HoxA9-regulated genes can be identified by nascent RNA-seq. (A) Schematic description of sample generation. HSPCs of WT mice transduced with a tamoxifen (TAM)-inducible HoxA9-ER fusion were used for these experiments. Cells were supplemented for 1 hour with 4-thiouridine at the indicated time points, and labeled RNA was isolated and sequenced to determine actual transcription rates in the presence of functional HoxA9 (0 hours), as well as after cessation of HoxA9 activity. (B) Venn diagram. Activated and repressed genes were defined at 24, 48, and 72 hours by a cumulated log2 fold change in transcription rate >1.5 (transcript up after inactivation of HoxA9 = repressed by HoxA9) or a cumulated log2 fold change in transcription rate less than −1.5 (transcript down in the absence of HoxA9 = activated by HoxA9). The overlap with 165 direct target genes of MLL-ENL, as identified previously,²³  is indicated. (C) The 65-gene HoxA9/MLL-ENL core signature. Heat map illustrates transcript changes 72 hours after inactivation of the respective oncogene. Red hues correspond to active genes, and blue colors designate repressed genes. (D) Gene set enrichment analysis of the HoxA9-on vs HoxA9-off (72 hours) gene-expression signatures. Normalized enrichment scores (NES) and false-discovery rates (FDS) are given.