MULTOVL: fast multiple overlaps of genomic regions.

We present the MULTOVL application suite that detects and statistically analyses multiple overlaps of genomic regions in a fast and efficient manner. The package supports the detection of multiple region intersections, unions and 'solitary' genomic regions. The significance of actually observed overlaps is estimated by comparing them with empirical null distributions generated by random shuffling of the input regions.

Intergenic Polycomb target sites are dynamically marked by non-coding transcription during lineage commitment.

Non-coding (nc) RNAs are involved both in recruitment of vertebrate Polycomb (PcG) proteins to chromatin, and in activation of PcG target genes. Here we investigate dynamic changes in the relationship between ncRNA transcription and recruitment of PcG proteins to chromatin during differentiation. Profiling of purified cell populations from different stages of a defined murine in vitro neural differentiation system shows that over 50% of regulated intergenic non-coding transcripts precisely correspond to PcG target sites. We designate these PcG recruiting elements as Transcribed Intergenic Polycomb (TIP) sites. The relationship between TIP transcription and PcG recruitment switches dynamically during differentiation between different states, in which transcription and PcG recruitment exclude each other, or in which both are present. Reporter assays show that transcribed TIP sites can repress a flanking gene. Knockdown experiments demonstrate that TIP ncRNAs are themselves required for repression of target genes both in cis and in trans. We propose that TIP transcription may ensure coordinated regulation of gene networks via dynamic switching and recruitment of PcG proteins both in cis and in trans during lineage commitment.

Analyzing the performance of conformational search programs on compound databases.

We have studied the sampling performance of conformational search programs using geometric and energetic criteria. Ideally, a conformational search algorithm should identify the largest possible number of low-energy structures (energy criterion) covering the widest possible range of molecular shapes (geometric criterion). Geometric analysis consisted in comparing the distribution of conformations within the generated ensembles by multidimensional scaling and by analysing the eigenvalue structure of the pairwise coordinate covariance matrices. The energetic comparison was carried out by assessing the energy distribution of conformers after minimizing them all using the same semi-empirical quantum mechanics optimization protocol. The present investigation focused on five conformational search programs: DGEOM, QXP, ROTATE, LMOD and OMEGA. We have applied these methodologies to a maximally diverse 604-compound subset of the LeadQuest library. The program LMOD performs best according to the energetic criterion, whereas a wider range of geometrically diverse conformations is sampled by the other programs, at the cost of higher median conformer energies. In terms of speed, OMEGA is fastest. We recommend the use of LMOD or OMEGA for high-quality conformational search applications.

Percutaneous absorption of drugs used in atopic eczema: pimecrolimus permeates less through skin than corticosteroids and tacrolimus.

For treatment of skin diseases with topical drugs, penetration of the agents into the relevant layers of the skin is required. Permeation through the skin should, however, be kept to a minimum, in order to avoid the risk of systemic side effects. Here we compared the in vitro skin penetration and permeation of two novel drugs used in the therapy of atopic eczema (pimecrolimus and tacrolimus) and three representative corticosteroids (betamethasone-17-valerate, clobetasol-17-propionate, and diflucortolon-21-valerate). Drug concentrations of pimecrolimus and corticosteroids in human skin were found to be in the same order of magnitude. Permeation of pimecrolimus through human skin was, however, lower by factors of 70-110 as compared to the steroids. When pimecrolimus was compared with tacrolimus in human, pig, or rat skin, similar concentrations of the two compounds were measured in the skin, whereas permeation of pimecrolimus through skin was consistently lower by factors of 9-10. Lipophilicity was found to be highest for pimecrolimus, its octanol-water distribution coefficient being higher by factors of 8 and 25-450 than that of tacrolimus and the corticosteroids, respectively. The low permeation of pimecrolimus may be explained by its higher lipophilicity (compared to tacrolimus and the corticosteroids) and higher molecular weight (compared to steroids). In conclusion, pimecrolimus appears to have a favourable skin penetration/permeation profile, featuring a low degree of percutaneous absorption.

H3K27me3 forms BLOCs over silent genes and intergenic regions and specifies a histone banding pattern on a mouse autosomal chromosome.

In mammals, genome-wide chromatin maps and immunofluorescence studies show that broad domains of repressive histone modifications are present on pericentromeric and telomeric repeats and on the inactive X chromosome. However, only a few autosomal loci such as silent Hox gene clusters have been shown to lie in broad domains of repressive histone modifications. Here we present a ChIP-chip analysis of the repressive H3K27me3 histone modification along chr 17 in mouse embryonic fibroblast cells using an algorithm named broad local enrichments (BLOCs), which allows the identification of broad regions of histone modifications. Our results, confirmed by BLOC analysis of a whole genome ChIP-seq data set, show that the majority of H3K27me3 modifications form BLOCs rather than focal peaks. H3K27me3 BLOCs modify silent genes of all types, plus flanking intergenic regions and their distribution indicates a negative correlation between H3K27me3 and transcription. However, we also found that some nontranscribed gene-poor regions lack H3K27me3. We therefore performed a low-resolution analysis of whole mouse chr 17, which revealed that H3K27me3 is enriched in mega-base-pair-sized domains that are also enriched for genes, short interspersed elements (SINEs) and active histone modifications. These genic H3K27me3 domains alternate with similar-sized gene-poor domains. These are deficient in active histone modifications, as well as H3K27me3, but are enriched for long interspersed elements (LINEs) and long-terminal repeat (LTR) transposons and H3K9me3 and H4K20me3. Thus, an autosome can be seen to contain alternating chromatin bands that predominantly separate genes from one retrotransposon class, which could offer unique domains for the specific regulation of genes or the silencing of autonomous retrotransposons.

SH2 binding site comparison: a new application of the SURFCOMP method.

To avoid side effects, it is often desirable to increase the specificity of a drug candidate when targeting one member of a family of related proteins, whereby one exploits small differences between the structures of the binding sites. Identification of such differences can be carried out by analyzing the distributions of physicochemical properties mapped onto molecular surfaces. Here we demonstrate that SURFCOMP, our local surface similarity detection method, is able to detect differences between the binding sites of two closely related proteins. We analyzed the SH2 domains of Sap and Eat-2, two highly similar signal transduction molecules involved in inflammatory processes and found differences between their binding sites that can possibly lead to a better understanding of the different specificities of the two proteins.

SURFCOMP: a novel graph-based approach to molecular surface comparison.

Analysis of the distributions of physicochemical properties mapped onto molecular surfaces can highlight important similarities or differences between compound classes, contributing to rational drug design efforts. Here we present an approach that uses maximal common subgraph comparison and harmonic shape image matching to detect locally similar regions between two molecular surfaces augmented with properties such as the electrostatic potential or lipophilicity. The complexity of the problem is reduced by a set of filters that implement various geometric and physicochemical heuristics. The approach was tested on dihydrofolate reductase and thermolysin inhibitors and was shown to recover the correct alignments of the compounds bound in the active sites.

mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure.

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.