Promoter elements and erythroid cell nuclear factors that regulate alpha-globin gene transcription in vitro.
We have previously purified four factors (alpha-IRP, alpha-CP1, alpha-CP2, and NF-E1) that interact with the promoter of the alpha-globin gene. One of these (NF-E1) is a tissue-restricted factor that has recently been cloned. The binding sites of these factors identify DNA sequence elements that might mediate the tissue-specific and inducible transcription of the alpha-globin gene. This possibility was tested in a series of in vitro transcription experiments. An examination of 5' truncated templates and synthetic promoters constituted from individual factor-binding sites apposed to the alpha-TATAA box showed that the binding elements of three factors (alpha-CP1, alpha-IRP, and NF-E1) mediate four- to sixfold activation of transcription in vitro. In contrast, one element (alpha-CP2) stimulated transcription less than twofold. The 5- to 10-fold stimulation of these latter templates upon addition of a DNA sequence affinity-purified factor suggests that alpha-CP2 is functionally limiting in nuclear extracts. Additional experiments further tested the effect of supplementing extracts with factors purified from erythroid cell nuclear extracts or, in the case of NF-E1, enriched from a bacterial cDNA expression system. Each factor tested stimulated transcription in vitro in a binding-site-dependent manner. Our results provide a comprehensive functional view of the murine alpha-globin promoter and suggest possible mechanisms for activation of alpha-globin gene transcription during induced differentiation of murine erythroleukemia cells.
Kim
C G
CG
DeWitt Wallace Research Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Swendeman
S L
SL
Barnhart
K M
KM
Sheffery
M
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D005914:Globins