Identification and characterization of an estrogen-responsive element binding protein repressed by estradiol.
Cytosolic proteins from uteri of 19-day-old rats were analyzed by an electrophoresis mobility shift assay (EMSA) using a 31 base pair DNA probe containing an estrogen-responsive element (ERE) from the vitellogenin A2 gene. EMSA identified three distinct cytosolic protein-DNA complexes that are separable by Q-Sepharose anion exchange chromatography into an estrogen receptor (ER)-containing fraction (150 mM NaCl eluate) and a non-ER-containing fraction (250 mM NaCl eluate). We thus refer to the non-ER fraction as the ERE binding protein (ERE-BP). The ERE-BP-containing fraction was repressed to 40-50% of its normal levels following a single injection of estradiol. In addition, ERE-BP levels were repressed to the same extent (greater than 50%) by day 20 of the rat's gestational period. Examination of the expression pattern of ERE-BP shows that this activity is differentially expressed in both estrogen-responsive and nonresponsive tissues, with the highest levels of expression occurring in the pituitary. We next examined the specificity of ERE-BP binding by competition analysis using DNA sequences corresponding to binding sites of several known transcription factors. ERE-BP was found to be specific for both the ER binding site (ERE) and TATA binding protein binding sites. Furthermore, saturation analysis demonstrated that ERE-BP binds to the ERE and TATA binding protein sequences with an apparent Kd of 1.2 and 0.12 nM, respectively. Partial purification of ERE-BP using three chromatography steps (Q-Sepharose, hydroxyapatite, and Sephacryl S300) followed by sodium dodecyl sulfate analysis indicated the presence of three major protein bands (p102, p81, and p48) as judged by Coomassie staining. UV cross-linking of the ERE-BP/DNA complex followed by sodium dodecyl sulfate analysis-polyacrylamide gel electrophoresis analysis indicates that the 48 kDa band seen in the final, partially purified fraction correlates with the ERE-BP activity. Thus, this study has identified a unique uterine cytosolic protein that binds to the ER binding site and may influence ER binding.
Gray
W G
WG
Department of Biochemistry, University of Wisconsin-Madison 53706-1569, USA.
Gorski
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D015870:Gene Expression; D008969:Molecular Sequence Data; D011247:Pregnancy; D051381:Rats; D011960:Receptors, Estrogen / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism; D014466:Ultraviolet Rays; D014599:Uterus / Q000378:metabolism; D014819:Vitellogenins / Q000235:genetics
D015342:DNA Probes; D004268:DNA-Binding Proteins; D011960:Receptors, Estrogen; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D014819:Vitellogenins; D004958:Estradiol; D004247:DNA