Mapping Drug Physico-Chemical Features to Pathway Activity Reveals Molecular Networks Linked to Toxicity Outcome

The identification of predictive biomarkers is at the core of modern toxicology. So far, a number of approaches have been proposed. These rely on statistical inference of toxicity response from either compound features (i.e., QSAR), in vitro cell based assays or molecular profiling of target tissues (i.e., expression profiling). Although these approaches have already shown the potential of predictive toxicology, we still do not have a systematic approach to model the interaction between chemical features, molecular networks and toxicity outcome. Here, we describe a computational strategy designed to address this important need. Its application to a model of renal tubular degeneration has revealed a link between physico-chemical features and signalling components controlling cell communication pathways, which in turn are differentially modulated in response to toxic chemicals. Overall, our findings are consistent with the existence of a general toxicity mechanism operating in synergy with more specific single-target based mode of actions (MOAs) and provide a general framework for the development of an integrative approach to predictive toxicology

Inferring a transcription regulatory network by directed perturbation

Transcription plays a key role in cellular processes and its regulation is of paramount importance. The aim of the work described in this thesis is to study the transcription regulatory network of Saccharomyces cerevisiae, employing genome-wide approaches. All the three presented research studies have in common that individual genes are deleted and resulting gene expression changes are monitored by DNA microarrays. It is first described how gene expression changes can be used as detailed molecular phenotypes to study the transcription regulatory network underlying a signalling pathway. Genome-wide expression changes of 91 viable deletion mutants of different glucose signalling and metabolic pathways are analysed. A gene signature is used to group pathway members with similar effects on transcription. A new network approach is developed that is designed to explain gene expression changes upon deletion of one pathway member through the transcriptional regulation of another pathway member. This new approach reveals hierarchy and feedback in the transcription regulatory network. In particular, it predicts that the different glucose pathways converge on the transcriptional regulation of the biosynthesis of storage carbohydrates. For understanding the transcription regulatory network, it is important to discern target genes of gene-specific transcription factors (GSTFs). Genome-wide gene expression changes of 183 viable GSTF deletion mutants are compared with available DNA binding data, re-evaluating the overlap between both data types as well as discerning direct target genes of GSTFs. Besides determining roles of previously uncharacterized GSTFs, for example Stp3, this comparison has led to the first systematic classification of GSTFs into activators and/or repressors. Of all surveyed gene-specific transcription factors that could be classified, activators account for less than 54%. The remaining 46% of gene-specific transcription factors are repressors (37%) or have a dual function (9%). The unanticipated high number of gene-specific repressors indicates that in the yeast S. cerevisiae, chromatin is not as restrictive to transcription as has previously been thought and indicates that a considerable part of the gene-specific machinery is aimed at restricting unwanted transcription. Recent studies have systematically exposed large numbers of non-additive genetic interactions, the majority of which are functionally uncharacterized. To investigate such genetic interactions between GSTFs, we systematically analysed 72 viable double deletion mutants and compared them to the respective single deletion mutants. By generating a high-resolution gene expression atlas, epistatic effects of GSTF pairs on the expression of individual genes are investigated. Known genetic interactions are confirmed, and new ones are revealed. The analysis also provides evidence for two previously uncharacterized mechanisms, one for a negative (“buffering by induced dependency” between Hac1 and Rpn4) and one for a positive genetic interaction (“alleviation by derepression” between Gln3 and Gzf3). The study provides general insight into the complex nature of epistasis and proposes new models for genetic interactions, the majority of which do not fall into easily recognizable within- or between-pathway relationships

Iterative error correction of long sequencing reads maximizes accuracy and improves contig assembly

Next-generation sequencers such as Illumina can now produce reads up to 300 bp with high throughput, which is attractive for genome assembly. A first step in genome assembly is to computationally correct sequencing errors. However, correcting all errors in these longer reads is challenging. Here, we show that reads with remaining errors after correction often overlap repeats, where short erroneous k-mers occur in other copies of the repeat. We developed an iterative error correction pipeline that runs the previously published String Graph Assembler (SGA) in multiple rounds of k-mer-based correction with an increasing k-mer size, followed by a final round of overlap-based correction. By combining the advantages of small and large k-mers, this approach corrects more errors in repeats and minimizes the total amount of erroneous reads. We show that higher read accuracy increases contig lengths two to three times. We provide SGA-Iteratively Correcting Errors (https://github.com/hillerlab/IterativeErrorCorrection/) that implements iterative error correction by using modules from SGA

p Convergent and lineage-specific genomic differences in limb regulatory elements in limbless reptile lineages

Loss of limbs evolved many times in squamate reptiles. Here we investigated the genomic basis of convergent limb loss in reptiles. We sequenced the genomes of a closely related pair of limbless-limbed gymnophthalmid lizards and performed a comparative genomic analysis including five snakes and the limbless glass lizard. Our analysis of these three independent limbless lineages revealed that signatures of shared sequence or transcription factor binding site divergence in individual limb regulatory elements are generally rare. Instead, shared divergence occurs more often at the level of signaling pathways, involving different regulatory elements associated with the same limb genes (such as Hand2 or Hox) and/or patterning mechanisms (such as Shh signaling). Interestingly, although snakes are known to have mutations in the Shh ZRS limb enhancer, this enhancer lacks relevant mutations in limbless lizards. Thus, different mechanisms could contribute to limb loss, and there are likely multiple evolutionary paths to limblessness in reptiles

Phenotype loss is associated with widespread divergence of the gene regulatory landscape in evolution

Detecting the genomic changes underlying phenotypic changes between species is a main goal of evolutionary biology and genomics. Evolutionary theory predicts that changes in cisregulatory elements are important for morphological changes. We combined genome sequencing, functional genomics and genome-wide comparative analyses to investigate regulatory elements in lineages that lost morphological traits. We first show that limb loss in snakes is associated with widespread divergence of limb regulatory elements. We next show that eye degeneration in subterranean mammals is associated with widespread divergence of eye regulatory elements. In both cases, sequence divergence results in an extensive loss of transcription factor binding sites. Importantly, diverged regulatory elements are associated with genes required for normal limb patterning or normal eye development and function, suggesting that regulatory divergence contributed to the loss of these phenotypes. Together, our results show that genome-wide decay of the phenotype-specific cis-regulatory landscape is a hallmark of lost morphological traits

The genome of the tegu lizard Salvator merianae: combining Illumina, PacBio, and optical mapping data to generate a highly contiguous assembly

Background Reptiles are a species-rich group with great phenotypic and life history diversity but are highly underrepresented among the vertebrate species with sequenced genomes. Results Here, we report a high-quality genome assembly of the tegu lizard, Salvator merianae, the first lacertoid with a sequenced genome. We combined 74X Illumina short-read, 29.8X Pacific Biosciences long-read, and optical mapping data to generate a high-quality assembly with a scaffold N50 value of 55.4 Mb. The contig N50 value of this assembly is 521 Kb, making it the most contiguous reptile assembly so far. We show that the tegu assembly has the highest completeness of coding genes and conserved non-exonic elements (CNEs) compared to other reptiles. Furthermore, the tegu assembly has the highest number of evolutionarily conserved CNE pairs, corroborating a high assembly contiguity in intergenic regions. As in other reptiles, long interspersed nuclear elements comprise the most abundant transposon class. We used transcriptomic data, homology- and de novo gene predictions to annotate 22,413 coding genes, of which 16,995 (76%) likely have human orthologs as inferred by CESAR-derived gene mappings. Finally, we generated a multiple genome alignment comprising 10 squamates and 7 other amniote species and identified conserved regions that are under evolutionary constraint. CNEs cover 38 Mb (1.8%) of the tegu genome, with 3.3 Mb in these elements being squamate specific. In contrast to placental mammal-specific CNEs, very few of these squamate-specific CNEs (<20 Kb) overlap transposons, highlighting a difference in how lineage-specific CNEs originated in these two clades. Conclusions The tegu lizard genome together with the multiple genome alignment and comprehensive conserved element datasets provide a valuable resource for comparative genomic studies of reptiles and other amniotes