Purification and characterization of an Ah receptor binding factor in chromatin.
Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We previously reported the binding of the Ah receptor, partially purified form rabbit liver, to receptor binding factors (termed AhRBFs) in chromatin. Rabbit liver chromatin proteins (CP) were isolated by absorption of chromatin to hydroxylapatite followed by sequential extraction with 3 M NaCl and 1-8 M guanidine hydrochloride (GdnHCl). In the present study, we continued the purification of the CP5 fraction, which exhibited AhRBF activity. The proteins in CP5 were separated by CL-Sepharose 6B column chromatography resolving lower molecular weight fractions. To assay for receptor binding, a portion of each Cl-Sepharose 6B fraction was reconstituted to rabbit double-stranded DNA (dsDNA) using a reverse gradient dialysis of 7.5 to 0.0 M GdnHCl. These reconstituted chromatins were then examined for binding to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter binding assay. Two protein fractions with a molecular weight in the range of 10,000-14,000 demonstrated high affinity binding to the Ah receptor. The binding of AhRBFs reconstituted to dsDNA was shown, by competition experiments with Ah receptor bound by unlabeled TCDD (TCDD-R), to be > 90% specific for [3H]TCDD-R. Further purification was achieved by preparative ADS-PAGE, and AhRBF activity was attributed to two fractions with molecular weights between 12,000 and 10,000. A kDa protein with AhRBF activity was found to have an isoelectric point (pI) of > or = 10. The 12 kDa AhRBF was sequenced by Edman degradation after cyanogen bromide cleavage and identified as histone H4. Although histone H4 has been postulated to interact with transcription factors in a variety of systems, this is the first report of a specific interaction of AhR with histone H4.
Dunn
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RT
St. Louis University Health Sciences Center, Department of Pharmacological and Physiological Science, MO 63104, USA.
Ruh
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Burroughs
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LK
Ruh
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MF
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D011817:Rabbits; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D013379:Substrate Specificity
D002352:Carrier Proteins; D002843:Chromatin; D006657:Histones; D010446:Peptide Fragments; D010750:Phosphoproteins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon