A cis-regulatory map of the Drosophila genome.

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.

The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed 'sausage bodies'. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon.

The impact of quantitative optimization of hybridization conditions on gene expression analysis.

BACKGROUND: With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. RESULTS: As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of up to 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays.For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We can ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirmed an unbiased determination of generally optimal experimental conditions. CONCLUSIONS: Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive pro filing of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks.

Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts.

Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during cell growth. Here we describe a polysaccharide microarray in form of a glycochip permitting simultaneous high-throughput monitoring of multiple transglycosylase activities in plant extracts. The glycochip, containing donor polysaccharides printed onto nitrocellulose-coated glass slides, was incubated with crude plant extracts, along with a series of fluorophore-labelled acceptor oligosaccharides. After removing unused labelled oligosaccharides by washing, fluorescence retained on the glycochip as a result of transglycosylase reaction was detected with a standard microarray scanner. The glycochip assay was used to detect transglycosylase activities in crude extracts from nasturtium (Tropaeolum majus) and mouse-ear cress (Arabidopsis thaliana). A number of previously unknown saccharide donor-acceptor pairs active in transglycosylation reactions that lead to the formation of homo- and hetero-glycosidic conjugates, were detected. Our data provide experimental support for the existence of diverse transglycosylase activities in crude plant extracts.

SimArray: a user-friendly and user-configurable microarray design tool.

BACKGROUND: Microarrays were first developed to assess gene expression but are now also used to map protein-binding sites and to assess allelic variation between individuals. Regardless of the intended application, efficient production and appropriate array design are key determinants of experimental success. Inefficient production can make larger-scale studies prohibitively expensive, whereas poor array design makes normalisation and data analysis problematic. RESULTS: We have developed a user-friendly tool, SimArray, which generates a randomised spot layout, computes a maximum meta-grid area, and estimates the print time, in response to user-specified design decisions. Selected parameters include: the number of probes to be printed; the microtitre plate format; the printing pin configuration, and the achievable spot density. SimArray is compatible with all current robotic spotters that employ 96-, 384- or 1536-well microtitre plates, and can be configured to reflect most production environments. Print time and maximum meta-grid area estimates facilitate evaluation of each array design for its suitability. Randomisation of the spot layout facilitates correction of systematic biases by normalisation. CONCLUSION: SimArray is intended to help both established researchers and those new to the microarray field to develop microarray designs with randomised spot layouts that are compatible with their specific production environment. SimArray is an open-source program and is available from http://www.flychip.org.uk/SimArray/.

Robotic spotting of cDNA and oligonucleotide microarrays.

DNA microarrays are a uniquely efficient method for simultaneously assessing the expression levels of thousands of genes. Owing to their flexibility and value, mechanically spotted microarrays remain the most popular platform. Here, we review recent technological advances with a focus on spotted arrays. Robotic spotting still poses numerous technical challenges. To reduce artefacts, many laboratories have recently investigated ways of improving the spotting process. We compare alternative options and discuss implications for next-generation systems. Together with modern approaches to data analysis, such developments bring greatly improved reliability to individual microarray experiments. Advancing towards the ultimate goal of delivering calibrated, truly quantitative gene-expression measurements on a genomic scale, microarray technology remains at the forefront of post-genomic systems biology.