The extraction by micrococcal nuclease of ghucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated

Abstract

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.

Control of gene expression by glucocorticoid hormones

“…This points to the importance, for hormone action, of the site of retrovirus integration and, presumably, of chromatin conformation. This is consistent with the observation (Andre et al, 1980) that in the normal host (mouse mammary tumour) cell, mild nuclease treatment of the nuclei preferentially releases the receptor together with a small proportion of MTV DNA. This fraction, containing the few glucocorticoidsensitive copies of MTV DNA, would correspond to 'active chromatin'.…”

The Action of Growth and Developmental Hormones

“…As one approach to demonstrating possible heterogeneity of nuclear receptors, several investigators are studying the distribution of receptors in different fractions of chromatin. Thus, fractionation of chromatin or nuclei by ion-exchange chromatography or nuclease digestion has suggested that the nuclear receptors for steroid and thyroid hormones are nonrandomly distributed on the chromatin (Charles et at., 197 5;de Boer et at., 1978;Franceschi and Kim, 1979;Andre et at., 1980;Cidlowski and Munck, 1980;Davies et at., 1980;Jump and Oppenheimer, 1980;Scott and Frankel, 1980;Pavlik and Katzenellenbogen, 1982). Other investigators have observed that components with the characteristics of steroid hormone receptors are also localized in non chromatin fractions derived from their target cell nuclei.…”

“…Thus, it was reported that estrogen treatment (and, therefore, the presence of hormone-receptor complexes in nuclei) enhances the overall sensitivity to DNase I of the genome (Senior and Frankel, 1978) as well as specifically of that of the ovalbumin gene (Garel and Axel, 1976;Weintraub and Groudine, 1976;. Micrococcal nuclease has also been reported to preferentially release from chromatin the nuclear receptor for estrogen (Andre et at., 1978(Andre et at., , 1980 and thyroid hormone (Gardner, 1978). Again, of course, the starting materials are complex and poorly characterized, and the results therefore difficult to interpret.…”

Identification of glucocorticoid-induced genes in rat hepatoma cells by isolation of cloned cDNA sequences.

“…It is likely that one of the principal effects of steroids in the nucleus is an alteration in the transcriptional rates of specific genes. Although numerous studies have been undertaken to characterize the in vivo binding of steroid hormone-receptor complexes to chromatin (10)(11)(12)(13)(14) and other nuclear components (15,16), the crucial nuclear acceptor molecules for the steroidreceptor complex have not yet been identified.…”