Glucocorticoid receptors recognize DNA sequences in and around murine mammary tumour virus DNA.

Abstract

In several rodent cell lines, glucocorticoids increase the transcription of murine mammary tumour virus (MMTV) proviral DNA in a process mediated by the glucocorticoid receptor. To investigate whether a direct interaction between the receptor and specific sequences on the induced genes can be implicated in the hormonal regulation of transcription, filter binding studies were performed with partially purified glucocorticoid receptor of rat liver and eight cloned MMTV proviral probes. Both the 40 000 and the 90 00 mol. wt. forms of the receptor do bind preferentially to restriction fragments containing the right 400‐500 nucleotides of the MMTV long terminal repeat units (LTR). Using LTR deletion mutants, we confirm that the right end of the LTR contains at least one binding site for the glucocorticoid receptor. In addition, the receptor binds preferentially to the mouse genomic sequences flanking at least three endogenous proviral copies, and to sequences within the env genes in some of the endogenous and exogenous proviruses. These findings are compatible with the hypothesis that steroid hormones regulate specific gene expression through a direct interaction of the hormone‐receptor complex with DNA sequences in and around the induced genes.

Control of gene expression by glucocorticoid hormones

“…The receptor also bound to MTV regions outside the LTR and to mouse genomic sequences flanking endogenous proviral copies (Geisse et al, 1982). The fact that a 45000 Mr receptor fragment, presumably devoid of the 'specifier' domain (see the Introduction), displayed DNA binding in these experiments casts doubt on the physiological significance of such receptor-DNA interactions.…”

The nucleotide sequences recognized by the glucocorticoid receptor in the rabbit uteroglobin gene region are located far upstream from the initiation of transcription.

“…To analyze what fragments in the uteroglobin gene region are bound by glucocorticoid receptor we first used nitrocellulose filter binding experiments (Geisse et al, 1982). In these experiments it is useful to compare DNA fragments of similar sizes as larger DNA fragments tend to bind the receptor nonspecifically.…”

“…In these experiments it is useful to compare DNA fragments of similar sizes as larger DNA fragments tend to bind the receptor nonspecifically. Equally important are assays conducted in the presence of competitor DNA or at salt concentrations higher than 60 mM as in these cases the non-specific binding of the receptor to DNA is markedly reduced (Geisse et al, 1982). With these precautions in mind, it is obvious from the results in Figure Id there is no preferential binding of the receptor to X DNA, we concluded that the sequences around the 5' end of the uteroglobin gene were responsible for the results shown in Figure Figure 1f).…”

“…wt. form of the glucocorticoid receptor was prepared from rat liver cytosol as previously described (Geisse et al, 1982). Routinely, the preparations are -507o pure as judged by the purity of the 90 000 mol.…”

Cross-talk between glucocorticoid receptor and AP-1

“…The active transcription factor GR is a dimer which is formed on its DNA element, the glucocorticoid response element GRE, in head-to-tail ± tail-to-head fashion. In addition to activating transcription of speci®c target genes which carry the palindromic GRE (Geisse et al, 1982;Hynes et al, 1983), the GR exerts negative in¯uences on the expression of certain genes. In principle, all nuclear receptors seem to possess this ability Beato et al, 1995).…”