Quantification of transcription factor-DNA binding affinity in a living cell.

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis.

The polyglutamine-expanded androgen receptor has increased DNA binding and reduced transcriptional activity.

Expansion of a polyglutamine-encoding trinucleotide CAG repeat in the androgen receptor (AR) to more than 37 repeats is responsible for the X-linked neuromuscular disease spinal and bulbar muscular atrophy (SBMA). Here we evaluated the effect of polyglutamine length on AR function in oocytes. This allowed us to correlate the nuclear AR concentration to its capacity for specific DNA binding and transcription activation .

Linker histone subtypes differ in their effect on nucleosomal spacing in vivo.

Linker histone H1 is located on the surface of the nucleosome where it interacts with the linker DNA region and stabilizes the 30-nm chromatin fiber. Vertebrates have several different, relatively conserved subtypes of H1; however, the functional reason for this is unclear. We have previously shown that H1 can be reconstituted in Xenopus oocytes, cells that lack somatic H1, by cytosolic mRNA injection and incorporated into in vivo assembled chromatin.

FoxA1 and glucocorticoid receptor crosstalk via histone H4K16 acetylation at a hormone regulated enhancer.

The forkhead transcription factor FoxA1 participates in many gene regulatory events with steroid hormone receptors, one example being the integrated mouse mammary tumor virus (MMTV) promoter. Its enhancer harbors several FoxA1 binding sites. FoxA1 promotes glucocorticoid receptor (GR)-DNA binding and transcription.

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.

FoxA1 binding directs chromatin structure and the functional response of a glucocorticoid receptor-regulated promoter.

Reconstitution of the glucocorticoid receptor (GR)-regulated mouse mammary tumor virus (MMTV) promoter in Xenopus oocytes was used to monitor the effects of different transcription factor contexts. Three constitutively binding factors, nuclear factor 1 (NF1), octamer transcription factor 1 (Oct1), and the Forkhead box A1 (FoxA1), were shown to act in concert, to direct the chromatin structure, and to enhance the GR response. FoxA1 has a dominant effect in the absence of hormone and induces a cluster of DNase I-hypersensitive sites in the segment comprising bp -400 to +25.

Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter.

Transcription from the mouse mammary tumor virus (MMTV) promoter is induced by the glucocorticoid receptor (GR). This switch was reconstituted in Xenopus oocytes. Previously, we showed that Nuclear Factor 1 (NF1) and Octamer Transcription Factor 1 (Oct1) bind constitutively to the MMTV promoter and thereby induce translational nucleosome positioning representing an intermediary, i.

A synthetic snRNA m3G-CAP enhances nuclear delivery of exogenous proteins and nucleic acids.

Accessing the nucleus through the surrounding membrane poses one of the major obstacles for therapeutic molecules large enough to be excluded due to nuclear pore size limits. In some therapeutic applications the large size of some nucleic acids, like plasmid DNA, hampers their access to the nuclear compartment. However, also for small oligonucleotides, achieving higher nuclear concentrations could be of great benefit.

Mechanism of histone H1-stimulated glucocorticoid receptor DNA binding in vivo.

Xenopus oocytes lack somatic linker histone H1 but contain an oocyte-specific variant, B4. The glucocorticoid receptor (GR) inducible mouse mammary tumor virus (MMTV) promoter was reconstituted in Xenopus oocytes to address the effects of histone H1. The expression of Xenopus H1o [corrected] (H1) via cytoplasmic mRNA injection resulted in H1 incorporation into in vivo assembled chromatin based on (i) the appearance of a chromatosome stop, (ii) the increased nucleosome repeat length (NRL), and (iii) H1-DNA binding assayed by chromatin immunoprecipitation (ChIP).

FoxA1 binding to the MMTV LTR modulates chromatin structure and transcription.

Novel binding sites for the forkhead transcription factor family member Forkhead box A (FoxA), previously referred to as Hepatocyte Nuclear Factor 3 (HNF3), were found within the mouse mammary tumor virus long terminal repeat (MMTV LTR). The effect of FoxA1 on MMTV LTR chromatin structure, and expression was evaluated in Xenopus laevis oocytes. Mutagenesis of either of the two main FoxA binding sites showed that the distal site, -232/-221, conferred FoxA1-dependent partial inhibition of glucocorticoid receptor (GR) driven MMTV transcription.

Nuclear factor 1 and octamer transcription factor 1 binding preset the chromatin structure of the mouse mammary tumor virus promoter for hormone induction.

When the mouse mammary tumor virus (MMTV) is integrated into the genome of a mammalian cell, its long terminal repeat (LTR) harbors six specifically positioned nucleosomes. Transcription from the MMTV promoter is regulated by the glucocorticoid hormone via the glucocorticoid receptor (GR). The mechanism of the apparently constitutive nucleosome arrangement has remained unclear.

Chromatin-mediated restriction of nuclear factor 1/CTF binding in a repressed and hormone-activated promoter in vivo.

Mouse mammary tumor virus (MMTV) promoter-driven transcription is induced by glucocorticoid hormone via binding of the glucocorticoid receptor (GR). The MMTV promoter also harbors a binding site for nuclear factor 1 (NF1). NF1 and GR were expressed in Xenopus oocytes; this revealed GR-NF1 cooperativity both in terms of DNA binding and chromatin remodeling but not transcription.

Structure and flexibility of individual immunoglobulin G molecules in solution.

In contrast to averaging methods of determining structure, such as X-ray diffraction, NMR, and single-particle tomography, cryo-electron tomography allows three-dimensional imaging of an individual object in solution. The method has previously been used to study cells and very large macromolecules. We have used cryo-electron tomography to analyze a monoclonal IgG, with a molecular weight of only 150 kDa.

Trichostatin A reduces hormone-induced transcription of the MMTV promoter and has pleiotropic effects on its chromatin structure.

The deacetylase inhibitor trichostatin A (TSA) has long been used to study the relationship between gene transcription and the acetylation status of chromatin. We have used Xenopus laevis oocytes to study the effects of TSA on glucocorticoid receptor (GR)-dependent transcription and we have related these effects to changes in the chromatin structure of a reporter mouse mammary tumor virus (MMTV) promoter. We show that TSA induces a low level of constitutive transcription.

The Chironomus tentans translation initiation factor eIF4H is present in the nucleus but does not bind to mRNA until the mRNA reaches the cytoplasmic perinuclear region.

In the cell nucleus, precursors to mRNA, pre-mRNAs, associate with a large number of proteins and are processed to mRNA-protein complexes, mRNPs. The mRNPs are then exported to the cytoplasm and the mRNAs are translated into proteins. The mRNAs containing in-frame premature stop codons are recognized and degraded in the nonsense-mediated mRNA decay process.