PURIFICATION AND CHARACTERIZATION OF AN AH RECEPTOR-BINDING FACTOR INCHROMATIN

Dioxin induces biological responses through interaction with a specifi c intracellular receptor, the Ah receptor, and the subsequent interact ion of the Ah receptor with chromatin. We previously reported the bind ing of the Ah receptor, partially purified from rabbit liver, to recep tor binding factors (termed AhRBFs) in chromatin. Rabbit liver chromat in proteins (CP) were isolated by adsorption of chromatin to hydroxyla patite followed by sequential extraction with 3 M NaCl and 1-8 M guani dine hydrochloride (GdnHCl). In the present study, we continued the pu rification of the CP5 fraction, which exhibited AhRBF activity. The pr oteins in CP5 were separated by CL-Sepharose 6B column chromatography resolving lower molecular weight fractions. To assay for receptor bind ing, a portion of each CL-Sepharose 6B fraction was reconstituted to r abbit double-stranded DNA (dsDNA) using a reverse gradient dialysis of 7.5 to 0.0 M GdnHCl. These reconstituted chromatins were then examine d for binding to [H-3]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([H-3]TCDD) -receptor complexes by the streptomycin filter binding assay. Two prot ein fractions with a molecular weight in the range of 10,000-14,000 de monstrated high affinity binding to the Ah receptor. The binding of Ah RBFs reconstituted to dsDNA was shown, by competition experiments with Ah receptor bound by unlabeled TCDD (TCDD-R), to be >90% specific for [H-3]TCDD-R. Further purification was achieved by preparative SDS-PAG E, and AhRBF activity was attributed to two fractions with molecular w eights between 12,000 and 10,000. A 12 kDa protein with AhRBF activity was found to have an isoelectric point (pi) of greater than or equal to 10. The 12 kDa AhRBF was sequenced by Edman degradation after cyano gen bromide cleavage and identified as histone H4. Although histone H4 has been postulated to interact with transcription factors in a varie ty of systems, this is the first report of a specific interaction of A hR with histone H4.