Summary The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1 induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus non-differentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1 bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that Polycomb Repressive Complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1 repressed genes. These data provide insights into GATA-1 mediated gene regulation in vivo.

Coregulator recruitment by DNA-bound factors results in chromatin modification and protein-protein interactions, which regulate transcription. However, the mechanism by which the Friend of GATA (FOG) coregulator mediates GATA factor-dependent transcription is unknown. We showed previously that GATA-1 replaces GATA-2 at an upstream region of the GATA-2 locus, and that this GATA switch represses GATA-2. Genetic complementation analysis in FOG-1-null hematopoietic precursors revealed that FOG-1 is not required for establishment or maintenance of the active GATA-2 domain, but is critical for the GATA switch. Analysis of GATA factor binding to additional loci also revealed FOG-1-dependent GATA switches. Thus, FOG-1 facilitates chromatin occupancy by GATA-1 at sites bound by GATA-2. We propose that FOG-1 is a prototype of a new class of coregulators termed chromatin occupancy facilitators, which confer coregulation in certain contexts via enhancing trans-acting factor binding to chromatin in vivo.

Summary Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study, we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ, and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiency generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via Polycomb Repressive Complex 2 (PRC2) independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

A complete understanding of the transcriptional regulation of developmental lineages requires that all relevant factors be identified. Here, we have taken a proteomic approach to identify novel proteins associated with GATA-1, a lineage-restricted zinc finger transcription factor required for terminal erythroid and megakaryocytic maturation. We identify the Krüppel-type zinc finger transcription factor ZBP-89 as being a component of multiprotein complexes involving GATA-1 and its essential cofactor Friend of GATA-1 (FOG-1). Using chromatin immunoprecipitation assays, we show that GATA-1 and ZBP-89 cooccupy cis-regulatory elements of certain erythroid and megakaryocyte-specific genes, including an enhancer of the GATA-1 gene itself. Loss-of-function studies in zebrafish and mice demonstrate an in vivo requirement for ZBP-89 in megakaryopoiesis and definitive erythropoiesis but not primitive erythropoiesis, phenocopying aspects of FOG-1-and GATA-1-deficient animals. These findings identify ZBP-89 as being a novel transcription factor involved in erythroid and megakaryocytic development and suggest that it serves a cooperative function with GATA-1 and/or FOG-1 in a developmental stage-specific manner.Lineage-specific transcription factors play essential roles in development. However, most of these factors have relatively small consensus DNA binding motifs, and by themselves, they are not likely to account for high-fidelity lineage-specific gene expression in higher organisms. Indeed, recent studies employing chromatin immunoprecipitation (IP) (ChIP) across extended loci or entire genomes show that only a small

The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.Over the past 2 decades, a number of transcription factors/ cofactors have been identified that play essential roles in megakaryocytic differentiation. These include GATA-1 (46, 57), GATA-2 (4), Friend of GATA-1 (FOG-1) (55), NF-E2 p45 (47), mafG and mafK (39), SCL/Tal1 (30), GABP␣ (41), FLI-1 (17, 49), ZBP-89 (62), and RUNX-1 (14, 18). Yet, how these transcription factors act together to coordinate terminal megakaryocytic maturation remains incompletely understood. Moreover, there is increasing evidence that terminal megakaryocyte maturation is coordinated with localization at vascular sinusoidal niches within the bone marrow (1,21,26). How signaling events related to these spatial cues, as well as more-traditional cytokine-mediated transduction pathways, intersect with these key megakaryocyte transcriptional regulators also remains unclear.The transcription factor RUNX-1 belongs to a family of proteins that share a conserved 128-amino-acid runt homology domain, which mediates DNA binding and interaction with the cofactor CBF-␤ (for a review, see reference 20). RUNX-1 Ϫ/Ϫ mice die between embryonic day 12.5 (E12.5) and E13.5 due to central nervous system hemorrhage and failure of all definitive hematopoiesis (38, 59). The latter cause of death is due to a defect in the emergence of hematopoietic stem cells from the aorta-gonadal-mesonephros region during embryogenesis (31,34,64). Conditional knockout studies of mice demonstrate a specific role for RUNX-1 in megakaryocyte differentiation during ...

The zinc fi nger transcription factor GATA-1 requires direct physical interaction with the cofactor friend of GATA-1 (FOG-1) for its essential role in erythroid and megakaryocytic development. We show that in the mast cell lineage, GATA-1 functions completely independent of FOG proteins. Moreover, we demonstrate that FOG-1 antagonizes the fate choice of multipotential progenitor cells for the mast cell lineage, and that its downregulation is a prerequisite for mast cell development. Remarkably, ectopic expression of FOG-1 in committed mast cell progenitors redirects them into the erythroid, megakaryocytic, and granulocytic lineages. These lineage switches correlate with transcriptional down-regulation of GATA-2 , an essential mast cell GATA factor, via switching of GATA-1 for GATA-2 at a key enhancer element upstream of the GATA-2 gene. These fi ndings illustrate combinatorial control of cell fate identity by a transcription factor and its co factor, and highlight the role of transcriptional networks in lineage determination. They also provide evidence for lineage instability during early stages of hematopoietic lineage commitment.

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Hematopoietic development occurs in complex microenvironments and is influenced by key signaling events. Yet how these pathways communicate with master hematopoietic transcription factors to coordinate differentiation remains incompletely understood. The transcription factor RUNX1 plays essential roles in definitive hematopoietic stem cell (HSC) ontogeny, HSC maintenance, megakaryocyte (Mk) maturation, and lymphocyte differentiation. It is also the most frequent target of genetic alterations in human leukemia. Here, we report that RUNX1 is phosphorylated by Src family kinases (SFKs) and that this occurs on multiple tyrosine residues located within its negative regulatory DNA-binding and autoinhibitory domains. Retroviral transduction, chemical inhibitor, and genetic studies demonstrate a negative regulatory role of tyrosine phosphorylation on RUNX1 activity in Mk and CD8 T-cell differentiation. We also demonstrate that the nonreceptor tyrosine phosphatase Shp2 binds directly to RUNX1 and contributes to its dephosphorylation. Last, we show that RUNX1 tyrosine phosphorylation correlates with reduced GATA1 and enhanced SWI/SNF interactions. These findings link SFK and Shp2 signaling pathways to the regulation of RUNX1 activity in hematopoiesis via control of RUNX1 multiprotein complex assembly.