Interaction of a protein factor within a thyroid hormone-sensitive region of rat alpha-myosin heavy chain gene

Using a gel mobility-shift assay, a nuclear protein factor was identified in cardiac myocyte which binds specifically to a DNA fragment from the 5' region of the alpha-myosin heavy chain gene shown previously to contain a thyroid hormone-sensitive element. Methylation interference experiments located the binding site within a 24-base pair sequence from positions -599 to -576. A double-stranded synthetic oligonucleotide containing this 24-base pair sequence bound to the factor and effectively competed with the natural binding site for factor binding. The factor was present in rat and human fibroblasts, and rat GH1 cells as well as L6E9 myoblasts and myotubes. The specificity with which this factor binds to DNA suggests that it could be involved in regulation of the alpha-myosin heavy chain gene.     

Domains of ERRgamma that mediate homodimerization and interaction with factors stimulating DNA binding.

The estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is an orphan member of the nuclear receptor superfamily closely related to the estrogen receptors. To explore the DNA binding characteristics, the protein-DNA interaction was studied in electrophoretic mobility shift assays (EMSAs). In vitro translated ERRgamma binds as a homodimer to direct repeats (DR) without spacing of the nuclear receptor half-site 5'-AGGTCA-3' (DR-0), to extended half-sites, and to the inverted estrogen response element. Using ERRgamma deletion constructs, binding was found to be dependent on the presence of sequences in the ligand binding domain (LBD). A far-Western analysis revealed that ERRgamma forms dimers even in the absence of DNA. Two elements, located in the hinge region and in the LBD, respectively, are necessary for DNA-independent dimerization. DNA binding of bacterial expressed ERRgamma requires additional factors present in the serum and in cellular extracts. Fusion proteins of the germ cell nuclear factor (GCNF/NR6A1) with ERRgamma showed that the characteristic feature to be stimulated by additional factors can be transferred to a heterologous protein. The stimulating activity was further characterized and its target sequence narrowed down to a small element in the hinge region.     

Identification of a cis-acting element and a novel trans-acting factor of the glutamine synthetase gene in liver cells

In the mammalian liver the expression of the enzyme glutamine synthetase (GS) is restricted to a small population of hepatocytes. In cells expressing the enzyme up to 3.5% of total cellular protein is GS. In order to identify enhancer elements contributing to this extraordinarily high level of expression we focused on a region roughly 2.5 kbp upstream of the GS promoter. Gel mobility shift assays revealed binding of an unknown protein within the most distal part of this region and reportergene assays demonstrated that roughly 60 bp downstream from position -2503 are indispensable for protein binding and the full effect of the enhancer. In UV cross-link analysis a 38 kDa nuclear protein that binds to the sequence was identified in rat hepatocytes. This nuclear protein, designated as upstream binding factor of the GS gene (UFGS) seems to play an important role in high-level expression of GS in liver.     

Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana.

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.