c Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine ␤-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult ␤-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult ␤major-globin gene promoter but did not affect expression of the ␤minor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult ␤major-globin gene promoter during erythroid cell differentiation.T ranscription of cell-type-specific genes is often regulated by multiple proximal and distal DNA regulatory elements (1, 2). Among the distal elements are enhancers, locus control regions (LCRs), and superenhancers (3, 4). Enhancers are single hypersensitive sites (HSs), usually 200 to 400 bp in size, that contain multiple binding sites for transcription factors that in some cases cooperate to form stable enhanceosomes (5). LCRs are often composite elements containing multiple HSs that operate together to regulate expression of single or several related genes within complex gene loci (3). Genes under the control of LCRs are normally expressed at extremely high levels in a cell-type-specific manner (6). Furthermore, LCRs have the remarkable capability of conferring position-independent and copy number-dependent expression to linked genes in transgenic assays. Superenhancers form a broad continuous region of transcription factor and coregulator binding and have also been associated with genes that are expressed at high levels (4).Transcription occurs not only at promoters but also at enhancers (7,8). Recent data demonstrate that both promoters and enhancers initiate bidirectional transcription but that only the coding sense transcripts at promoters are stable, whereas the antisense transcripts and the bidirectional transcripts originating from enhancers (enhancer RNAs [eRNAs]) are unstable (7,8). Despite the observation that most eRNAs are unstable, some eRNAs have been shown to be involved in the regulation of gene expression (9), while in other cases, like the human growth hormone gene locus, the process of transcription, initiated at a distal regulatory element, rather than the nongenic transcript itself, was...