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.
Kim
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School of Biochemistry and Molecular Genetics, University of New South Wales, NSW 2052, Sydney, Australia.
Murray
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D000880:Anthraquinones / Q000737:chemistry; D001761:Bleomycin / Q000737:chemistry; D004249:DNA Damage; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D019901:Locus Control Region / Q000235:genetics*; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D055544:beta-Globins / Q000235:genetics*
D000880:Anthraquinones; D009707:Nucleosomes; D055544:beta-Globins; C001328:hedamycin; D001761:Bleomycin; D003850:Deoxyribonuclease I