Structural basis of VDR-DNA interactions on direct repeat response elements

The vitamin D receptor (VDR) forms homo- or heterodimers on response elements composed of two hexameric half-sites separated by 3 bp of spacer DNA. We describe here the crystal structures at 2.7-2.8 A resolution of the VDR DNA-binding region (DBD) in complex with response elements from three different promoters: osteopontin (SPP), canonical DR3 and osteocalcin (OC). These structures reveal the chemical basis for the increased affinity of VDR for the SPP response element, and for the poor stability of the VDR-OC complex, relative to the canonical DR3 response element. The homodimeric protein-protein interface is stabilized by van der Waals interactions and is predominantly non-polar. An extensive alpha-helix at the C-terminal end of the VDR DBD resembles that found in the thyroid hormone receptor (TR), and suggests a mechanism by which VDR and TR discriminate among response elements. Selective structure-based mutations in the asymmetric homodimeric interface result in a VDR DBD protein that is defective in homodimerization but now forms heterodimers with the 9-cis retinoic acid receptor (RXR) DBD.     

Dynamic interactions between transposable elements and their hosts

Transposable elements (TEs) have a unique ability to mobilize to new genomic locations, and the major advance of second-generation DNA sequencing has provided insights into the dynamic relationship between TEs and their hosts. It now is clear that TEs have adopted diverse strategies - such as specific integration sites or patterns of activity - to thrive in host environments that are replete with mechanisms, such as small RNAs or epigenetic marks, that combat TE amplification. Emerging evidence suggests that TE mobilization might sometimes benefit host genomes by enhancing genetic diversity, although TEs are also implicated in diseases such as cancer. Here, we discuss recent findings about how, where and when TEs insert in diverse organisms.     

Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene.

The Adh (alcohol dehydrogenase, EC 1.1.1.1.) gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments.     

Identification of structural elements critical for inter-domain interactions in a group II self-splicing intron.

Thus far, conventional biophysical techniques, such as NMR spectroscopy or X-ray crystallography, allow the determination, at atomic resolution, of only structural domains of large RNA molecules such as group I introns. Determination of their overall spatial organization thus still relies on modeling. This requires that a relatively high number of tertiary interactions are defined in order to get sufficient topological constraints. Here, we report the use of a modification interference assay to identify structural elements involved in interdomain interactions. We used this technique, in a group II intron, to identify the elements involved in the interactions between domain V and the rest of the molecule. Domain V contains many of the active site components of these ribozymes. In addition to a previously identified 11 nucleotide motif involved in the binding of the domain V terminal GAAA tetraloop, a small number of elements were shown to be essential for domain V binding. In particular, we show that domain III is specifically required for the interaction with sequences encompassing the conserved 2 nucleotide bulge of domain V.     

Long-distance RNA-RNA interactions and conserved sequence elements affect potato virus X plus-strand RNA accumulation.

Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5' end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5' nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA-RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation.     

DNA protein interactions at the interferon-responsive promoter elements: potential for an H-DNA conformation.

The regions of several genes (IFI-56K, HLA-A3, HLA-DR and 6-16) containing the (putative) ISRE (Interferon Stimulatable Response Element) were tested for their ability to be recognized by HeLa cells nuclear extract proteins. In a band shift assay, all probes yielded two B1 and B2 DNA-protein complexes of similar mobilities. Unexpectedly the titration of the B1 complex with a synthetic ISRE core (OL1), promoted the formation of B2. Both the probe and OL1 were recovered in B2. For each probe, the possibility of the part of the sequence involved in B1 complex to form a H-DNA structure with the part of the sequence involved in B2 exists. Such a structure was favored by the colinearity of the pairing regions and requires ATP. Although probes seemed to have a secondary structure, the formal existence of a H-DNA structure has not been demonstrated. Such a model could be extended to other interferon inducible gene promoters and may account for their binding properties and differential inducibility after 5' deletion or point mutations.     

Cytotype regulation by telomeric P elements in Drosophila melanogaster: interactions with P elements from M' strains

P strains of Drosophila are distinguished from M strains by having P elements in their genomes and also by having the P cytotype, a maternally inherited condition that strongly represses P-element-induced hybrid dysgenesis. The P cytotype is associated with P elements inserted near the left telomere of the X chromosome. Repression by the telomeric P elements TP5 and TP6 is significantly enhanced when these elements are crossed into M′ strains, which, like P strains, carry P elements, but have little or no ability to repress dysgenesis. The telomeric and M′ P elements must coexist in females for this enhanced repression ability to develop. However, once established, it is transmitted maternally to the immediate offspring independently of the telomeric P elements themselves. Females that carry a telomeric P element but that do not carry M′ P elements may also transmit an ability to repress dysgenesis to their offspring independently of the telomeric P element. Cytotype regulation therefore involves a maternally transmissible product of telomeric P elements that can interact synergistically with products from paternally inherited M′ P elements. This synergism between TP and M′ P elements also appears to persist for at least one generation after the TP has been removed from the genotype.     

Formation of cytoskeletal elements during mouse embryogenesis. IV. Ultrastructure of primary mesenchymal cells and their cell-cell interactions

The ultrastructure of the day 8.5 mouse embryo has been studied by transmission electron microscopy, with special emphasis on the primary mesenchymal cells and their interaction with cells of the embryonic ectoderm and the proximal endoderm. The organization of the two polar epithelial cell layers (embryonic ectoderm and proximal endoderm), the isolated cells of the distal endoderm and the primary mesenchymal cells is described. Primary mesenchymal cells are different from embryonic ectoderm cells, from which they are derived, not only by the absence of desmosomes and intermediate-sized filaments of the cytokeratin type but also by their variable morphology not exhibiting stable polar architecture, and their numerous cytoplasmic processes which make contacts with the basal lamina of the ectoderm, the basal cell surface of the proximal endoderm, and other mesenchymal cells. Over most of the embryo the embryonic ectoderm is covered by a typical basal lamina, except for certain regions that are frequently characterized by cytoplasmic projections ("blebs') from the basal cell surface membrane. In contrast, the basal surface of the proximal endoderm is not covered by a continuous basal lamina and reveals mushroom-like protrusions of the cortical cytoplasm. Junctions between primary mesenchymal cells are numerous and include adhaerens-type formations of various sizes as well as gap junctions. Occasionally, a special type of junction between mesenchymal cells and embryonic ectoderm has been found, resulting in local interruptions of the basal lamina. The observations are discussed in relation to possible mechanisms of mesoderm formation and the drastic changes of cell character that accompany this process, including cytoskeletal changes such as the disappearance of cytokeratin filaments and the expression of vimentin.     

Imprinting and looping: epigenetic marks control interactions between regulatory elements

Gene regulation involves various cis-regulatory elements that can act at a distance. They may physically interact each other or with their target genes to exert their effects. Such interactions are beginning to be uncovered in the imprinted Igf2/H19 domain.(1) The differentially methylated regions (DMRs), containing insulators, silencers and activators, were shown to have physical contacts between them. The interactions were changeable depending on their epigenetic state, presumably enabling Igf2 to move between an active and a silent chromatin domain. The study gives us a novel view on how regulatory elements influence gene expression and how epigenetic modifications modulate their long-range effects.     

Cytoplasmic domain of protocadherin-alpha enhances homophilic interactions and recognizes cytoskeletal elements

Cell adhesion molecules of the protocadherin-alpha (pcdh-alpha), -beta, and -gamma families have been proposed to be synaptic specifiers. Pcdh-alpha and -gamma family members localize in part to synapses, and deletion of all pcdh-gammas in mouse affects synaptogenesis. Little is known, however, about the binding specificities and intracellular signaling of protocadherins. Using heterologous expression of tagged constructs, immunostaining, and biotinylation of surface components followed by Western blots we demonstrate that pcdh-alphas undergo homophilic interactions that are significantly enhanced by the cytoplasmic domain. Pcdh-alphas cloned from chick ciliary ganglion have one of two cytoplasmic constant regions (A- and B-types). Screening a yeast two-hybrid library of ciliary ganglion cDNA with the A-type domain yielded a fragment of neurofilament M (NFM); screening with B-type domain yielded a fragment of the actin-bundling protein fascin. Cotransfection of HEK cells with the constructs indicated that the NFM and A-type fragments codistributed as did the fascin and B-type fragments, and the latter could be coimmunoprecipitated. Antibody-induced clustering of full-length pcdh-alphas on the surface of transfected HEK cells induced coclustering of the interacting NFM fragment. Native full-length NFM in tissue extracts bound specifically to the A-type domain on beads, while native full-length fascin in tissue extracts specifically coimmunoprecipitated with pcdh-alpha. Immunostaining neurons demonstrated codistribution of full-length pcdh-alpha with both NFM and actin filaments. These findings suggest cytoskeletal links for pcdh-alphas and identify candidate targets. They also demonstrate homophilic interactions for pcdh-alphas as described for classical cadherins.     

Structure and interactions of NCAM modules 1 and 2, basic elements in neural cell adhesion.

The structure in solution of the second Ig-module fragment of residues 117-208 of NCAM has been determined. Like the first Ig-module of residues 20-116, it belongs to the I set of the immunogloblin superfamily. Module 1 and module 2 interact weakly, and the binding sites of this interaction have been identified. The two-module fragment NCAM(20-208) is a stable dimer. Removal of the charged residues in these sites in NCAM(20-208) abolishes the dimerization. Modeling the dimer of NCAM(20-208) to fit the interactions of these charges produces one coherent binding site for the formation of two antiparallel strands of the first two NCAM modules. This mode of binding could be a major element in trans-cellular interactions in neural cell adhesion.     

Transposable elements.

Transposable elements comprise a major fraction of eukaryotic genomes. They are studied both because of their intrinsic biological interest and because they can be exploited as valuable research tools. Many interesting papers dealing with various aspects of the biology of these elements have been published during the past year and a number of new elements have been reported. Four areas in which particularly valuable contributions have been made are the mechanisms of transposition, the regulation of transposition, the use of transposable elements as research tools, and the biological function of transposable elements.     

Morphine-element interactions – The influence of selected chemical elements on neural pathways associated with addiction

Addiction is a pressing social problem worldwide and opioid dependence can be considered the strongest and most difficult addiction to treat. Mesolimbic and mesocortical dopaminergic pathways play an important role in modulation of cognitive processes and decision making and, therefore, changes in dopamine metabolism are considered the central basis for the development of dependence. Disturbances caused by excesses or deficiency of certain elements have a significant impact on the functioning of the central nervous system (CNS) both in physiological conditions and in pathology and can affect the cerebral reward system and therefore, may modulate processes associated with the development of addiction. In this paper we review the mechanisms of interactions between morphine and zinc, manganese, chromium, cadmium, lead, fluoride, their impact on neural pathways associated with addiction, and on antinociception and morphine tolerance and dependence.