DNA cruciform arms nucleate through a correlated but non-synchronous cooperative mechanism

Inverted repeat (IR) sequences in DNA can form non-canonical cruciform structures to relieve torsional stress. We use Monte Carlo simulations of a recently developed coarse-grained model of DNA to demonstrate that the nucleation of a cruciform can proceed through a cooperative mechanism. Firstly, a twist-induced denaturation bubble must diffuse so that its midpoint is near the centre of symmetry of the IR sequence. Secondly, bubble fluctuations must be large enough to allow one of the arms to form a small number of hairpin bonds. Once the first arm is partially formed, the second arm can rapidly grow to a similar size. Because bubbles can twist back on themselves, they need considerably fewer bases to resolve torsional stress than the final cruciform state does. The initially stabilised cruciform therefore continues to grow, which typically proceeds synchronously, reminiscent of the S-type mechanism of cruciform formation. By using umbrella sampling techniques we calculate, for different temperatures and superhelical densities, the free energy as a function of the number of bonds in each cruciform along the correlated but non-synchronous nucleation pathways we observed in direct simulations.Comment: 12 pages main paper + 11 pages supplementary dat

Real-time detection of cruciform extrusion by single-molecule DNA nanomanipulation

During cruciform extrusion, a DNA inverted repeat unwinds and forms a four-way junction in which two of the branches consist of hairpin structures obtained by self-pairing of the inverted repeats. Here, we use single-molecule DNA nanomanipulation to monitor in real-time cruciform extrusion and rewinding. This allows us to determine the size of the cruciform to nearly base pair accuracy and its kinetics with second-scale time resolution. We present data obtained with two different inverted repeats, one perfect and one imperfect, and extend single-molecule force spectroscopy to measure the torque dependence of cruciform extrusion and rewinding kinetics. Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting. We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem. These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states

Non-B DB: a database of predicted non-B DNA-forming motifs in mammalian genomes

Although the capability of DNA to form a variety of non-canonical (non-B) structures has long been recognized, the overall significance of these alternate conformations in biology has only recently become accepted en masse. In order to provide access to genome-wide locations of these classes of predicted structures, we have developed non-B DB, a database integrating annotations and analysis of non-B DNA-forming sequence motifs. The database provides the most complete list of alternative DNA structure predictions available, including Z-DNA motifs, quadruplex-forming motifs, inverted repeats, mirror repeats and direct repeats and their associated subsets of cruciforms, triplex and slipped structures, respectively. The database also contains motifs predicted to form static DNA bends, short tandem repeats and homo(purine•pyrimidine) tracts that have been associated with disease. The database has been built using the latest releases of the human, chimp, dog, macaque and mouse genomes, so that the results can be compared directly with other data sources. In order to make the data interpretable in a genomic context, features such as genes, single-nucleotide polymorphisms and repetitive elements (SINE, LINE, etc.) have also been incorporated. The database is accessed through query pages that produce results with links to the UCSC browser and a GBrowse-based genomic viewer. It is freely accessible at http://nonb.abcc.ncifcrf.gov

The Unconserved Groucho Central Region Is Essential for Viability and Modulates Target Gene Specificity

Groucho (Gro) is a Drosophila corepressor required by numerous DNA-binding repressors, many of which are distributed in gradients and provide positional information during development. Gro contains well-conserved domains at its N- and C-termini, and a poorly conserved central region that includes the GP, CcN, and SP domains. All lethal point mutations in gro map to the conserved regions, leading to speculation that the unconserved central domains are dispensable. However, our sequence analysis suggests that the central domains are disordered leading us to suspect that the lack of lethal mutations in this region reflects a lack of order rather than an absence of essential functions. In support of this conclusion, genomic rescue experiments with Gro deletion variants demonstrate that the GP and CcN domains are required for viability. Misexpression assays using these same deletion variants show that the SP domain prevents unrestrained and promiscuous repression by Gro, while the GP and CcN domains are indispensable for repression. Deletion of the GP domain leads to loss of nuclear import, while deletion of the CcN domain leads to complete loss of repression. Changes in Gro activity levels reset the threshold concentrations at which graded repressors silence target gene expression. We conclude that co-regulators such as Gro are not simply permissive components of the repression machinery, but cooperate with graded DNA-binding factors in setting borders of gene expression. We suspect that disorder in the Gro central domains may provide the flexibility that allows this region to mediate multiple interactions required for repression

Groucho binds two conserved regions of LEF-1 for HDAC-dependent repression

<p>Abstract</p> <p>Background</p> <p><it>Drosophila </it>Groucho and its human Transducin-like-Enhancer of Split orthologs (TLEs) function as transcription co-repressors within the context of Wnt signaling, a pathway with strong links to cancer. The current model for how Groucho/TLE's modify Wnt signaling is by direct competition with β-catenin for LEF/TCF binding. The molecular events involved in this competitive interaction are not defined and the actions of Groucho/TLEs within the context of Wnt-linked cancer are unknown.</p> <p>Methods</p> <p>We used <it>in vitro </it>protein interaction assays with the LEF/TCF family member LEF-1, and <it>in vivo </it>assays with Wnt reporter plasmids to define Groucho/TLE interaction and repressor function.</p> <p>Results</p> <p>Mapping studies reveal that Groucho/TLE binds two regions in LEF-1. The primary site of recognition is a 20 amino acid region in the Context Dependent Regulatory domain. An auxiliary site is in the High Mobility Group DNA binding domain. Mutation of an eight amino acid sequence within the primary region (RFSHHMIP) results in a loss of Groucho action in a transient reporter assay. <it>Drosophila </it>Groucho, human TLE-1, and a truncated human TLE isoform Amino-enhancer-of-split (AES), work equivalently to repress LEF-1•β-catenin transcription in transient reporter assays, and these actions are sensitive to the HDAC inhibitor Trichostatin A. A survey of Groucho/TLE action in a panel of six colon cancer cell lines with elevated β-catenin shows that Groucho is not able to repress transcription in a subset of these cell lines.</p> <p>Conclusion</p> <p>Our data shows that Groucho/TLE repression requires two sites of interaction in LEF-1 and that a central, conserved amino acid sequence within the primary region (F S/T/P/xx y I/L/V) is critical. Our data also reveals that AES opposes LEF-1 transcription activation and that both Groucho and AES repression require histone deacetylase activity suggesting multiple steps in Groucho competition with β-catenin. The variable ability of Groucho/TLE to oppose Wnt signaling in colon cancer cells suggests there may be defects in one or more of these steps.</p

Differential Modulation of TCF/LEF-1 Activity by the Soluble LRP6-ICD

The canonical Wnt/β-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

Complement activation in myasthenia gravis (MG) may damage muscle endplate and complement regulatory proteins such as decay-accelerating factor (DAF) or CD55 may be protective. We hypothesize that the increased prevalence of severe extraocular muscle (EOM) dysfunction among African MG subjects reported earlier may result from altered DAF expression. To test this hypothesis, we screened the DAF gene sequences relevant to the classical complement pathway and found an association between myasthenics with EOM paresis and the DAF regulatory region c.-198C>G SNP (odds ratio=8.6; P=0.0003). This single nucleotide polymorphism (SNP) results in a twofold activation of a DAF 5′-flanking region luciferase reporter transfected into three different cell lines. Direct matching of the surrounding SNP sequence within the DAF regulatory region with the known transcription factor-binding sites suggests a loss of an Sp1-binding site. This was supported by the observation that the c.-198C>G SNP did not show the normal lipopolysaccharide-induced DAF transcriptional upregulation in lymphoblasts from four patients. Our findings suggest that at critical periods during autoimmune MG, this SNP may result in inadequate DAF upregulation with consequent complement-mediated EOM damage. Susceptible individuals may benefit from anti-complement therapy in addition to immunosuppression

The Stress Response Factors Yap6, Cin5, Phd1, and Skn7 Direct Targeting of the Conserved Co-Repressor Tup1-Ssn6 in S. cerevisiae

Maintaining the proper expression of the transcriptome during development or in response to a changing environment requires a delicate balance between transcriptional regulators with activating and repressing functions. The budding yeast transcriptional co-repressor Tup1-Ssn6 is a model for studying similar repressor complexes in multicellular eukaryotes. Tup1-Ssn6 does not bind DNA directly, but is directed to individual promoters by one or more DNA-binding proteins, referred to as Tup1 recruiters. This functional architecture allows the Tup1-Ssn6 to modulate the expression of genes required for the response to a variety of cellular stresses. To understand the targeting or the Tup1-Ssn6 complex, we determined the genomic distribution of Tup1 and Ssn6 by ChIP-chip. We found that most loci bound by Tup1-Ssn6 could not be explained by co-occupancy with a known recruiting cofactor and that deletion of individual known Tup1 recruiters did not significantly alter the Tup1 binding profile. These observations suggest that new Tup1 recruiting proteins remain to be discovered and that Tup1 recruitment typically depends on multiple recruiting cofactors. To identify new recruiting proteins, we computationally screened for factors with binding patterns similar to the observed Tup1-Ssn6 genomic distribution. Four top candidates, Cin5, Skn7, Phd1, and Yap6, all known to be associated with stress response gene regulation, were experimentally confirmed to physically interact with Tup1 and/or Ssn6. Incorporating these new recruitment cofactors with previously characterized cofactors now explains the majority of Tup1 targeting across the genome, and expands our understanding of the mechanism by which Tup1-Ssn6 is directed to its targets