A large "footprint" at the boundary of the human beta-globin locus control region hypersensitive site-2

The 5'-boundary region of the human beta-globin locus control region hypersensitive site-2 (HS-2) was examined for protein-DNA interactions. The HS-2 is an erythroid specific DNase I hypersensitive site that extends for approximately 600 bp. Erythroid K562 cells and non-erythroid HeLa cells were damaged by bleomycin and hedamycin--these agents are able to "footprint" nucleosome cores and proteins bound to DNA. The fragments generated by DNA damage were amplified by the ligation-mediated polymerase chain reaction with primers specific for the 5'-boundary region of HS-2 and examined at base pair resolution on DNA sequencing gels. The intensity of damage in intact cells was compared with that in purified DNA. The comparison between intact cells and purified DNA revealed a protected region of 226 bp with bleomycin and 182 bp with hedamycin in K562 cells. The length of the protected region was consistent with the presence of a nucleosome core. We postulate that an erythroid-specific protein binds next to the positioned nucleosome at the boundary of HS-2 to prevent sliding of the nucleosome into the hypersensitive site--this would also account for the large size of the protected region. HeLa cells (lacking a hypersensitive site in the beta-globin cluster) did not have an area of protection in this region.     

Chromatin structure at the flanking regions of the human beta-globin locus control region DNase I hypersensitive site-2: proposed nucleosome positioning by DNA-binding proteins including GATA-1

The human beta-globin locus control region DNase I hypersensitive site-2 (LCR HS-2) is erythroid-specific and is located 10.9 kb upstream of the epsilon-globin gene. Most studies have only examined the core region of HS-2. However, previous studies in this laboratory indicate that positioned nucleosomes are present at the 5'- and 3'-flanking regions of HS-2. In addition, footprints were observed that indicated the involvement of DNA-binding proteins in positioning the nucleosome cores. A consensus GATA-1 site exists in the region of the 3'-footprint. In this study, using an electrophoretic mobility shift assay (EMSA) and DNase I footprinting, we confirmed that GATA-1 binds in vitro at the 3'-end of HS-2. An additional GATA-1 site was found to bind GATA-1 in vitro at a site positioned 40 bp upstream. At the 5'-end of HS-2, DNase I footprinting revealed a series of footprints showing a marked correlation with the in vivo footprints. EMSA indicated the presence of several erythroid-specific complexes in this region including GATA-1 binding. Sequence alignment for 12 mammalian species in HS-2 confirmed that the highest conservation to be in the HS-2 core. However, a second level of conservation extends from the core to the sites of the proposed positioning proteins at the HS-2 flanking regions, before declining rapidly. This indicates the importance of the HS-2 flanking regions and supports the proposal of nucleosome positioning proteins in these regions.     

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.     

Sequences downstream of the erythroid promoter are required for high level expression of the human alpha-spectrin gene

Alpha-spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the alpha-spectrin gene. Previously, we demonstrated that the core promoter of the human alpha-spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3' of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the alpha-spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3' of the alpha-spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased alpha-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis.     

DNase I footprint of ABC excinuclease

The incision and excision steps of nucleotide excision repair in Escherichia coli are mediated by ABC excinuclease, a multisubunit enzyme composed of three proteins, UvrA, UvrB, and UvrC. To determine the DNA contact sites and the binding affinity of ABC excinuclease for damaged DNA, it is necessary to engineer a DNA fragment uniquely modified at one nucleotide. We have recently reported the construction of a 40 base pair (bp) DNA fragment containing a psoralen adduct at a central TpA sequence (Van Houten, B., Gamper, H., Hearst, J. E., and Sancar, A. (1986a) J. Biol. Chem. 261, 14135-14141). Using similar methodology a 137-bp fragment containing a psoralen-thymine adduct was synthesized, and this substrate was used in DNase I-footprinting experiments with the subunits of ABC excinuclease. It was found that the UvrA subunit binds specifically to the psoralen modified 137-bp fragment with an apparent equilibrium constant of K8 = 0.7 - 1.5 X 10(8) M-1, while protecting a 33-bp region surrounding the DNA adduct. The equilibrium constant for the nonspecific binding of UvrA was Kns = 0.7 - 2.9 X 10(5) M-1 (bp). In the presence of the UvrB subunit, the binding affinity of UvrA for the damaged substrate increased to K8 = 1.2 - 6.7 X 10(8) M-1 while the footprint shrunk to 19 bp. In addition the binding of the UvrA and UvrB subunits to the damaged substrate caused the 11th phosphodiester bond 5' to the psoralen-modified thymine to become hypersensitive to DNase I cleavage. These observations provide evidence of an alteration in the DNA conformation which occurs during the formation of the ternary UvrA.UvrB.DNA complex. The addition of the UvrC subunit to the UvrA.UvrB.DNA complex resulted in incisions on both sides of the adduct but did not cause any detectable change in the footprint. Experiments with shorter psoralen-modified DNA fragments (20-40 bp) indicated that ABC excinuclease is capable of incising a DNA fragment extending either 3 or 1 bp beyond the normal 5' or 3' incision sites, respectively. These results suggest that the DNA beyond the incision sites, while contributing to ABC excinuclease-DNA complex formation, is not essential for cleavage to occur.     

Structural analysis of human pyruvate kinase L-gene and identification of the promoter activity in erythroid cells.

The human pyruvate kinase (PK) L-gene is organized in 12 exons over 9.5 kilobases, and the first and second exons are specifically transcribed to the R- and L-type PK mRNA. The 5'-flanking region upstream the first exon has two CAC boxes and four GATA motifs within 250 bp from the translational initiation codon. Comparison with the rat L-gene revealed four well-conserved elements in the region. The transient transfection demonstrated that the 270-bp upstream region was a powerful promoter in K562 cells, whereas deletion of the distal 150-bp sequences, which included three GATA motifs, resulted in drastic reduction of the activity. When the hypersensitive site 2 of the human beta-globin gene locus was joined to the promoter, the activity of the proximal 120-bp region was enhanced. We concluded that the proximal 120-bp region had a basal promoter activity and that the distal 150-bp region acted as an enhancer in erythroid cells.     

Hypersensitive site 4 of the human beta globin locus control region.

The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2.