Evidence for distinct DNA binding forms of the erythroid-specific transcription factor NF-E2.
The transcriptional activity of the beta-globin genes is regulated by a complex genetic element, the locus control region (LCR), at the 5'-end of the beta-globin locus. Tandem binding sites for the erythroid-specific transcription factor NF-E2 are important for the transcriptional activation function of the LCR. We discovered that vanadate strongly stimulates the DNA binding activity of NF-E2 in crude and fractionated nuclear extracts. The other oxyanions, molybdate and tungstate, do not affect NF-E2 DNA binding. Quantitative DNA binding experiments indicated that vanadate stimulates NF-E2 DNA binding by increasing the number of NF-E2 molecules that are competent to bind to DNA, rather than influencing the affinity of binding. Gel filtration analysis revealed a similar Stokes' radius for NF-E2, in the absence or presence of vanadate, inconsistent with a role for vanadate in stabilizing the heteromeric NF-E2 complex. Distinct NF-E2 forms, which were either weakly or strongly induced by vanadate, were resolved by cation and anion exchange chromatography. A model is proposed in which two conformers of NF-E2 share an identical subunit composition, but differ in DNA binding activity. Vanadate may interact directly with one of the conformers to generate the high-affinity DNA binding state. The presence of a non-DNA binding pool of NF-E2 suggests that the formation of an active NF-E2 heteromer may be a regulated step in the cell.
Lam
L T
LT
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706, USA.
Bresnick
E H
EH
D001483:Base Sequence; D002850:Chromatography, Gel; D004273:DNA, Neoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D050981:Erythroid-Specific DNA-Binding Factors; D005914:Globins / Q000235:genetics*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D008969:Molecular Sequence Data; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D011485:Protein Binding / Q000187:drug effects; D013347:Subcellular Fractions / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology*
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors; D014638:Vanadates; D005914:Globins; D010744:Phosphoric Monoester Hydrolases