Correlation between patterns of DNase I-hypersensitive sites and upstream promoter activity of the human epsilon-globin gene at different stages of erythroid development.

DNA 5' to the human epsilon-globin gene exhibits unique patterns of DNase I-hypersensitive sites (DHS) in three human erythroleukemic cell lines which represent the embryonic (K562), fetal (HEL), and adult (KMOE) stages of erythroid development. We have mapped 10 epsilon-globin DHS in K562 cells, in which the epsilon-globin gene is maximally active. Major sites are located -11.7, -10.5, -6.5, -2.2 kilobase pairs (kbp) and -200 base pairs (bp) upstream of the gene and directly over the major cap site. Minor sites are located -5.5, -4.5, and -1.48 kbp and -900 bp upstream of the cap site. In HEL cells, in which the epsilon-globin gene is expressed at extremely low levels, the -11.7-, -10.5-, -5.5-, -4.5-, and -2.2-kbp DHS are no longer detectable; the -200-bp site is approximately 300-fold less sensitive to DNase I; and the -1.48-kbp, -900-bp, and major cap site DHS are 3- to 4-fold less sensitive. Only the DHS located -6.5 kbp relative to the major cap site is detectable at all three stages of erythroid development, including KMOE cells in which epsilon-globin synthesis is undetectable. We suggest that this site may be implicated in maintaining the entire beta-globin cluster in an active chromatin conformation. The five DHS downstream of the -6.5-kbp element possess associated promoters. Thus two distinct types of DHS exist--promoter positive and promoter negative. In HEL cells, all the upstream promoters are inactivated, although the -1.48-kbp and -900- and -200-bp DHS are still present. This suggests that the maintenance of DHS and regulation of their associated promoters occur by independent mechanisms. The inactivation of the upstream promoters in HEL cells while the major cap site remains active represents a unique pattern of expression and suggests that HEL cells possess regulatory factors which specifically down regulate the epsilon-globin upstream promoters.     

The beta-globin dominant control region: hypersensitive site 2.

The Dominant Control Region (DCR) of the human beta-globin gene locus consists of four strong hypersensitive sites (HSS) upstream of the epsilon-globin gene. Addition of these sites confers copy number dependent expression on the human beta-globin gene in murine erythroleukaemia cells and transgenic mice, at levels comparable with the endogenous mouse globin genes. We have shown previously that a 1.9 kb fragment comprising HSS 2 accounts for 40-50% of the full effect of the DCR. In this paper we describe a deletional analysis of HSS 2. We show that a 225 bp fragment is sufficient to direct high levels of expression of the human beta-globin gene which is copy number dependent and integration site independent. This 225 bp fragment overlaps the major region that is hypersensitive 'in vivo'. DNase I footprinting shows the presence of four binding sites for the erythroid specific protein NF-E1; the three other footprinted regions display a remarkable redundancy of the sequence GGTGG and bind a number of proteins including Sp1 and the CACC box protein. The significance of these results for the regulation of globin gene expression is discussed.     

Restriction fragment length polymorphism of the human beta-globin gene cluster: analysis of a beta-thalassemic family in Taiwan

We have analysed seven polymorphic restriction sites of the human beta-globin gene cluster of six members of a Chinese family with a beta +-thalassemic sibling. The seven polymorphic sites analysed are the HincII site at the 5'-end of the epsilon-globin gene, the HindIII sites in the two gamma-globin genes, two HincII sites within and at the 3'-end of the psi beta 1 pseudogene, the AvaII site in the beta-globin gene and the BamHI site located at the 3' side of the beta-globin gene. The beta thal chromosome has been identified to have a haplotype of +----++ with respect to these seven polymorphic sites. This is also the most predominant haplotype associated with beta +-thalassemia in Mediterranean and Chinese populations (Chen et al., 1984; Orkin et al., 1982). Of the seven sites analysed in this family, four will be useful in prenatal diagnosis of beta-thalassemia in subsequent pregnancies in the family.     

Nuclease hypersensitivity in the beta-globin gene region of K562 cells

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.     

A 46 base pair enhancer sequence within the locus activating region is required for induced expression of the gamma-globin gene during erythroid differentiation.

The locus activating region (LAR), contained within 30 kb of chromatin flanking the human beta-globin gene cluster, has recently been shown to be essential for high level beta-globin gene expression. To determine the effect of fragments containing LAR sequences on globin gene expression, mRNA from a marked gamma-globin gene linked to LAR fragments was assayed in stably transfected K562 erythroleukemia cells. DNaseI hypersensitive site II (HS II), located 10.9 kb upstream of the epsilon-globin gene, was required for high level gamma-globin gene expression. We also showed that a 46 bp enhancer element within HS II was necessary and sufficient for the increased gamma-globin gene expression observed with hemin induced erythroid maturation of K562 cells. These results localize a distant regulatory element important for activation of globin genes during human erythroid cell maturation.