Örjan Wrange

Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region

Glucocorticoid receptor protein stimulates transcription initiation within murine mammary tumor virus (MTV) DNA sequences in vivo, and interacts selectively with MTV DNA in vitro. We mapped and compared five regions of MTV DNA that are bound specifically by purified receptor; one resides upstream of the transcription start site, and the others are distributed within transcribed sequences between 4 and 8 kb from the initiation site. Each region contains at least two strong binding sites for receptor, which itself appears to be a tetramer of 94,000 dalton hormone-binding subunits. Three of the five binding regions contain nine nuclease footprints that lack extensive homology, although a family of related octanucleotides can be discerned. Receptor interacts with the different regions with similar efficiencies, suggesting that receptor affinity for upstream and internal regions may differ by less than one order of magnitude. Moreover, each region appears to be bound independent of the others. A restriction fragment containing four footprint sequences from one of the regions has previously been shown to act in vivo as a receptor-dependent transcriptional enhancer element, implying that the binding sites detected in vitro may be biologically functional.

Glucocorticoid receptor-glucocorticoid response element binding stimulates nucleosome disruption by the SWI/SNF complex.

The organization of DNA in chromatin is involved in repressing basal transcription of a number of inducible genes. Biochemically defined multiprotein complexes such as SWI/SNF (J. Côté, J. Quinn, J. L. Workman, and C. L. Peterson, Science 265:53-60, 1994) and nucleosome remodeling factor (T. Tsukiyama and C. Wu, Cell 83:1011-1020, 1995) disrupt nucleosomes in vitro and are thus candidates for complexes which cause chromatin decondensation during gene induction. In this study we show that the glucocorticoid receptor (GR), a hormone-inducible transcription factor, stimulates the nucleosome-disrupting activity of the SWI/SNF complex partially purified either from HeLa cells or from rat liver tissue. This GR-mediated stimulation of SWI/SNF nucleosome disruption depended on the presence of a glucocorticoid response element. The in vitro-reconstituted nucleosome probes used in these experiments harbored 95 bp of synthetic DNA-bending sequence in order to rotationally position the DNA. The GR-dependent stimulation of SWI/SNF-mediated nucleosome disruption, as evaluated by DNase I footprinting, was 2.7- to 3.8-fold for the human SWI/SNF complex and 2.5- to 3.2-fold for the rat SWI/SNF complex. When nuclear factor 1 (NF1) was used instead of GR, there was no stimulation of SWI/SNF activity in the presence of a mononucleosome containing an NF1 binding site. On the other hand, the SWI/SNF nucleosome disruption activity increased the access of NF1 for its nucleosomal binding site. No such effect was seen on binding of GR to its response element. Our results suggest that GR, but not NF1, is able to target the nucleosome-disrupting activity of the SWI/SNF complex.

Accessibility of a glucocorticoid response element in a nucleosome depends on its rotational positioning.

Gene expression requires binding of transcription factors to their cognate DNA response elements, the latter being often integrated into sequence-specifically positioned nucleosomes. To investigate the constraints imposed on factor-DNA recognition by the nucleosomal organization, we studied the binding of glucocorticoid receptor to a single glucocorticoid response element (GRE) displaying four different rotational frames in three different translational positions in reconstituted nucleosomes. We demonstrate that rotational setting of the GRE per se is important for its accessibility. Furthermore, the effects of rotational positioning of the GRE are different for different translational positions of the GRE in the nucleosome. A GRE placed near the nucleosomal dyad is totally blocked by rotating it 180 degrees so that the major groove of the GRE faces the histone octamer. If, on the other hand, the GRE is placed about 40 bp from the nucleosome dyad, then the 180 degrees rotation of the GRE still allows glucocorticoid receptor binding, albeit with a sixfold lower affinity than the peripherally oriented GRE. This suggests that both the rotational positioning and the translational positioning function as a framework for transcription factor response elements in gene regulation.

Hormone activation induces nucleosome positioning in vivo

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone‐ and receptor‐dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)‐binding region was detected. The presence of only a single GR‐binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone.

Estrogen receptor concentrations in 269 cases of histologically classified human breast cancer

This report provides a histological review of 264 female primary breast cancers analyzed for estrogen receptor protein (ER). We also describe 5 cases of male breast cancer all of which bound estradiol specifically. Generally the ER concentrations were lower in the tumors of premenopausal women than in those of postmenopausal women. Three types of cancer with specific morphological features were shown to have ER concentrations that differed significantly from the other types of cancer. Medullary and comedo carcinoma had very low and papillary carcinoma very high levels of ER. No obvious trend in ER concentration was found in unspecified ductal carcinoma, colloid carcinoma, nor in lobular carcinoma. A positive correlation was demonstrated between ER content and degree of differentiation in ductal carcinoma. Cancer with lymphoid infiltration generally showed low ER levels.

Formation and characterisitcs of hepatic dexamethasone-receptor complexes of different molecular weight

The dexamethasone-binding receptor protein in rat liver cytosol has a Stokes radius of 61 A and a sedimentation coefficient of 4.0 S. In contrast, cell nuclei labelled with [3H]dexamethasone in vivo or in vitro (reconstitution experiments with [3H]dexamethasone-labelled cytosol and isolated unlabelled nuclei) contain a high-salt-extractable dexamethasone-receptor complex with a Stokes radius of 30-36 A and a sedimentation coefficient of 3.2 S. Exposure of liver homogenate or 1000 X g homogenate supernatant to low ionic strength during preparation of cytosol resulted in conversion of the 61 A to a 36 A complex very similar to the intranuclear form of dexamethasone receptor. 61 leads to 36 A complex-converting activity was present in both the 100 X g-10 000 X g sediment of liver homogenate, from which it could be extracted by hypotonic media, and in the liver cell nuclei, from which it could be extracted by hypertonic media. Mild digestion of the 61 A dexamethasone-receptor complex with trypsin also gave rise to a complex with a Stokes radius of 36 A. Reconstitution experiments with isolated liver cell nuclei indicated that both the 61 A and 36 A dexamethasone-receptor complexes were taken up by the nuclei; reextraction of the nuclei incubated with the 61 A complex revealed that this form had been converted to the 30-36 A complex. Further digestion of the 61 and 36 A [3H]dexamethasone-receptor complexes with hypotonic extract of the 1000 X g-10 000 X g sediment of liver homogenate or with trypsin resulted in formation of a third complex with Stokes radius of 19 A and a sedimentation coefficient of 2.5 S. The approximate molecular weights of the 61, 36 and 19 A dexamethasone-receptor complexes were calculated as 102 000, 46 000 and 19 000, respectively, and the frictional ratios of the molecules as 1.84, 1.38 and 1.00, respectively. It is concluded that the nuclear 30-36 A dexamethasone-receptor complex is formed from the cytosol 61 A complex by proteolytic digestion and that this latter protein contains at least two sites with a relatively high sensitivity to protelytic cleavage.

Identification of SLC38A7 (SNAT7) Protein as a Glutamine Transporter Expressed in Neurons

The SLC38 family of transporters has in total 11 members in humans and they encode amino acid transporters called sodium-coupled amino acid transporters (SNAT). To date, five SNATs have been characterized and functionally subdivided into systems A (SLC38A1, SLC38A2, and SLC38A4) and N (SLC38A3 and SLC38A5) showing the highest transport for glutamine and alanine. Here we present identification of a novel glutamine transporter encoded by the Slc38a7 gene, which we propose should be named SNAT7. This transporter has l-glutamine as the preferred substrate but also transports other amino acids with polar side chains, as well as l-histidine and l-alanine. The expression pattern and substrate profile for SLC38A7 shows highest similarity to the known system N transporters. Therefore, we propose that SLC38A7 is a novel member of this system. We used in situ hybridization and immunohistochemistry with a custom-made antibody to show that SLC38A7 is expressed in all neurons, but not in astrocytes, in the mouse brain. SLC38A7 is unique in being the first system N transporter expressed in GABAergic and also other neurons. The preferred substrate and axonal localization of SLC38A7 close to the synaptic cleft indicates that SLC38A7 could have an important function for the reuptake and recycling of glutamate.

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [3H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [3H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [3H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [3H]TA-GR-antibody complex compared to that of the [3H]TA-GR complex, and (III) an increased molecular size of the [3H]TA-GR-antibody complex when compared to that of the [3H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptors, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat or to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physicochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.

Hormone-induced nucleosome positioning in the MMTV promoter is reversible

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone via the glucocorticoid receptor (GR). The hormone‐triggered effects on MMTV transcription and chromatin structure were studied in Xenopus oocytes. We previously showed that the nucleosomes organizing the MMTV promoter became translationally positioned upon hormone induction. A single GR‐binding site was necessary and sufficient for the chromatin events to occur, while transcription and basal promoter elements were dispensable. Here we show that addition of the hormone antagonists RU486 or RU43044 to the previously hormone‐induced MMTV promoter results in cessation of transcription and loss of chromatin remodeling and nucleosome positioning. In vivo footprinting demonstrated agonist‐ and RU486‐induced GR binding to its DNA response element (GRE), while the other antagonist, RU43044, did not promote GR–GRE interaction. These results demonstrate that induction and maintenance of nucleosome positioning is an active process that requires constant ‘pressure’ of agonist–GR‐recruited chromatin‐modifying factor(s) rather than GR–DNA binding itself.

Estrogen and progestin receptors in intracranial meningiomas

Tissue samples from 16 meningioma patients were analysed for the cytosolic estrogen receptor concentration using isoelectric focusing and 11 of the 16 cases were also analysed for the cytosolic progestin receptor concentration using sucrose gradient centrifugation. In this study, 15 of the 16 (94%) and 9 of the 11 (82%) meningiomas had detectable estrogen and progestin receptors, respectively. The mean estrogen receptor concentration in female subjects was 8.9 fmol/mg protein and in males 3.0 fmol/mg protein. This sexual difference in receptor content was statistically significant. When the progestin receptor concentrations were plotted against the estrogen receptor concentrations in the same tumors, a relatively good positive correlation was obtained in female patients (r = 0.87). These findings confirm and extend previous reports on the existence of sex steroid receptors in human brain tumors and might open up the possibility of selective endocrine therapy against meningiomas, especially in case of poor-risk patients.