DNase I hypersensitivity and epsilon-globin transcriptional enhancement are separable in locus control region (LCR) HS1 mutant human beta-globin YAC transgenic mice.
Expression of the five beta-like globin genes (epsilon, Ggamma, Agamma, delta, beta) in the human beta-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5'HS1 through 5'HS5). We report here a novel enhancer activity in 5'HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5'HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human beta-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, epsilon-, but not gamma-globin, gene expression in primitive erythroid cells was severely attenuated, while adult beta-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5'HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5'HS1 is critical for efficient epsilon-globin gene expression, hypersensitive site formation per se is independent of 5'HS1 function, if any, in definitive erythroid cells.
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
Motoshi
M
Shimotsuma
Eiichi
E
Okamura
Hitomi
H
Matsuzaki
Akiyoshi
A
Fukamizu
Keiji
K
Tanimoto
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D041905:Erythroid Cells / Q000378:metabolism; D050982:GATA1 Transcription Factor / Q000378:metabolism; D006801:Humans; D019901:Locus Control Region / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D055544:beta-Globins / Q000235:genetics* / Q000378:metabolism; D055547:epsilon-Globins / Q000235:genetics* / Q000378:metabolism; D055545:gamma-Globins / Q000235:genetics* / Q000378:metabolism
D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D012333:RNA, Messenger; D055544:beta-Globins; D055547:epsilon-Globins; D055545:gamma-Globins; D003850:Deoxyribonuclease I