Hypersensitive Site 2 Specifies a Unique Function within the Human β-Globin Locus Control Region To Stimulate Globin Gene Transcription

Locus control regions (LCRs) are highly specialized tissue-specific DNA regulatory elements that are able to confer position-independent and copy number-dependent expression of cis-linked genes when examined in transgenic mice. Since the discovery of the human β-globin LCR (13, 20), a growing number of genes or loci have been found to be regulated by LCR-like activities. Most LCRs appear to be composite elements and, perhaps not coincidentally, contain several DNase I hypersensitive sites (HS). The examples of genes regulated by such elements include the human β-globin (45), the T-cell-specific CD2 (18), the T-cell receptor α/δ (41), and the chicken lysozyme loci (2). Higgs et al. (23) and Montoliu et al. (35) have shown that single HS located upstream of the α-globin or tyrosinase genes also bear multiple activities normally attributed to an LCR. The human β-globin LCR, located from approximately 8 to 22 kbp upstream of the ɛ-globin gene (13, 14, 47, 48) is composed of four erythroid cell-specific (HS1 to HS4) and one ubiquitous (HS5) DNase I HS. This region mediates chromatin opening over the whole β-globin gene locus and also is responsible for stimulating high-level expression of the globin genes throughout erythroid cell development (12). Perhaps most remarkably, the LCR enhances transcription over a considerable distance, more than 60 kbp in the adult human β-globin gene. Characteristically, it is able to confer position-independent and copy number-dependent expression to linked globin genes in transgenic mice. In addition, the human β-globin LCR is required for altered timing of DNA replication of the locus, which initiates in erythroid cells from an origin located between the adult δ- and β-globin genes (1, 27). The mechanism(s) by which the LCR elicits its multiple activities is unclear. One model has proposed that the LCR acts only indirectly, by providing an open, accessible chromatin environment and that gene-proximal regulatory elements mediate the developmental switches as well as high levels of globin gene expression (32). In a second, mutually exclusive model, it was proposed that the LCR, after inducing chromatin opening, directly interacts by DNA looping to stimulate high-level, stage-specific expression of individual globin genes (10, 49). The contribution of individual HS elements to the overall function of the LCR is also somewhat controversial. Previous studies showed that human β-globin LCR element HS2 is a potent enhancer when analyzed in both transient- and stable-transfection assays, showing that HS2 functions as a classical enhancer in the absence or presence of chromatin (7, 46, 48). In contrast, HS3 and HS4 were shown to function as potent activators only when the constructs were integrated into chromosomal DNA, indicating that for these two elements, chromatin environment plays an important role in their activity (7, 26, 36). Subsequent studies revealed that HS2, HS3, and HS4 act as strong individual enhancers in transgenic mice when linked to cosmid constructs bearing the human γ- and β-globin genes (16). More importantly, Fraser et al. (16) showed that individual HS elements exhibit stage-specific preferences for enhancing particular globin genes in the various hematopoietic compartments: only HS2 appeared to be equally active at the embryonic, fetal, and adult stages. It was originally concluded that HS2 alone was able to provide copy number-dependent and position-independent transcription to linked genes (5, 15), but this view was challenged when it was shown that HS2 was able to protect from position-of-integration effects only in multiply integrated transgene copies (8). The entire transcriptional stimulatory activity of LCR HS2 was originally mapped to a 375-bp HindIII-XbaI restriction fragment (46). This region contains a cluster of binding sites for ubiquitous as well as tissue-specific transcription factors. Several of these sites within the HS2 core element are highly conserved among different species (22). It was shown that one of these highly conserved elements is a 60-bp motif encompassing a tandem maf-responsive element (MARE) (37; also referred to as an NF-E2 binding site). This element was shown to harbor most of the enhancer activity of HS2, and a similar configuration of tandem MAREs is not found in other LCR HS sites (39). Here we report the analysis of seven transgenic lines containing intact human β-globin yeast artificial chromosomes (YACs) bearing a deletion of the 375-bp core enhancer of HS2. The results show that deletion of HS2 results in a severe reduction of ɛ-globin gene expression in the embryonic yolk sac and that expression of the adult β-globin gene is significantly reduced in fetal liver and adult spleen. Moreover, in three of the lines bearing single-copy transgenes, expression of all of the genes is much more severely affected than in the context of multiple tandemly integrated copies. We also analyzed four transgenic lines carrying intact human β-globin YACs in which the 220-bp core of HS3 was substituted for the 375-bp HS2 core enhancer. These results show that HS3 is unable to functionally compensate for the loss of HS2 and demonstrate that both HS2 and HS3 provide unique contributions to LCR activity. Moreover, the substitution mutants are expressed in a far more consistent manner from line to line than are the deletion mutants, suggesting that although transcription is impaired, the substitution mutations protect the transgenes from position-of-integration effects. These experiments extend our earlier observations showing that deletion of single core elements for HS3 or HS4 from intact, single-copy transgenic YACs severely diminishes LCR-mediated transcriptional activation (4, 29). Together, the data show that deletion of any single-core HS element (HS2, HS3, or HS4) abrogates the activity of the entire LCR. Deletion of any of these core elements also causes variable DNase I hypersensitivity within the LCR that is dependent on the site of integration into the mouse genome. We interpret these results as being most compatible with a model in which an LCR holocomplex provides a unique chromatin architecture that protects transgenes from position-of-integration effects. This architectural complex includes both the core elements and their flanking sequences, and it can be partially restored if the core element plus flanking sequences of any single LCR HS site are deleted, if one HS core element is used to replace another, or, indeed, even if multiple copies of a particular transgene have integrated in tandem into the mouse genome. Additionally, the core HS elements appear to be required to cooperatively form a subdomain, or active site, within the superstructure provided by the holocomplex, and it is this active site within the holocomplex that is required specifically for transcriptional stimulation.