The mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions.

In the studies reported here, we have examined the properties of the Mcp element from the Drosophila melanogaster bithorax complex (BX-C). We have found that sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and that they silence adjacent reporters by a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a "pairing-sensitive" silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression.     

Chromatin looping mediates boundary element promoter interactions

One facet of the control of gene expression is long-range promoter regulation by distant enhancers. It is an important component of the regulation of genes that control metazoan development and has been appreciated for some time but the molecular mechanisms underlying this regulation have remained poorly understood. A recent study by Cleard and colleagues1 reports the first in vivo evidence of chromatin looping and boundary element promoter interaction. Specifically, they studied the function of a boundary element within the cis-regulatory region of the Abdominal-B (Abd-B) gene of Drosophila melanogaster.     

The cadherin Flamingo mediates level-dependent interactions that guide photoreceptor target choice in Drosophila

Quantitative differences in cadherin activity have been proposed to play important roles in patterning connections between pre- and postsynaptic neurons. However, no examples of such a function have yet been described, and the mechanisms that would allow such differences to direct growth cones to specific synaptic targets are unknown. In the Drosophila visual system, photoreceptors are genetically programmed to make a complex, stereotypic set of synaptic connections. Here we show that the atypical cadherin Flamingo functions as a short-range, homophilic signal, passing between specific R cell growth cones to influence their choice of postsynaptic partners. We find that individual growth cones are sensitive to differences in Flamingo activity through opposing interactions between neighboring cells and require these interactions to be balanced in order to extend along the appropriate trajectory.     

Drosophila PS2 integrin mediates RGD-dependent cell-matrix interactions.

Integrins are a family of transmembrane glycoproteins that mediate cell-matrix and cell-cell interactions. We have transfected cultured Drosophila cells with genes that express the Drosophila PS2 integrin. We demonstrate that this integrin is expressed on the surface of the cells and can mediate cell spreading on an undefined component of fetal calf serum or on the purified vertebrate matrix molecules vitronectin and fibronectin. Additionally, PS2 integrin can cause cell spreading on RGD peptide. The spreading on matrix components or RGD peptide can be inhibited by soluble RGD peptide and is dependent on divalent cations.     

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.     

The RNA transport element of the murine musD retrotransposon requires long-range intramolecular interactions for function

Retrovirus replication requires specialized transport mechanisms to export genomic mRNA from the nucleus to the cytoplasm of the infected cell. This regulation is mediated by a combination of viral and/or cellular factors that interact with cis-acting RNA export elements linking the viral RNA to the cellular CRM1 or NXF1 nuclear export pathways. Endogenous type D murine LTR retrotransposons (musD) were reported to contain an RNA export element located upstream of the 3'-LTR. Although functionally equivalent, the musD export element, termed the musD transport element, is distinct from the other retroviral RNA export elements, such as the constitutive transport element of simian/Mason-Pfizer monkey retroviruses and the RNA transport element found in rodent intracisternal A-particle LTR retrotransposons. We demonstrate here that the minimal RNA transport element (musD transport element) of musD comprises multiple secondary structure elements that presumably serve as recognition signals for the cellular export machinery. We identified two classes of tertiary interactions, namely kissing loops and a pseudoknot. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export.     

Recognition and long-range interactions of a minimal nanos RNA localization signal element

Localization of nanos (nos) mRNA to the germ plasm at the posterior pole of the Drosophila embryo is essential to activate nos translation and thereby generate abdominal segments. nos RNA localization is mediated by a large cis-acting localization signal composed of multiple, partially redundant elements within the nos 3' untranslated region. We identify a protein of approximately 75 kDa (p75) that interacts specifically with the nos +2' localization signal element. We show that the function of this element can be delimited to a 41 nucleotide domain that is conserved between D. melanogaster and D. virilis, and confers near wild-type localization when present in three copies. Two small mutations within this domain eliminate both +2' element localization function and p75 binding, consistent with a role for p75 in nos RNA localization. In the intact localization signal, the +2' element collaborates with adjacent localization elements. We show that different +2' element mutations not only abolish collaboration between the +2' and adjacent +1 element but also produce long-range deleterious effects on localization signal function. Our results suggest that higher order structural interactions within the localization signal, which requires factors such as p75, are necessary for association of nos mRNA with the germ plasm.     

The iStem, a long-range RNA secondary structure element required for efficient exon inclusion in the Drosophila Dscam pre-mRNA

The Drosophila Dscam gene encodes 38,016 different proteins, due to alternative splicing of 95 of its 115 exons, that function in axon guidance and innate immunity. The alternative exons are organized into four clusters, and the exons within each cluster are spliced in a mutually exclusive manner. Here we describe an evolutionarily conserved RNA secondary structure we call the Inclusion Stem (iStem) that is required for efficient inclusion of all 12 variable exons in the exon 4 cluster. Although the iStem governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons.     

The long-range electrostatic interactions control tRNA-aminoacyl-tRNA synthetase complex formation

In most cases aminoacyl-tRNA synthetases (aaRSs) are negatively charged, as are the tRNA substrates. It is apparent that there are driving forces that provide a long-range attraction between like charge aaRS and tRNA, and ensure formation of "close encounters." Based on numerical solutions to the nonlinear Poisson-Boltzmann equation, we evaluated the electrostatic potential generated by different aaRSs. The 3D-isopotential surfaces calculated for different aaRSs at 0.01 kT/e contour level reveal the presence of large positive patches-one patch for each tRNA molecule. This is true for classes I and II monomers, dimers, and heterotetramers. The potential maps keep their characteristic features over a wide range of contour levels. The results suggest that nonspecific electrostatic interactions are the driving forces of primary stickiness of aaRSs-tRNA complexes. The long-range attraction in aaRS-tRNA systems is explained by capture of negatively charged tRNA into "blue space area" of the positive potential generated by aaRSs. Localization of tRNA in this area is a prerequisite for overcoming the barrier of Brownian motion.     

Importance of long-range interactions in protein folding

Long-range interactions play an active role in the stability of protein molecules. In this work, we have analyzed the importance of long-range interactions in different structural classes of globular proteins in terms of residue distances. We found that 85% of residues are involved in long-range contacts. The residues occurring in the range of 4-10 residues apart contribute more towards long-range contacts in all-alpha proteins while the range is 11-20 in all-beta proteins. The hydrophobic residues Cys, Ile and Val prefer the 11-20 range and all other residues prefer the 4-10 range. The residues in all-beta proteins have an average of 3-8 long-range contacts whereas the residues in other classes have 1-4 long-range contracts. Furthermore, the preference of residue pairs to the folding and stability will be discussed.     

A novel cis-regulatory element, the PTS, mediates an anti-insulator activity in the Drosophila embryo

The Abd-B Hox gene contains an extended 3' cis-regulatory region that is subdivided into a series of separate lab domains. The lab-7 domain activates Abd-B in parasegment 12 (ps12), whereas lab-8 controls expression in ps13. iab-7 is flanked by two insulators, Fab-7 and Fab-8, which are thought to prevent regulatory factors, such as Polycomb silencers, from influencing neighboring iab domains. This organization poses a potential paradox, since insulator DNAs can work in a dominant fashion to block enhancer-promoter interactions over long distances. Here, we present evidence for a novel cis-regulatory sequence located within lab-7, the promoter targeting sequence (PTS), which permits distal enhancers to overcome the blocking effects of Fab-8 and the heterologous su(Hw) insulator. We propose that the PTS converts dominant, long-range insulators into local regulatory elements that separate neighboring lab domains.     

Functional architecture of long-range perceptual interactions

The pattern of lateral interactions in the primary visual cortex, which has emerged from recent studies, conforms to the grouping rules of similarity, proximity, smoothness and closure. The goal of this paper is to understand the perceptual salience of oriented elements that are specifically organized to form a smooth contour. An overview of recent studies, in combination with new experimental results, is presented here to emphasis the idea that visual responses depend on input from both the center and the surround of the classical receptive field (CRF). It is assumed that normal lateral interactions produce a neuronal network that is formed by two antagonistic mechanisms: (i) excitation, that is spatially organized along the optimal orientation (collinear), and is predominant near the contrast threshold of the neuron, and (ii) inhibition, that is less selective and is distributed diffusely around the cell's response field. Thus, the inputs from the CRF and the anisotropic surround are summated non-linearly. The specificity of the facilitation and suppression along the collinear direction suggests the existence of second-order elongated collinear filters, which may increase the response similarity between neurons responding to elongated stimulus, thus may enhance the perceptual salience of anisotropic configurations such as contours. This causal connection is particularly evident in amblyopes, where abnormal development of the network results in the abnormal perception of contours.     

Insulators, not Polycomb response elements, are required for long-range interactions between Polycomb targets in Drosophila melanogaster

The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb "bodies" or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.     

Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1

The miRNA pathway has been shown to regulate developmentally important genes. Dicer-1 is required to cleave endogenously encoded microRNA (miRNA) precursors into mature miRNAs that regulate endogenous gene expression. RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) that protects organisms from parasitic nucleic acids. In Drosophila, Dicer-2 cleaves dsRNA into 21 base-pair small interfering RNA (siRNA) that are loaded into RISC (RNA induced silencing complex) that in turn cleaves mRNAs homologous to the siRNAs. Dicer-2 co-purifies with R2D2, a low-molecular weight protein that loads siRNA onto Ago-2 in RISC. Loss of R2D2 results in defective RNAi. However, unlike mutants in other RNAi components like Dicer-2 or Ago-2, we report here that r2d2¹ mutants have striking developmental defects. r2d2¹ mutants have reduced female fertility, producing less than 1/10 the normal number of progeny. These escapers have normal morphology. We show R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects are dramatically enhanced when one copy of the dcr-1 gene is missing and Dicer-1 protein co-immunoprecipitates with R2D2 antisera. These data show that r2d2¹ mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function, and Dicer-1 impacts this process. We conclude that R2D2 functions beyond its role in RNA interference to include ovarian development in Drosophila.