ESTROGEN-RECEPTOR INTERACTION WITH SPECIFIC HISTONES - BINDING TO GENOMIC DNA AND AN ESTROGEN RESPONSE ELEMENT

Chromosomal proteins that impart high affinity and specificity to the binding of the estrogen receptor (ER) to DNA are termed estrogen recep tor binding factors (ERBFs). Certain partially purified chromosomal pr otein fractions obtained from rabbit uterine chromatin by extraction w ith various molarities of GdnHCl when reconstituted to double-stranded DNA demonstrated high affinity binding for the ER. We report the puri fication and characterization of ERBFs in the chromosomal protein frac tion extracted with 4 M GdnHCl (CP4) after large scale purification. T hese protein fractions were further purified by CL-Sepharose 6B column chromatography which resolved fractions from CP4 that recognized the ER bound by estrogen only or antiestrogen only. Thus, these hydrophobi c chromosomal proteins enhanced the binding of the ER to reconstituted chromatin. To further investigate the interaction of ERBFs with ER, g el mobility shift assays were performed. The highly purified CP4 fract ion with ERBF activity in the binding assay with reconstituted chromat in caused an increase in the formation of the retarded ER-estrogen res ponsive element (ERE) band. Thus, chromatin contains specific ERBFs fo r ER bound by estrogen which enhance the binding of ER to genomic DNA and a target ERE sequence. Further purification of the CL-Sepharose fr action with ERBF activity was achieved by preparative SDS-PAGE. ERBF a ctivity was attributed to proteins with approximate molecular weights of 16,000, 13,000, and 12,000 and a pI of > 9.0. Peptides were partial ly sequenced by Edman degradation and were found to have identity with histones H2B and H4. A 17 kDa protein without ERBF activity was ident ified as H3. Since these histones were not readily extracted from chro matin with 3 M NaCl or 1-3 M GdnHCl, we postulate that some ERBFs may be histone variants or modified histones that display a very high affi nity for DNA and ER.