Pituitary-specific chromatin structure of the rat prolactin distal enhancer element.

The location of target DNA sequences within chromatin may affect the ability of trans-acting factors to bind cis-elements and regulate gene transcription. To examine the effect of chromatin structure on the ability of the estrogen-estrogen receptor complex (E2R) to bind its respective DNA binding element within the rat prolactin (rPRL) gene and modulate rPRL gene expression, we have developed cell lines derived from the rPRL-expressing (rPRL+) rat pituitary cell line GH3 and the rPRL-non- expressing (rPRL-) rat embryo fibroblast cell line Rat1. These cell lines contain mini-chromosomes composed of the 5' upstream regulatory region of the rPRL gene driving expression of a reporter gene, Tn5, within a bovine papillomavirus (BPV) vector. The rPRL-Tn5 gene retains the characteristics of cell-specific expression and estrogen inducibility of transcription displayed by the endogenous rPRL gene. The distal enhancer region, which contains an estrogen response element, was found to exist in a nucleosome-free region in pituitary-derived cells even in the absence of estrogen. In contrast, the rPRL distal enhancer in fibroblast cells was found to be randomly packaged into nucleosomes. These results indicate that DNA sequence is not sufficient to position nucleosomes in the rPRL gene. Rather, it suggests that cell-specific factors are present in pituitary cells that modify the chromatin structure of the distal enhancer which allow E2R to bind to its response element.

Estrogen-mediated induction of rat prolactin gene transcription requires the formation of a chromatin loop between the distal enhancer and proximal promoter regions.

Rat PRL (rPRL) gene transcription is controlled by proteins that interact with two DNA elements located in the 5'-upstream regulatory region. These elements, the distal enhancer and the proximal promoter, are separated by nearly 1500 bp of DNA. Induction of rPRL transcription by the estrogen 17 beta-estradiol (E2) is conferred through the estrogen response element located in the distal enhancer. To activate transcription, the estrogen-estrogen receptor complex must "communicate" with RNA polymerase II located at the proximal promoter. Using a novel nuclear ligation assay, it is shown that estrogen treatment of rat pituitary GH3 cells stimulates the formation of chromatin loops between the distal enhancer and proximal promoter of the rPRL gene, juxtaposing these two transcriptional control regions. The formation of these chromatin loops was observed in both the endogenous rPRL gene and in a rPRL-Tn5 minichromosome. Induction of rPRL and rPRL-Tn5 expression by E2 was reduced by coincubation of cells with the antiestrogen 4-hydroxytamoxifen. Likewise, 4-hydroxytamoxifen was found to block the E2-induced formation of chromatin loops between the distal and proximal regions. Concurrent treatment with the synthetic glucocorticoid dexamethasone reduced the E2-induced transcription rate of the rPRL and rPRL-Tn5 gene to near basal levels. Similarly, dexamethasone treatment inhibited the ability of E2 to enhance the formation of the chromatin loops. These data suggest that the activation of rPRL gene transcription by E2 involves the stabilization of a chromatin loop that facilitates protein-protein interactions between transcription factors that are associated with the distal enhancer and the proximal promoter.

Interaction between transcription regulatory regions of prolactin chromatin.

The regulation of transcription requires complex interactions between proteins bound to DNA sequences that are often separated by hundreds of base pairs. As demonstrated by a nuclear ligation assay, the distal enhancer and the proximal promoter regions of the rat prolactin gene were found to be juxtaposed. By acting through its receptor bound to the distal enhancer, estrogen stimulated the interaction between the distal and proximal regulatory regions two- to threefold compared to control values. Thus, the chromatin structure of the prolactin gene may facilitate the occurrence of protein-protein interactions between transcription factors bound to widely separated regulatory elements.

An interaction between the 5' flanking distal and proximal regulatory domains of the rat prolactin gene is required for transcriptional activation by estrogens.

In vitro studies have demonstrated that the estrogen receptor (ER) can bind to the rat PRL estrogen response element (ERE) located 1700 basepairs upstream of the transcriptional start site. However, the mechanism by which the receptor-DNA complex influences the activity of RNA polymerase located in the promoter region is not understood. To begin investigating this process, we developed cell lines derived from GH3 cells that contain steroid-responsive bovine papillomavirus minichromosomes. Within these minichromosomes is a hybrid gene composed of the 5' flanking region of the PRL gene, driving the expression of the Tn5 gene. The episomal PRL DNA sequences responded to 17 beta-estradiol (E2) by increasing the rate of Tn5 gene transcription. Nucleosome mapping experiments using micrococcal nuclease demonstrated that nucleosome-like structures were assembled on the minichromosome in an ordered array separated by 150-200 basepairs of DNA. Novel S1 nuclease as well as DNase-I-hypersensitive sites in the chromatin of the promoter and distal regulatory regions of the episomal PRL gene were detected by indirect end-labeling studies. The nuclease hypersensitive sites in the distal region containing the ERE were modified after treatment of the cells with either E2 or the antiestrogen 4-hydroxytamoxifen. However, only E2 treatment of cells resulted in an increase in the nuclease hypersensitivity of the promoter region and induced gene expression, while antiestrogen treatment had no effect on either parameter. This suggests that complex interactions between factors located at the distal and proximal regulatory regions ultimately determine the transcriptional response of the PRL gene to E2.

Transcriptional regulation by estrogen of episomal prolactin gene regulatory elements.

As a first step in defining the role of chromatin structure in steroid-regulated gene transcription, we have established a steroid-responsive minichromosome system that contains the 5' upstream regulatory region of the rat PRL gene (PRL) from -10 to -1960-basepairs fused to the antibiotic resistance gene, Tn5. The hybrid gene was inserted into a bovine papilloma virus (BPV) vector and then transfected into GH3 cells. Southern analysis of total genomic DNA revealed that the PRL-Tn5-BPV DNA existed in the cells as unrearranged episomes or minichromosomes at a level of 25-100 copies/cell. We monitored the estrogen responsiveness of the minichromosome-based PRL regulatory regions by measuring Tn5 mRNA levels. Treatment of GH3 cells for 48 h with 10 nM 17 beta-estradiol (E2) increased Tn5 mRNA levels 3- to 6-fold over those in untreated cells. Concurrently, endogenous PRL mRNA levels were induced 8- to 15-fold. Using nuclear run-on assays, it was found that E2 increased PRL-Tn5 transcription rates approximately 3-fold over those in untreated cells. The induced transcription was mediated through the PRL elements and not through any other cis-acting elements within the minichromosome. The PRL elements that contain a functional enhancer are located 3' downstream of the BPV early gene promoters in the minichromosome. However, there was no detectable effect of the PRL enhancer on BPV early gene transcription. Thus, we have established a minichromosome system containing the transcriptional regulatory elements of the rat PRL gene that responds to E2 in a manner very similar to the endogenous rat PRL gene.

Characterization of D-myo-inositol 1,4,5-trisphosphate phosphatase in rat liver plasma membranes.

D-myo-Inositol 1,4,5-trisphosphate has been previously demonstrated to act as a second messenger for the hormonal mobilization of intracellular calcium in rat liver. In this study, the breakdown of D-myo-inositol 1,4,5-trisphosphate by a phosphatase activity was characterized. Using partially purified subcellular fractions, it was found that D-myo-inositol 1,4,5-trisphosphate phosphatase (I-P3ase) specific activity was highest in the plasma membrane fraction, while D-myo-inositol 1,4-bisphosphate phosphatase specific activity was highest in the cytosolic and microsomal fractions. The plasma membrane I-P3ase was Mg2+-dependent with optimal activity observed at 0.5-1.5 mM free Mg2+. The enzyme had a neutral pH optimum, suggesting that it was neither an acid nor alkaline phosphatase. Neither LiCl nor NaF inhibited the I-P3ase activity. However, both L-cysteine and dithiothreitol stimulated the activity 2-fold. Spermine (2.0 mM) inhibited the I-P3ase activity by 50%, while putrescine and spermidine had little or no effect.

Rapid diglyceride phosphorylation in isolated bovine rod outer segments.

When isolated bovine rod outer segment fragments were incubated with [gamma-32P]ATP, 32P, as revealed by autoradiography, was rapidly incorporated into rhodopsin bands on sodium dodecyl sulfate polyacrylamide gels, and into a low Mr lipid band. Incorporation of 32P into rhodopsin was light-dependent, but labeling of the lipid band was not. A single phosphorylated product, phosphatidic acid, was identified by 2-dimensional thin layer chromatography and by high pressure liquid chromatography of the corresponding glycerophosphate ester. Incorporation of label into phosphatidic acid was detected as early as 15 sec following start of incubation and the product was stable for at least 30 min. No other products were detected, indicating that under the experimental conditions phosphatidic acid was not metabolized to other phospholipids. Up to 1 mol phosphatidic acid was formed per 18 to 40 mol rhodopsin present.

Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes.

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.

Subcellular site and mechanism of vasopressin-stimulated hydrolysis of phosphoinositides in rat hepatocytes.

The intracellular site of vasopressin-induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 s of vasopressin treatment of hepatocytes prelabeled with 32Pi, the levels of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4,5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%, then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P] PI-P and [32P]PI-P2 were observed in the other subcellular fractions, and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol in the plasma membrane decreased by 15% after 45 s of vasopressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes, it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphosphoinositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (200 nM) of free calcium when assayed at low ionic strength, but the calcium requirement shifted to micromolar concentrations under isosmotic, intracellular-like, ionic conditions. Addition of vasopressin (200 nM) to the 32P-labeled plasma membranes stimulated the breakdown of 20% of the [32P]PI-P2 present in the plasma membranes in 1 min when assayed under isosmotic conditions in the presence of 2 nM MgCl2 and approximately 200 nM free calcium. This suggests that the phosphoinositide-specific phosphodiesterase is not active under normal cellular conditions, but is activated upon the addition of vasopressin to the intact cell.