Journal of Steroid Biochemistry & Molecular Biology 99 (2006) 1–8 Reconstitution of the type II [ 3 H]estradiol binding site with recombinant histone H4 Kevin Shoulars a,∗ , Mary Ann Rodriguez a , Jan Crowley c , John Turk c , Trellis Thompson a , Barry M. Markaverich a,b a Department of Molecular and Cellular Biology, Baylor College of Medicine One Baylor Plaza, Houston, TX 77030, USA b Center for Comparative Medicine, Baylor College of Medicine One Baylor Plaza, Houston, TX 77030, USA c Medicine Department Mass Spectrometry Facility, Washington University Medical School, St. Louis, MO 63110, USA Received 17 August 2005; accepted 3 November 2005 Abstract Previously, we identified the rat uterine nuclear type II [ 3 H]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 [ 3 H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [ 3 H]estradiol with a low affinity (K d ∼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 [ 3 H]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. © 2006 Elsevier Ltd. All rights reserved. Keywords: Type II [ 3 H] estradiol binding site; Histone H4; Luteolin; Bioflavonoid; Methyl-p-hydroxyphenyllactate (MeHPLA); Recombinant protein 1. Introduction Early studies from our laboratory described a second nuclear binding site for [ 3 H]estradiol, designated type II, that is distinct from the estrogen receptor (ER; type I site) in the rat uterus, breast and prostatic cancer cells [1–4]. Type II sites bind [ 3 H]estradiol with a lower affinity (K d ∼20 nM) than the ER or ER (K d < 1 nM), are ubiquitous, and appear to be involved in the regulation of normal and malignant cel- lular growth and proliferation in a variety of tissue and cell types [5–8]. Methyl-p-hydroxyphenyllactate (MeHPLA), a bioflavonoid metabolite, was identified as the endogenous ∗ Corresponding author. Tel.: +1 713 798 5022 (office); fax: +1 713 790 1275. E-mail address: shoulars@bcm.tmc.edu (K. Shoulars). ligand for type II sites [9–12] and inhibits the estrogenic response and blocks cell proliferation. Similarly, naturally occurring bioflavonoids such as luteolin [13] and quercetin [6] also bind to type II sites with a high affinity or covalently and antagonize the estrogenic response in the uterus, prostate and malignant cell lines [5]. These ligands do not bind to recombinant ER or ER and thus control cell growth and proliferation through independent mechanisms [14]. Luteolin is a catechol-containing bioflavonoid that binds covalently to type II sites [15] and studies with this compound facilitated the purification of [ 3 H]luteolin-labeled type II sites by a wide variety chromatographic techniques [16]. Fluoro- graphic studies with [ 3 H]luteolin-labeled type II site from rat uterine nuclear extracts identified two labeled proteins with molecular weights of 11 and 35 kDa. The 11 kDa pro- tein was sequenced and identified as histone H4. Sequence 0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.11.009