Combinatorial interactions among trans-acting factors establish transcriptional circuits that orchestrate cellular differentiation, survival, and development. Unlike circuits instigated by individual factors, efforts to identify gene ensembles controlled by multiple factors simultaneously are in their infancy. A paradigm has emerged in which the important regulators of hematopoiesis GATA-1 and GATA-2 function combinatorially with Scl/TAL1, another key regulator of hematopoiesis. The underlying mechanism appears to involve preferential assembly of a multimeric complex on a composite DNA element containing WGATAR and E-box motifs. Based on this paradigm, one would predict that GATA-2 and Scl/TAL1 would commonly co-occupy such composite elements in cells. However, chromosome-wide analyses indicated that the vast majority of conserved composite elements were occupied by neither GATA-2 nor Scl/TAL1. Intriguingly, the highly restricted set of GATA-2-occupied composite elements had characteristic molecular hallmarks, specifically Scl/TAL1 occupancy, a specific epigenetic signature, specific neighboring cis elements, and preferential enhancer activity in GATA-2-expressing cells. Genes near the GATA-2-Scl/TAL1-occupied composite elements were regulated by GATA-2 or GATA-1, and therefore these fundamental studies on combinatorial transcriptional mechanisms were also leveraged to discover novel GATA factor-mediated cell regulatory pathways.Combinatorial interactions among trans-acting factors establish transcriptional circuits that control fundamental biological processes. In the context of metazoans, these interactions often occur at regulatory elements far from genes and within introns. Many genes require a complex collection of trans-acting factors, coregulator complexes, and long-range regulation, and therefore considerable challenges exist in forging general principles to explain combinatorial transcriptional control. We investigated combinatorial transcriptional mechanisms in the context of GATA factors, which interact with an assortment of regulatory factors to control differentiation, survival, and development (12, 42).GATA-1 and GATA-2 have unique and essential roles to control hematopoiesis. GATA-2 is required for maintenance and expansion of hematopoietic stem cells (HSCs) (78, 79), while GATA-1 promotes the development of erythrocytes (20,62,63,72), megakaryocytes (70), eosinophils (92), and mast cells (54). GATA-2 is also expressed in endothelial cells (17,48,56), and conditional GATA-2 expression in embryonic stem (ES) cells increases the genesis of hemangioblasts, precursors to hematopoietic and endothelial cells (50). GATA-2 deregulation is associated with early-onset coronary artery disease (15), atherosclerosis (69), and chronic myelogenous leukemia (94), whereas GATA-1 mutations cause megakaryoblastic leukemia (85) and additional blood disorders (16,58).Both GATA-1 and GATA-2 bind an identical DNA motif (WGATAR) (45, 52), but the majority of these motifs are unoccupied in cells (8,26,27,34,36,51). Despit...