Sequences located 3' to the breakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhancer activity and can modify the developmental expression of the human fetal A gamma-globin gene in transgenic mice.

Expression of fetal gamma-globin genes in individuals with the deletion forms of hereditary persistence of fetal hemoglobin (HPFH) has been attributed either to enhancement by 3' regulatory elements juxtaposed to gamma-globin genes or to deletion of gamma-gene silencers normally residing within the beta-globin gene cluster. In the present study, we tested the hypothesis of imported enhancers downstream of beta-globin gene using the HPFH-3 deletion as a model. The abnormal bridging fragment of 13.6 kilobases (kb) containing the A gamma-gene with its flanking sequences and 6.2 kb of the juxtaposed region was microinjected into fertilized mouse eggs. Twelve transgenic mice positive for the fragment were generated. Samples from 11.5-day yolk sacs, 16-day fetal liver, and adult blood were analyzed for A gamma-mRNA using RNase protection assays. Three mice lacked A gamma expression in the yolk sac indicating non-optimal integration site. Four expressed A gamma-mRNA at the embryonic stage only, while two expressed A gamma-mRNA in both embryonic and fetal liver erythroid cells. Since the A gamma-gene with its normal flanking sequences and in the absence of the locus control region is expressed only in embryonic cells of transgenic mice, these data suggest that the juxtaposed sequences have altered the developmental specificity of the fetal gamma-globin gene. These sequences were further tested for the presence of an enhancer element, by their ability to activate a fusion reporter gene consisting of the CAT gene linked to the gamma-globin gene promoter, in erythroid (K562) and non-erythroid (HeLa) cells. A 0.7-kb region located immediately 3' to the breakpoint, enhanced chloramphenicol acetyltransferase activity by 3-fold in erythroid cells. The enhancer also activated the embryonic epsilon-globin gene promoter by 2-fold but not the adult beta- or delta-globin gene promoters. The enhancer represents a region of previously known complex tandem repeats; in this study we have completed the sequencing of the region encompassing the 0.7-kb enhancer element. Multiple areas of the enhancer region exhibit homology to the core element of the simian virus 40 enhancer and to the sequences of the human 3' A gamma- and the chicken 3' beta-globin enhancers. A consensus binding site for the erythroid specific GATA-1 transcription factor and seven consensus sites for the ubiquitous CP1 transcription factor are also included within the enhancer. These data suggest that these sequences located immediately 3' to the breakpoint of the HPFH-3 deletion, exhibit both the structure and the function of an enhancer, and can modify the developmental specificity of the fetal gamma-globin genes, resulting in their continued expression during adult life.

The MHC class II E beta promoter: a complex arrangement of positive and negative elements determines B cell and interferon-gamma (IFN-gamma) regulated expression.

The 5' proximal region of the E beta gene was studied with respect to B lymphoid expression and responsiveness to cytokines, revealing a complex array of general and cell type specific cis-elements and factors. Full lymphoid activity and response to interferon-gamma (IFN-gamma) is generated by the concerted action of the MHC boxes (H, X and Y) and additional elements. Combinatorial interactions between elements and their cognate factors are indicated by several lines of evidence. Thus, mutations within the X box in the promoter context are strongly deleterious to both B lymphoid activity and IFN-gamma regulation. However, the X box alone has minimal lymphoid activity upon heterologous promoters. Data from deletion, insertion and site directed mutagenesis demonstrate that sequences extending approximately 35 bp 5' of the X box (designated as Cytokine Response Sequence--CRS) have a dual role: they are required for cytokine-regulated expression as well as serving as an enhancer element for cell-specific constitutive expression. A region that carries X and CRS permits both lymphoid activity and IFN-gamma response. In contrast, sequences that include X and the downstream Y box are constitutively active in all cell types tested. Combination of the sequences both upstream and downstream of the X box results in a tissue-specific and cytokine-regulated enhancer of full strength. In vivo competition studies show that titratable trans-acting factors, shared by Class I and Class II promoters, mediate the CRS-dependent IFN-gamma response. We report here the identification of novel nuclear complexes that bind to the CRS and recognize sites which correlate with its negative or positive elements. One of these complexes is present in B lymphoid cells only. Three other CRS complexes that are upregulated by either IFN-alpha and IFN-gamma are competed by a non-Class II, IFN-alpha stimulated response element (ISRE), providing evidence for the functional interconnection of these cytokines.