CluGene: A Bioinformatics Framework for the Identification of Co-Localized, Co-Expressed and Co-Regulated Genes Aimed at the Investigation of Transcriptional Regulatory Networks from High-Throughput Expression Data

The full understanding of the mechanisms underlying transcriptional regulatory networks requires unravelling of complex causal relationships. Genome high-throughput technologies produce a huge amount of information pertaining gene expression and regulation; however, the complexity of the available data is often overwhelming and tools are needed to extract and organize the relevant information. This work starts from the assumption that the observation of co-occurrent events (in particular co-localization, co-expression and co-regulation) may provide a powerful starting point to begin unravelling transcriptional regulatory networks. Co-expressed genes often imply shared functional pathways; co-expressed and functionally related genes are often co-localized, too; moreover, co-expressed and co-localized genes are also potential targets for co-regulation; finally, co-regulation seems more frequent for genes mapped to proximal chromosome regions. Despite the recognized importance of analysing co-occurrent events, no bioinformatics solution allowing the simultaneous analysis of co-expression, co-localization and co-regulation is currently available. Our work resulted in developing and valuating CluGene, a software providing tools to analyze multiple types of co-occurrences within a single interactive environment allowing the interactive investigation of combined co-expression, co-localization and co-regulation of genes. The use of CluGene will enhance the power of testing hypothesis and experimental approaches aimed at unravelling transcriptional regulatory networks. The software is freely available a

Evolution of gene expression levels in the male reproductive organs of Anopheles mosquitoes

Modifications in gene expression determine many of the phenotypic differentiations between closely related species. This is particularly evident in reproductive tissues, where evolution of genes is more rapid, facilitating the appearance of distinct reproductive characteristics which may lead to species isolation and phenotypic variation. Large-scale, comparative analyses of transcript expression levels have been limited until recently by lack of inter-species data mining solutions. Here, by combining expression normalisation across lineages, multivariate statistical analysis, evolutionary rate, and protein–protein interaction analysis, we investigate ortholog transcripts in the male accessory glands and testes across five closely related species in the Anopheles gambiae complex. We first demonstrate that the differentiation by transcript expression is consistent with the known Anopheles phylogeny. Then, through clustering, we discover groups of transcripts with tissue-dependent expression patterns conserved across lineages, or lineage-dependent patterns conserved across tissues. The strongest associations with reproductive function, transcriptional regulatory networks, protein–protein subnetworks, and evolutionary rate are found for the groups of transcripts featuring large expression differences in lineage or tissue-conserved patterns

Regulation of Anopheles gambiae male accessory gland genes influences postmating response in female

In Drosophila, the accessory gland proteins (Acps) secreted from the male accessory glands (MAGs) and transferred along with sperm into the female reproductive tract have been implicated in triggering postmating behavioral changes, including refractoriness to subsequent mating and propensity to egg laying. Recently, Acps have been found also in Anopheles, suggesting similar functions. Understanding the mechanisms underlying transcriptional regulation of Acps and their functional role in modulating Anoph-eles postmating behavior may lead to the identification of novel vector control strategies to reduce mosquito populations. We identified heat-shock factor (HSF) binding sites within the Acp promoters of male Anoph-eles gambiae and discovered three distinct Hsf isoforms; one being significantly up-regulated in the MAGs after mating. Through genome-wide transcription analysis of Hsf-silenced males, we observed significant down-regulation in 50% of the Acp genes if compared to control males treated with a construct directed against an unrelated bacterial sequence. Treated males retained normal life span and reproductive behavior compared to control males. However, mated wild-type females showed a 46% reduction of egg deposition rate and a 23% reduction of hatching rate (58% combined reduction of progeny). Our results highlight an unsus-pected role of HSF in regulating Acp transcription in A. gambiae and provide evidence that Acp down-regulation in males leads a significant reduction of progeny, thus opening new avenues toward the development of novel vector control strategies.—Dottorini, T., Persampieri, T.

Co-localization of TF target genes positively affects co-regulation.

<p>(<b>a</b>). Comparison of expression profiles of genes showing both co-localization patterns and recognition patterns by a specific DLBCL annotated transcription factor (Status = 0) from those that although being co-localized were not recognized by the specified transcription factor (Status = 1). The comparison of the gene expression profiles between the two groups was done using the Mann-Whitney U test at a threshold of p<0.05 and results were illustrated as boxplot. Only TF co-localized target genes differing in their expression patterns with respect to those not recognized by the specific TFs have been detailed (Oct-1 and IK-1). (<b>b</b>). Both co-regulation by a common TF and co-localization patterns determine co-expression of TF target genes. The expression profiles of Oct-1 co-localized target genes (Status = 0) were compared to those that although recognized by Oct-1did not cluster together (Status = 1); notably Oct-1 co-localized target genes differed in their expression from those that did not cluster as shown by the Mann-Whitney U test at a threshold of p<0.05.</p

Characterization of the immune/inflammatory response genes of the HR subset.

<p>(<b>a</b>). Total and average number of TFBSs present in the promoters of genes expressed in the HR subset. Frequencies were calculated on the genes clustered using the functional distance metric weighted by local density and TF similarity. In bold are shown TFs (Oct-1 and NF-KAPPA B) with an average number value exceeding 2 sites/promoter. (<b>b</b>). Analysis of localization within clusters of HR genes having the signature of immune/inflammatory response. The <i>Show dataset into clusters</i> utility of CluGene (top panel), allows to identify which clusters (red) out of the total (blue) contain a specified list of genes (immune annotated genes). The list of genes contained in clusters and their location is given on the right part of the panel. The tables (bottom) include the identified clustered immunity genes (bold), their function and the size and what other genes are grouped within the same cluster.</p

Analysis of co-operative regulatory effect of Oct-1, c-REL, NF-KappaB, IK-1, BSAP and CP2 in shared DLBCL genomic sites.

<p>Co-operative regulatory regions in target genes were identified using the functional distance metric weighted by local density and TF similarity. The panels refer to an investigation aimed at identifying TFs appearing more often together in clusters. The values in the contingency tables (right panels) are the frequencies of clusters featuring either both TFs of a given pair (true), or only one of them (false). The tables contain also the p-values of the Fisher’s exact test, indicating whether there is some statistically significant difference between the co-occurrences of TF pairs within clusters. Panels refer specifically to the (a). Oxphos; (b). BRC and (c). HR subsets. Only statistically significant results are shown. These results emphasize the role of c-REL, CP2 and Oct-1 association in shared diffuse large B-cell lymphoma regulatory regions.</p

Percentage of of co-localized positive annotated B-cell transcription factors.

<p>Percentage of Oct-1, NF-KAPPA B, IK-1, c-REL, BSAP and CP2 target genes found in clusters obtained with the functional distance metric weighted by local gene density and TF similarity and with the simple positional distance metric, out of the total amount of recognized TF target genes.</p

Different clustering behaviours of the DLBCL subsets subjected to sequential clustering and different distance metrics.

<p>(<b>a</b>). The one sample t-test was used to validate the statistical significance of the number of clusters found in the test datasets, vs. number of clusters found in random datasets. (<b>b</b>). % target genes found in clusters with: simple positional distance metric, functional with local density and functional with local density and TF-similarity. (<b>c</b>) A histogram showing the frequency distribution of clusters is shown.</p

Rapid evolution of female-biased genes among four species of Anopheles

Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can reveal important insights into the biology and evolution of a species. Among Anopheles mosquito species, these phenotypic differences include vectorial capacity, as it is only females that blood feed and thus transmit human malaria. Here, we use RNA-seq data from multiple tissues of four vector species spanning the Anopheles phylogeny to explore the genomic and evolutionary properties of sex-biased genes. We find that, in these mosquitoes, in contrast to what has been found in many other organisms, female-biased genes are more rapidly evolving in sequence, expression, and genic turnover than male-biased genes. Our results suggest that this atypical pattern may be due to the combination of sex-specific life history challenges encountered by females, such as blood feeding. Furthermore, female propensity to mate only once in nature in male swarms likely diminishes sexual selection of post-reproductive traits related to sperm competition among males. We also develop a comparative framework to systematically explore tissue- and sex-specific splicing to document its conservation throughout the genus and identify a set of candidate genes for future functional analyses of sex-specific isoform usage. Finally, our data reveal that the deficit of male-biased genes on the X Chromosomes in Anopheles is a conserved feature in this genus and can be directly attributed to chromosome-wide transcriptional regulation that de-masculinizes the X in male reproductive tissues

Large-cage assessment of a transgenic sex-ratio distortion strain on populations of an African malaria vector

Abstract Background Novel transgenic mosquito control methods require progressively more realistic evaluation. The goal of this study was to determine the effect of a transgene that causes a male-bias sex ratio on Anopheles gambiae target populations in large insectary cages. Methods Life history characteristics of Anopheles gambiae wild type and Ag(PMB)1 (aka gfp124L-2) transgenic mosquitoes, whose progeny are 95% male, were measured in order to parameterize predictive population models. Ag(PMB)1 males were then introduced at two ratios into large insectary cages containing target wild type populations with stable age distributions and densities. The predicted proportion of females and those observed in the large cages were compared. A related model was then used to predict effects of male releases on wild mosquitoes in a west African village. Results The frequency of transgenic mosquitoes in target populations reached an average of 0.44 ± 0.02 and 0.56 ± 0.02 after 6 weeks in the 1:1 and in the 3:1 release ratio treatments (transgenic male:wild male) respectively. Transgenic males caused sex-ratio distortion of 73% and 80% males in the 1:1 and 3:1 treatments, respectively. The number of eggs laid in the transgenic treatments declined as the experiment progressed, with a steeper decline in the 3:1 than in the 1:1 releases. The results of the experiment are partially consistent with predictions of the model; effect size and variability did not conform to the model in two out of three trials, effect size was over-estimated by the model and variability was greater than anticipated, possibly because of sampling effects in restocking. The model estimating the effects of hypothetical releases on the mosquito population of a West African village demonstrated that releases could significantly reduce the number of females in the wild population. The interval of releases is not expected to have a strong effect. Conclusions The biological data produced to parameterize the model, the model itself, and the results of the experiments are components of a system to evaluate and predict the performance of transgenic mosquitoes. Together these suggest that the Ag(PMB)1 strain has the potential to be useful for reversible population suppression while this novel field develops