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.
Davies
Neil
N
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia.
Freebody
John
J
Murray
Vincent
V
D024509:3' Flanking Region; D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000737:chemistry*; D017124:Conserved Sequence; D021901:DNA, Intergenic / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005914:Globins / Q000235:genetics*; D006801:Humans; D019901:Locus Control Region; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism*; D010802:Phylogeny; D014157:Transcription Factors / Q000378:metabolism
D002843:Chromatin; D021901:DNA, Intergenic; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D009707:Nucleosomes; D014157:Transcription Factors; D005914:Globins; D003850:Deoxyribonuclease I