Context-dependent EKLF responsiveness defines the developmental specificity of the human epsilon-globin gene in erythroid cells of YAC transgenic mice.
We explored the mechanism of definitive-stage epsilon-globin transcriptional inactivity within a human beta-globin YAC expressed in transgenic mice. We focused on the globin CAC and CAAT promoter motifs, as previous laboratory and clinical studies indicated a pivotal role for these elements in globin gene activation. A high-affinity CAC-binding site for the erythroid krüppel-like factor (EKLF) was placed in the epsilon-globin promoter at a position corresponding to that in the adult beta-globin promoter, thereby simultaneously ablating a direct repeat (DR) element. This mutation led to EKLF-independent epsilon-globin transcription during definitive erythropoiesis. A second 4-bp substitution in the epsilon-globin CAAT sequence, which simultaneously disrupts a second DR element, further enhanced ectopic definitive erythroid activation of epsilon-globin transcription, which surprisingly became EKLF dependent. We finally examined factors in nuclear extracts prepared from embryonic or adult erythroid cells that bound these elements in vitro, and we identified a novel DR-binding protein (DRED) whose properties are consistent with those expected for a definitive-stage epsilon-globin repressor. We conclude that the suppression of epsilon-globin transcription during definitive erythropoiesis is mediated by the binding of a repressor that prevents EKLF from activating the epsilon-globin gene.
Tanimoto
K
K
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Liu
Q
Q
Grosveld
F
F
Bungert
J
J
Engel
J D
JD
D000367:Age Factors; D000818:Animals; D001665:Binding Sites; D051836:COUP Transcription Factors; D007446:Chromosome Inversion; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000235:genetics*; D006801:Humans; D020014:K562 Cells / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic; D011987:Receptors, Steroid; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D011987:Receptors, Steroid; D012097:Repressor Proteins; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor; D005914:Globins