Enhanced maps of transcription factor binding sites improve regulatory networks learned from accessible chromatin data

Determining where transcription factors (TFs) bind in genomes provides insight into which transcriptional programs are active across organs, tissue types, and environmental conditions. Recent advances in high-throughput profiling of regulatory DNA have yielded large amounts of information about chromatin accessibility. Interpreting the functional significance of these data sets requires knowledge of which regulators are likely to bind these regions. This can be achieved by using information about TF-binding preferences, or motifs, to identify TF-binding events that are likely to be functional. Although different approaches exist to map motifs to DNA sequences, a systematic evaluation of these tools in plants is missing. Here, we compare four motif-mapping tools widely used in the Arabidopsis (Arabidopsis thaliana) research community and evaluate their performance using chromatin immunoprecipitation data sets for 40 TFs. Downstream gene regulatory network (GRN) reconstruction was found to be sensitive to the motif mapper used. We further show that the low recall of Find Individual Motif Occurrences, one of the most frequently used motif-mapping tools, can be overcome by using an Ensemble approach, which combines results from different mapping tools. Several examples are provided demonstrating how the Ensemble approach extends our view on transcriptional control for TFs active in different biological processes. Finally, a protocol is presented to effectively derive more complete cell type-specific GRNs through the integrative analysis of open chromatin regions, known binding site information, and expression data sets. This approach will pave the way to increase our understanding of GRNs in different cellular conditions

TF2Network : predicting transcription factor regulators and gene regulatory networks in Arabidopsis using publicly available binding site information

A gene regulatory network (GRN) is a collection of regulatory interactions between transcription factors (TFs) and their target genes. GRNs control different biological processes and have been instrumental to understand the organization and complexity of gene regulation. Although various experimental methods have been used to map GRNs in Arabidop-sis thaliana, their limited throughput combined with the large number of TFs makes that for many genes our knowledge about regulating TFs is incomplete. We introduce TF2Network, a tool that exploits the vast amount of TF binding site information and enables the delineation of GRNs by detecting potential regulators for a set of co-expressed or functionally related genes. Validation using two experimental benchmarks reveals that TF2Network predicts the correct regulator in 75-92% of the test sets. Furthermore, our tool is robust to noise in the input gene sets, has a low false discovery rate, and shows a better performance to recover correct regulators compared to other plant tools. TF2Network is accessible through a web interface where GRNs are interactively visualized and annotated with various types of experimental functional information. TF2Network was used to perform systematic functional and regulatory gene annotations, identifying new TFs involved in circadian rhythm and stress response

HB-PLS: A statistical method for identifying biological process or pathway regulators by integrating Huber loss and Berhu penalty with partial least squares regression

Gene expression data features high dimensionality, multicollinearity, and non-Gaussian distribution noise, posing hurdles for identification of true regulatory genes controlling a biological process or pathway. In this study, we integrated the Huber loss function and the Berhu penalty (HB) into partial least squares (PLS) framework to deal with the high dimension and multicollinearity property of gene expression data, and developed a new method called HB-PLS regression to model the relationships between regulatory genes and pathway genes. To solve the Huber-Berhu optimization problem, an accelerated proximal gradient descent algorithm with at least 10 times faster than the general convex optimization solver (CVX), was developed. Application of HB-PLS to recognize pathway regulators of lignin biosynthesis and photosynthesis in Arabidopsis thaliana led to the identification of many known positive pathway regulators that had previously been experimentally validated. As compared to sparse partial least squares (SPLS) regression, an efficient method for variable selection and dimension reduction in handling multicollinearity, HB-PLS has higher efficacy in identifying more positive known regulators, a much higher but slightly less sensitivity/(1-specificity) in ranking the true positive known regulators to the top of the output regulatory gene lists for the two aforementioned pathways. In addition, each method could identify some unique regulators that cannot be identified by the other methods. Our results showed that the overall performance of HB-PLS slightly exceeds that of SPLS but both methods are instrumental for identifying real pathway regulators from high-throughput gene expression data, suggesting that integration of statistics, machine leaning and convex optimization can result in a method with high efficacy and is worth further exploration

Unique networks: a method to identity disease-specific regulatory networks from microarray data

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.The survival of any organismis determined by the mechanisms triggered in response to the inputs received. Underlying mechanisms are described by graphical networks that can be inferred from different types of data such as microarrays. Deriving robust and reliable networks can be complicated due to the microarray structure of the data characterized by a discrepancy between the number of genes and samples of several orders of magnitude, bias and noise. Researchers overcome this problem by integrating independent data together and deriving the common mechanisms through consensus network analysis. Different conditions generate different inputs to the organism which reacts triggering different mechanisms with similarities and differences. A lot of effort has been spent into identifying the commonalities under different conditions. Highlighting similarities may overshadow the differences which often identify the main characteristics of the triggered mechanisms. In this thesis we introduce the concept of study-specific mechanism. We develop a pipeline to semiautomatically identify study-specific networks called unique-networks through a combination of consensus approach, graphical similarities and network analysis. The main pipeline called UNIP (Unique Networks Identification Pipeline) takes a set of independent studies, builds gene regulatory networks for each of them, calculates an adaptation of the sensitivity measure based on the networks graphical similarities, applies clustering to group the studies who generate the most similar networks into study-clusters and derives the consensus networks. Once each study-cluster is associated with a consensus-network, we identify the links that appear only in the consensus network under consideration but not in the others (unique-connections). Considering the genes involved in the unique-connections we build Bayesian networks to derive the unique-networks. Finally, we exploit the inference tool to calculate each gene prediction-accuracy across all studies to further refine the unique-networks. Biological validation through different software and the literature are explored to validate our method. UNIP is first applied to a set of synthetic data perturbed with different levels of noise to study the performance and verify its reliability. Then, wheat under stress conditions and different types of cancer are explored. Finally, we develop a user-friendly interface to combine the set of studies by using AND and NOT logic operators. Based on the findings, UNIP is a robust and reliable method to analyse large sets of transcriptomic data. It easily detects the main complex relationships between transcriptional expression of genes specific for different conditions and also highlights structures and nodes that could be potential targets for further research

Integrative methods for reconstruction of dynamic networks in chondrogenesis

Application of human mesenchymal stem cells represents a promising approach in the field of regenerative medicine. Specific stimulation can give rise to chondrocytes, osteocytes or adipocytes. Investigation of the underlying biological processes which induce the observed cellular differentiation is essential to efficiently generate specific tissues for therapeutic purposes. Upon treatment with diverse stimuli, gene expression levels of cultivated human mesenchymal stem cells were monitored using time series microarray experiments for the three lineages. Application of gene network inference is a common approach to identify the regulatory dependencies among a set of investigated genes. This thesis applies the NetGenerator V2.0 tool, which is capable to deal with multiple time series data, which investigates the effect of multiple external stimuli. The applied model is based on a system of linear ordinary differential equations, whose parameters are optimised to reproduce the given time series datasets. Several procedures in the inference process were adapted in this new version in order to allow for the integration of multiple datasets. Network inference was applied on in silico network examples as well as on multi-experiment microarray data of mesenchymal stem cells. The resulting chondrogenesis model was evaluated on the basis of several features including the model adaptation to the data, total number of connections, proportion of connections associated with prior knowledge and the model stability in a resampling procedure. Altogether, NetGenerator V2.0 has provided an automatic and efficient way to integrate experimental datasets and to enhance the interpretability and reliability of the resulting network. In a second chondrogenesis model, the miRNA and mRNA time series data were integrated for the purpose of network inference. One hypothesis of the model was verified by experiments, which demonstrated the negative effect of miR-524-5p on downstream genes

Gene regulatory network modelling with evolutionary algorithms -an integrative approach

Building models for gene regulation has been an important aim of Systems Biology over the past years, driven by the large amount of gene expression data that has become available. Models represent regulatory interactions between genes and transcription factors and can provide better understanding of biological processes, and means of simulating both natural and perturbed systems (e.g. those associated with disease). Gene regulatory network (GRN) quantitative modelling is still limited, however, due to data issues such as noise and restricted length of time series, typically used for GRN reverse engineering. These issues create an under-determination problem, with many models possibly fitting the data. However, large amounts of other types of biological data and knowledge are available, such as cross-platform measurements, knockout experiments, annotations, binding site affinities for transcription factors and so on. It has been postulated that integration of these can improve model quality obtained, by facilitating further filtering of possible models. However, integration is not straightforward, as the different types of data can provide contradictory information, and are intrinsically noisy, hence large scale integration has not been fully explored, to date. Here, we present an integrative parallel framework for GRN modelling, which employs evolutionary computation and different types of data to enhance model inference. Integration is performed at different levels. (i) An analysis of cross-platform integration of time series microarray data, discussing the effects on the resulting models and exploring crossplatform normalisation techniques, is presented. This shows that time-course data integration is possible, and results in models more robust to noise and parameter perturbation, as well as reduced noise over-fitting. (ii) Other types of measurements and knowledge, such as knock-out experiments, annotated transcription factors, binding site affinities and promoter sequences are integrated within the evolutionary framework to obtain more plausible GRN models. This is performed by customising initialisation, mutation and evaluation of candidate model solutions. The different data types are investigated and both qualitative and quantitative improvements are obtained. Results suggest that caution is needed in order to obtain improved models from combined data, and the case study presented here provides an example of how this can be achieved. Furthermore, (iii), RNA-seq data is studied in comparison to microarray experiments, to identify overlapping features and possibilities of integration within the framework. The extension of the framework to this data type is straightforward and qualitative improvements are obtained when combining predicted interactions from single-channel and RNA-seq datasets