Reconstitution of the type II [3H]estradiol binding site with recombinant histone H4

Kevin Shoulars, Mary Ann Rodriguez, Jan Crowley, John Turk, Trellis Thompson, Barry M. Markaverich

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Previously, we identified the rat uterine nuclear type II [3H]estradiol binding site as histone H4 and an unknown 35 kDa protein with histone H4 immunoreactivity. Studies using calf thymus histones indicated that the 35 kDa protein was likely a dimer of histone H3 and H4. Further study of the type II site required methodology for producing sufficient quantities of recombinant histones, which retained ligand-binding properties. A variety of production methods produce sufficient quantities of histone for binding analyses were evaluated prior to finding a successful technique. The present studies describe techniques for the production of recombinant histones that retain the ligand binding properties of type II binding site. Binding studies with recombinant protein mirrored [3H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [3H]estradiol with a low affinity (Kd ∼20 nM) and in a cooperative fashion (curvilinear Scatchard plot; Hill coefficient ∼4). Although histone H3 does not appear to bind ligand, regeneration of the histone H3/H4 pair produced a 35 kDa protein equivalent to the 35 kDa protein labeled with [3H]luteolin in rat uterine nuclear extracts and calf thymus histones. These data confirm the identification of histone H4 as a key component of the type II site. Future studies with recombinant proteins will lead to the identification of the "nucleosomal ligand-binding domain" for methyl-p-hydroxyphenyllactate (MeHPLA) and related ligands and delineation of their epigenetic control of gene expression and cell proliferation.

Original languageEnglish
Pages (from-to)1-8
Number of pages8
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume99
Issue number1
DOIs
StatePublished - Apr 2006

Keywords

  • Bioflavonoid
  • Histone H4
  • Luteolin
  • Methyl-p-hydroxyphenyllactate (MeHPLA)
  • Recombinant protein
  • Type II [H] estradiol binding site

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