An N-terminal deletion mutant of estrogen receptor exhibits increased synergism with upstream activators and enhanced binding to the estrogen response element.
To study the role of the N-terminal region of the estrogen receptor (ER) in transcription activation and in DNA binding, we constructed a mutant of the Xenopus laevis ER which lacks amino acids 1-159 (XER160/586). In transient transfections, XER160/586 exhibited < 10% of the activity of wild-type XER on a synthetic promoter containing two estrogen response elements (EREs). To examine transcriptional synergism by XER and by XER160/586, we determined the activity of promoters containing EREs and binding sites for either the vitellogenin activator, NF1, or AP1 upstream activator protein. For the three promoters transcription by XER was 2.8-fold greater than expected for additive activities, and transcription by XER160/586 was 6.2-fold greater. These data demonstrate that an upstream activator protein bound near the promoter can partially compensate for the loss of the internal N-terminal (AF1) transactivation domain in XER160/586. Using a promoter interference assay to study the intracellular interaction between ER and the estrogen response element, we found that XER160/586 exhibited a significant increase in affinity for the ERE. Its low basal activity and enhanced affinity for the ERE make XER160/586 an effective dominant negative mutant. When co-expressed with wild-type XER at 1:1 and 5:1 ratios, XER160/586 suppressed the activity of wild-type XER by 57% and > 80%, respectively.
Xing
H
H
Department of Biochemistry, University of Illinois, Urbana 61801.
Mattick
S
S
Lew
D
D
Shapiro
D J
DJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008099:Liver; D008969:Molecular Sequence Data; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D015533:Transcriptional Activation; D014162:Transfection; D014819:Vitellogenins / Q000235:genetics*; D014982:Xenopus laevis
D004268:DNA-Binding Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D014819:Vitellogenins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase