The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus

Abstract

Glucocorticoids are known to induce the transcription of integrated proviral mouse mammary tumour virus (MMTV) genes in a variety of cell lines derived from mouse mammary tumours. Chimaeric genes in which selectable markers are linked to the long terminal repeat (LTR) region of MMTV can be induced by the synthetic glucocorticoid dexamethasone after introduction into mouse fibroblasts. This suggests that the regulatory elements required for hormonal induction are located within the cloned LTR fragments. The idea is supported by the observation that glucocorticoid receptors bind to certain cloned fragments of MMTV DNA in vitro. Using filter binding studies and monoclonal antibodies to the glucocorticoid receptor we have now delimited the receptor binding region to a DNA segment of 152 base pairs (bp) that has been shown to be relevant for hormonal induction. In nuclease protection experiments we have identified partially homologous receptor binding sequences located in this region, all of which share the hexanucleotide 5'-TGTTCT-3'.

Control of gene expression by glucocorticoid hormones

“…Experiments with LTR deletion mutants showed that the receptor binding region was restricted to a DNA segment located between 202 and 50bp upstream from the cap (i.e. initiation) site (Scheidereit et al, 1983). It is precisely within this segment that one finds the GRE defined by the transfection experiments discussed above.…”

“…Recent experiments with the chimaeric metallothionein-thymidine kinase constructs described above showed that the purified rat receptor binds preferentially to, and protects against nuclease digestion, a DNA sequence (-248 to -263) located within the GRE. This sequence has a high degree of homology with two regions (-186 to -170 and -129 to -1 13) of the MTV-LTR that are protected against nuclease digestion (Scheidereit et al, 1983). A comparison of these segments yields the consensus sequence T(C)GGTA(T)CAA(C)A(T)TGTT(C)CT.…”

“…Specific binding of purified LTR-containing DNA to the receptor in crude cell extracts could also be shown by using a DNA-cellulose competition assay (Pfahl, 1982) or by 'fishing out' the DNA-receptor complex with an anti-receptor antibody (Scheidereit et al, 1983). This allowed one to show that inhibition of receptor transformation by molybdate inhibited DNA binding and that mouse receptor mutants deficient in nuclear transfer displayed decreased affinity for DNA.…”

Dual function of a nuclear factor I binding site in MMTV transcription regulation

“…While steroids like progestins and androgens have recently been shown to also affect MMTV transcription in mammary cells (11,12,13,14,15), it is the response to glucocorticoids in cultured fibroblasts that has been studied in greatest detail (16,17,18,19). In vitro binding of purified glucocorticoid receptor to MMTV DNA has been demonstrated, showing several binding sites within the glucocorticoid regulatory region (20,21,22).…”

“…B (distal) and C (proximal): sequence of regulatory regions and mutants therein (coding strand). Below the wild-type sequence (7), two lines indicate the DNase I footprints (on both strands) of purified glucocorticoid receptor (20). Triangles (open for protection, filled for enhancement) point to residues involved in methylation protection experiments, and squares to residues involved in methylation interference (22).…”