Structural and Functional Cross-Talk between a Distant Enhancer and the ɛ-Globin Gene Promoter Shows Interdependence of the Two Elements in Chromatin

A central question in developmental biology is how enhancers activate gene transcription in a tissue- and developmental-stage-specific fashion, a complex process which takes place in the chromatin environment of the nucleus. We have addressed this issue by studying components of the human β-globin gene locus, the locus control region (LCR) DNase I-hypersensitive site 2 (HS2) enhancer and the embryonic ɛ-globin gene. The locus consists of five genes expressed sequentially during development and a multicomponent, far-upstream regulatory element, the LCR (for a review, see reference 6). In naturally occurring thalassemias with large deletions encompassing the LCR and upstream sequences, expression of the downstream globin genes is abolished, and the chromatin structure of the locus becomes resistant to DNase I cleavage (20). Therefore, it has long been thought that the LCR mediates both decondensation of the chromatin of the globin locus and activation of transcription of the genes at different stages in development. However, recent experiments in which the mouse or human β-globin LCR was deleted in its natural chromosomal context demonstrate that while the LCR is required for high-level transcription, it may not be required for decondensation of the locus or correct developmental regulation of the globin genes (16, 51). Thus, the LCR, at a minimum, fulfills the role of a traditional enhancer. The LCR contains four HS sites (HS1 to HS4) detected exclusively in the chromatin of erythroid cells (21, 55). Of these, only HS2 has enhancer activity in transient transfection assays, although in transgenic mice all four of the HS sites can activate the various β-globin genes to different extents (22, 56). During development in the mouse, HS2 and the other HS sites form before the globin genes are activated for transcription (33). Subsequently, when the genes are activated, their individual promoters become DNase I hypersensitive as well (31). A small number of transcription factor recognition sequences recur throughout the globin promoters and/or LCR cores including HS2. These include GATA-1 motifs, Maf recognition elements (recognized by NF-E2 and other factors), and CACCC class motifs (recognized by Kruppel-like proteins) (2, 8, 17, 24). Two mechanistic explanations for the activation of globin promoters by the LCR have been proposed. In the dominant chromatin opening model, regulation of the individual genes is autonomous and dependent on the changing transcription factor milieu during development (39). In this view, the LCR is simply responsible for creating a decondensed, or otherwise favorable, chromatin environment in which the globin gene promoters can interact with stage-specific transcription factors. In the mutual interaction model, the LCR physically contacts the individual promoters and activates them in sequence, switching in response to stage-specific factors (14, 42). While there is evidence for autonomous regulation, several lines of investigation provide indirect support for a mutual interaction model for enhancer-promoter cross-talk. In situ hybridization data indicate that only a single globin gene on a chromosome is active at any one time (58). Studies of transgenic mice suggest that individual globin promoters may compete for interaction with the LCR (14). Moreover, it has been observed that the chick β/ɛ 3′ enhancer alone, without a promoter, is not sufficient to form an open chromatin structure (52). Finally, in vitro experiments show that the transcription factors which bind to HS2 and the other LCR HS sites and globin promoters can homo- and heterodimerize (13, 41, 61) and can interact with TAFII130 or CREB binding protein/p300, components of the transcriptional machinery (1, 9, 12, 64). Enhancers work, at least in part, by altering repressive chromatin structure (19). In vitro approaches and transient transfection assays lack a physiological chromatin context, while random chromosomal integrants in transgenic mouse experiments may be confounded by position effects, particularly when mutant regulatory elements are studied (34). To circumvent these problems and to focus on the role of particular transcription factors in mediating enhancer-dependent promoter remodeling and transcription activation in chromatin, we have used Epstein-Barr virus-derived episomes that are stably maintained in human erythroid K562 cells (62). Such minichromosomes have been used extensively in Saccharomyces cerevisiae to obtain refined structural analyses of chromatin transitions accompanying transcription activation and in mammalian cells to dissect the immunoglobulin heavy-chain LCR enhancer (18, 38). We have previously reported that transcription of the human ɛ-globin gene on minichromosomes is dependent on the HS2 enhancer, and the nucleosomal structure of the gene correlates well with that of the endogenous locus (28). In those studies we observed that the loss or alteration of a particular nucleosome in the ɛ-globin promoter depended on linking HS2 to ɛ-globin in the same minichromosome. The studies presented here demonstrate that the structure of HS2 depends on the ɛ-globin promoter and is mediated by GATA-1 and CACCC binding factors. Furthermore, the interaction of enhancer and globin promoter is dependent on the presence of an intact TATA box.