Chromatin domain activation via GATA-1 utilization of a small subset of dispersed GATA motifs within a broad chromosomal region

Cis elements that mediate transcription factor binding are abundant within genomes, but the rules governing occupancy of such motifs in chromatin are not understood. The transcription factor GATA-1 that regulates red blood cell development binds with high affinity to GATA motifs, and initial studies suggest that these motifs are often unavailable for occupancy in chromatin. Whereas GATA-2 regulates the differentiation of all blood cell lineages via GATA motif binding, the specificity of GATA-2 chromatin occupancy has not been studied. We found that conditionally active GATA-1 (ER-GATA-1) and GATA-2 occupy only a small subset of the conserved GATA motifs within the murine beta-globin locus. Kinetic analyses in GATA-1-null cells indicated that ER-GATA-1 preferentially occupied GATA motifs at the locus control region (LCR), in which chromatin accessibility is largely GATA-1-independent. Subsequently, ER-GATA-1 increased promoter accessibility and occupied the betamajor promoter. ER-GATA-1 increased erythroid Krüppel-like factor and SWI/SNF chromatin remodeling complex occupancy at restricted LCR sites. These studies revealed three phases of beta-globin locus activation: GATA-1-independent establishment of specific chromatin structure features, GATA-1-dependent LCR complex assembly, and GATA-1-dependent promoter complex assembly. The differential utilization of dispersed GATA motifs therefore establishes spatial/temporal regulation and underlies the multistep activation mechanism.