While many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly co-expressed genes, some of which are restricted to a single lineage, but most are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.

In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, which recruit much of the cell’s transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits it’s H3K27 acetylase binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, suggesting a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells.

SUMMARY The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3 and RUNX1. We show that TAL1 forms a positive interconnected auto-regulatory loop with GATA3 and RUNX1, and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Summary BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, while expression of the erythroid regulator GATA1 directs SMAD1 loss on non-erythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.

Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.Supplemental material is available at http://www.genesdev.org.Received September 16, 2008; revised version accepted November 4, 2008. Chromosomal translocations involving the mixed-lineage leukemia gene (MLL) are a frequent occurrence in human acute leukemias of both children and adults (Eguchi et al. 2005). In over half of all infant acute leukemias, the MLL protein fuses to one of >50 identified partner genes, resulting in a MLL fusion protein that acts as a potent oncogene (Krivtsov and Armstrong 2007). While extensive gene expression signatures have been determined for primary human leukemia samples (Armstrong et al. 2002;Yeoh et al. 2002;Ferrando et al. 2003;Ross et al. 2003;Rozovskaia et al. 2003;Haferlach et al. 2005), the direct genomic targets of MLL fusion proteins remain unknown. This information is essential to determine how MLL fusion proteins impose oncogenic transcriptional programs and to identify targets for therapeutic intervention in human disease.Distinct chromatin-modifying complexes and histone modifications are associated with distinct phases of transcription (Li et al. 2007). The trithorax group proteins, including MLL, catalyze histone H3-Lys-4 trimethyl (H3K4me3) modifications at the start sites of transcriptionally engaged genes (Ruthenburg et al. 2007). These H3K4me3-modified regions are largely constrained to the transcription start site regions of genes that are transcriptionally initiated, but not necessarily fully transcribed (Bernstein et al. 2006;Barski et al. 2007;Guenther et al. 2007). As a gene becomes fully transcribed, elongating RNA Polymerase II (Pol II) molecules proceed through gene coding regions along with associated elongation factors including DOT1, which catalyzes dimethylation of histone H3-Lys-79 (H3K79me2) (Li et al. 2007). Physical interactions between the most common MLL partner proteins and transcriptional elongation components suggest that defects in H3K4 and H3K79 methylation might be a key factor in MLL leukemogenesis In order to define the portion of gene regulatory circuitry that is controlled directly by MLL fusion proteins in human leukemia, we determined the binding patterns of an MLL fusion protein and chromatin modifications across the entire human genome. We performed this mapping in leukemic cells harboring the MLL-AF4 fusion gene, because this rearran...