Distribution of Acceptor Sites for Androgen-Receptor Complexes Between Transcriptionally Active and Inactive Fractions of Rat Ventral Prostate Chromatin

Abstract

Rat ventral prostate chromatin was separated into two main fractions by controlled digestion with micrococcal nuclease. The soluble fraction obtained after lysis of digested nuclei with EDTA (1 mmol/l), the S2 fraction, represented approximately 17% of the original nuclear DNA, and showed properties consistent with transcriptional activity, i.e. enrichment in nascent RNA, non-histone protein and endogenous RNA polymerase B activity as well as depletion in histones. The fraction sedimented after lysis of nuclei, fraction P, comprised approximately 60% of nuclear DNA, was depleted in nascent RNA, non-histone proteins and endogenous RNA polymerase B activity, but had a higher content of histones. In an attempt to relate the concentration of acceptor sites for androgen-receptor complexes with transcriptional activity, it was shown that the S2 fraction was enriched in these acceptor sites. However, if measurements were based on the intact cell the transcriptionally inactive portion contained 2.5--3 times as many 'acceptor' sites, although these sites had lower affinity for androgen-receptor complexes than had those in the transcriptionally active fraction.

Association of glucocorticoid receptors with prostate nuclear sites for androgen receptors and with androgen response elements

“…3b). In concordance with AR distribution within prostate chromatin (Rennie, 1979;Davies et al 1980), GR was associ¬ ated with dinucleosomes and higher oligomers, but not mononucleosomes, although the diffuse pattern of GR preceding the monomer peak in each case was suggestive of association with particles smaller than mononucleosomes. This was verified using a differ¬ ent sedimentation regime (co2t=l-26x 1012 rad2.s2) in the absence and presence of 0-6 M KC1 (Fig.…”

“…Preparation of nuclei and nuclear fractions Details of homogenization of tissue and the buffers employed have been given elsewhere (Davies, Thomas, Borthwick & Giles, 1980;Davies, Thomas & Giles, 1982;Davies, 1983;Davies & Thomas, 1984). These conditions of homogenization yield predominantly epithelial cells.…”

“…These conditions of homogenization yield predominantly epithelial cells. Purification of nuclei and digestion with micrococcal nuclease have also been described previously (Davies et al 1980(Davies et al , 1982Davies, Thomas & Manning, 1986). Input of nuclei and yield of fractions were estimated spectrophotometrically at 260 nm and verified colorimetrically or fluorimetrically.…”

“…Information regarding digestion procedures in individual experiments is given where necessary in the text. Fractions result¬ ing from controlled nucleolysis by our procedure have been characterized extensively (Davies et al 1980(Davies et al , 1982(Davies et al , 1986. Briefly, the fractions are desig¬ nated SI (rapidly released, some by shearing, and acid-soluble fragments), S2 (EDTA-solubilized oligomeric chromatin) and P (those nuclear regions refractory to solubilization at a given concentration of nuclease or over a defined digestion period).…”

“…Glucocorticoid receptors (GR) in rat ventral prostate chromatin. Ventral prostate nuclei from rats castrated (a) 24 h or (b) 72 h previously were digested with micrococcal nuclease, and S2 fractions (see Materials and Methods, and Davies et al 1980) analysed on isokinetic gradients of sucrose (Davies & Thomas, 1984) Davies et al 1980Davies et al , 1982Davies et al , 1986 analysed on isokinetic gradients of sucrose (Davies & the presence of KC1 all radioactivity dissociated from the nucleosomal and prenucleosomal regions of the gradient to a single peak in the area of the gradient occupied by free [3H]GR (Fig. 4b).…”