Nuclear type II [3H]estradiol binding sites: a histone H3-H4 complex.
[(3)H]luteolin covalently labels two forms (11kDa and 35kDa proteins) of type II binding sites in rat uterine nuclear extracts [K. Shoulars, T. Brown, M. Alejandro, J. Crowley, B. Markaverich, Identification of rat uterine nuclear type II [(3)H]estradiol binding sites as histone H4, Biochem. Biophys. Res. Commun. 296 (2002) 1083-1090]. The 11kDa protein was identified as histone H4. Levels of the 35kDa protein were insufficient for sequencing; however, this protein was recognized by anti-histone H4 antibodies. Histones H3 and H4 exist as dimers in vivo (mw>>35kDa) and we suspected the 35kDa [(3)H]luteolin-labeled protein in uterine nuclear extracts might be a complex of histones H3 and H4. This manuscript describes methods for the purification of commercially available calf thymus core histones that retain [(3)H]luteolin binding activity and are of sufficient purity for recombination studies. Mixing experiments with pure H3 and H4 from calf thymus demonstrate that a 35kDa H3-H4 dimer capable of binding [(3)H]luteolin is generated and this protein appears equivalent to the 35kDa [(3)H]luteolin binding protein in rat uterine nuclear extracts. If this is the case, type II site ligands including MeHPLA, luteolin, and other bioflavonoids and phytoestrogens may control histone-dependent gene transcription and cellular proliferation via binding to and modulating core histone/nucleosome function.
Shoulars
Kevin
K
Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498, USA.
Rodrigues
Mary Ann
MA
Crowley
Jan R
JR
Turk
John
J
Thompson
Trellis
T
Markaverich
Barry M
BM
D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D020033:Protein Isoforms / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D014599:Uterus / Q000378:metabolism*
D006657:Histones; D020033:Protein Isoforms; D004958:Estradiol