Interactions between glucocorticoid receptor-bearing chromatin and antireceptor antibody preparations.
Recent evidence indicates that the control of gene expression by steroid hormones is mediated by hormone-receptor complexes bound at specific chromosomal locations. The isolation of these in vivo sites of binding would be useful in an analysis of the mechanism of this control. We have therefore examined the interaction between two different antiglucocorticoid receptor antibody preparations and a defined chromatin fraction containing bound glucocorticoid receptors in order to test the feasibility of this approach. Both antibody preparations, when attached to sepharose, removed nucleosome-bound and nucleosome free receptor from solution, indicating that chromatin-bound receptor was exposed and available for reaction with antibody. The bulk of the chromatin, not containing receptor, was mainly unaffected during these reactions, showing that the antibodies exhibited significant specificity. To determine whether the nucleosome-bound receptor remained attached to the nucleosome during reaction with antibody, studies using soluble antibody were performed. One of the antibodies caused a shift in the sedimentation rate of the nucleosome-bound receptor from 11S to 11.5S, suggesting that an intact ternary complex of antibody-receptor-nucleosome had formed. The other antibody produced various-sized aggregates of the free and bound receptors. Surprisingly, we found that one of the antibodies reacted strongly with free and nucleosome-bound estrogen receptors as well as glucocorticoid receptors. These studies suggest that an antibody preparation with appropriate characteristics should permit isolation of chromosomal receptor binding sites.
D000368:Aged; D000818:Animals; D000937:Antigen-Antibody Reactions; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000276:immunology / Q000378:metabolism*; D014221:Triamcinolone / Q000378:metabolism; D014599:Uterus / Q000378:metabolism
D002843:Chromatin; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D011965:Receptors, Glucocorticoid; D014221:Triamcinolone