Histones are important in gene regulation, especially in determining the chromatin structure necessary for binding of ligand-activated intracellular receptors and for recruitment of transcription factors. Estrogen receptors (ER) bound by estradiol differ from ER bound by antiestrogens in receptor conformation as well as DNA binding characteristics and ability to recruit transcription factors necessary to induce gene expression. It is presumed that in the intact cell the ligand-induced differences in receptor conformation lead to different interactions with the nuclear apparatus. Steroid hormone receptors are thought to disrupt specific nucleosomes, resulting in altered local chromatin structure which increases promoter accessibility and recruitment of transcription factors. We have recently determined that modified or variant forms of histone H4 and H2B, isolated and purified from chromatin, interact with ER and impart high affinity binding of the ER to an oligonucleotide containing a consensus ERE. The experiments outlined in this proposal will characterize these histone isoforms with respect to amino acid sequence and post-translational modifications. Since other chromatin protein fractions also contain proteins that bind ER, we will isolate and characterize the protein components of these fractions to determine if other modifications of histones are also responsible for enhanced interaction with ER. Since histone acetylation and/or ADP-ribosylation may be important factors in determining the ability of ER to interact with histone isoforms (estrogen receptor binding factor (ERBF) histones), we will particularly study these protein modifications. ERBF histones with these modifications (or lacking such modification) will be reconstituted in nucleosomes and tested for ER binding. In addition, we will be able to demonstrate in MCF-7 cells if the state of acetylation/ADP-ribosylation of the histones can be directed by estrogen and if this affects responsiveness to estrogen and/or antiestrogen. We will also determine whether a specific histone modification affects cell responsiveness to estrogen using expression of mutant ERBF histones.
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