Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.
We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA.
Perlmann
T
T
Department of Molecular Genetics, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
Wrange
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O
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D066298:In Vitro Techniques; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009707:Nucleosomes; D011965:Receptors, Glucocorticoid; D004247:DNA; D003850:Deoxyribonuclease I