 in TetR-PAF1 and n = 2 independent experiments for Enl in TetR-DOT1L, Paf1 in TetR-DOT1L, Ssrp1 in TetR-DOT1L. Source data are provided as a Source Data file. l Model indicating direct or indirect in vivo interactions demonstrated in (k).