Predicting functional transcription factor binding through alignment-free and affinity-based analysis of orthologous promoter sequences

Motivation: The identification of transcription factor (TF) binding sites and the regulatory circuitry that they define is currently an area of intense research. Data from whole-genome chromatin immunoprecipitation (ChIP–chip), whole-genome expression microarrays, and sequencing of multiple closely related genomes have all proven useful. By and large, existing methods treat the interpretation of functional data as a classification problem (between bound and unbound DNA), and the analysis of comparative data as a problem of local alignment (to recover phylogenetic footprints of presumably functional elements). Both of these approaches suffer from the inability to model and detect low-affinity binding sites, which have recently been shown to be abundant and functional

Conservation and Evolution of Cis-Regulatory Systems in Ascomycete Fungi

Relatively little is known about the mechanisms through which gene expression regulation evolves. To investigate this, we systematically explored the conservation of regulatory networks in fungi by examining the cis-regulatory elements that govern the expression of coregulated genes. We first identified groups of coregulated Saccharomyces cerevisiae genes enriched for genes with known upstream or downstream cis-regulatory sequences. Reasoning that many of these gene groups are coregulated in related species as well, we performed similar analyses on orthologs of coregulated S. cerevisiae genes in 13 other ascomycete species. We find that many species-specific gene groups are enriched for the same flanking regulatory sequences as those found in the orthologous gene groups from S. cerevisiae, indicating that those regulatory systems have been conserved in multiple ascomycete species. In addition to these clear cases of regulatory conservation, we find examples of cis-element evolution that suggest multiple modes of regulatory diversification, including alterations in transcription factor-binding specificity, incorporation of new gene targets into an existing regulatory system, and cooption of regulatory systems to control a different set of genes. We investigated one example in greater detail by measuring the in vitro activity of the S. cerevisiae transcription factor Rpn4p and its orthologs from Candida albicans and Neurospora crassa. Our results suggest that the DNA binding specificity of these proteins has coevolved with the sequences found upstream of the Rpn4p target genes and suggest that Rpn4p has a different function in N. crassa

Mating type and pheromone genes in the Gibberella fujikuroi species complex : an evolutionary perspective

Reproductive isolation is an essential stage in speciation. In Ascomycetes, the ubiquitous distribution of many species suggests that sympatric speciation through assertive mating should be an important factor. The MAT locus and the pheromone/receptor system could both potentially contribute to the development of such sexual isolation. Alterations at the MAT loci could lead to distinct reproductive habits or a change in mating system, both of which can reduce gene-flow between species. However, if deliberate pre-mating sexual preferences exist, they are more likely to be determined by the pheromone/receptor system. This study of Fusarium reproductive genes, and comparisons with other Ascomycetes, has yielded numerous interesting findings regarding the evolution of these mate-recognition mechanisms and the implications thereof. The G. fujikuroi and F. graminearum species complexes have offered an interesting comparison between heterothallic and homothallic MAT locus evolution. The value of comparative sequence analysis has been demonstrated in the discovery of a previously unknown gene, MAT1-2-3, which may be specific to members of the Order Hypocreales. While all MAT genes share similar regulatory elements, this is the first report of evidence that a transition to homothallism can be accompanied by the recruitment of distinct elements that could facilitate alternate expression of MAT genes. The MAT genes are also highly divergent between Fusarium spp., largely due to relaxed selective constraint, particularly in homothallic species. However, inter-specific gene-flow could curb MAT gene divergence among homothallic species. Despite strong reproductive barriers in the G. fujikuroi complex, the F. sacchari MAT1-1 sequence appears to have been acquired through lateral transfer from a distant relative. Analytical analysis of the MAT locus novelties reported here, including the new MAT gene, will be necessary to determine their biological significance. To investigate the extent of pheromone diversity in the Ascomycetes, and to gain clues as to its biological importance, pheromone peptides from seventy ascomycete species were compared. A number of reproductively incompatible species, such as those in the G. fujikuroi complex, share identical pheromones; which implies that another mechanism must be responsible for the observed reproductive barriers. However, on the whole, pheromones are highly divergent among species. Both adaptive and non-adaptive evolution could have contributed to this pattern. In fact the structure of the á-class pheromone precursor gene, which consists of multiple repeats of the pheromone module, could facilitate rapid diversification through “birth-and-death” evolution. Within species, selection maintains pheromone peptides, implying that much of the inter-specific variation is functionally relevant. This further suggests that pheromone evolution could contribute to the generation of reproductive isolation between species. The most general trend in the findings of this study is that ascomycete reproductive genes are highly divergent. This is in agreement with findings in other Kingdoms. A number of evolutionary forces are probably involved but weaker selective constraint, resulting from the fact that reproduction is not essential in these fungi, appears to be a common factor. This reproductive gene variability could be directly linked to speciation and, therefore, the great diversity in Ascomycetes. Additional information on the appendices is available on a CD, stored at the Merensky Library on Level 3Dissertation (MSc)--University of Pretoria, 2011.GeneticsUnrestricte

Evaluating the impact of horizontally acquired genes on the metabolism of nonconventional yeast lineage

The Wickerhamiella/Starmerella (W/S) clade is a group of non-conventional yeast with atypical metabolic functions that enable the colonization of specialized niches, such as flowers or the gut of insects that visit flowers. Comparative genomics demonstrates that one of the main forces that drives metabolic evolution in the W/S clade is the unusual high frequency of horizontally acquired genes, some of which have been deeply studied and characterized. Yet, a high proportion of acquired genes with unknown impact in the W/S-clade metabolism still remains. This work aimed to advance the current understanding of the metabolic evolution in the W/S clade, by analyzing the complete transcriptomes of W. versatilis, W. domercqiae and S. bombicola grown in two different conditions. Comparative transcriptomic analyses between native and acquired genes across the three species were performed in order to provide a first high-throughput evaluation of the impact of the acquired genes on the host metabolism. Quantitative levels of gene expression and patterns of differential expression were studied and analyzed together with functional annotation and the role of acquired genes evaluating e.g. whether it enabled a function normally absent in yeasts. The results indicate that, depending on the species analyzed, the expression levels of acquired genes can either be similar or sig-nificantly lower than native genes. Yet, in all instances, an important proportion of these genes are actively regulated. Expressed acquired genes tend to be fixed by replacing pre-existing genes in the genomes, which were often involved in the assimilation of carbon and nitrogen from minority resources. The whole transcriptome analysis is a tool that perfectly complements current knowledge of whole genome evolution in the W/S clade, especially in understanding the evolutionary impact of horizontal gene transfer events in these yeasts.O clado Wickerhamiella/Starmerella (W/S) é um grupo de leveduras não convencionais com funções metabólicas atípicas que permitem a colonização de nichos especializados, como flores ou intestino de insetos que visitam flores. A genómica comparativa demonstra que uma das principais forças que impulsionam a evolução metabólica no clado W/S é a grande frequência de genes adquiridos horizontalmente, alguns dos quais já caracterizados em detalhe. No entanto, ainda persiste uma elevada proporção de genes horizontalmente adquiridos com impacto desconhecido no metabolismo do clado W/S. Este trabalho teve como objetivo avançar no conhecimento da evolução metabólica no clado W/S, analisando os transcriptomas completos de W. versatilis, W. domercqiae e S. bombicola cultivados em duas condições diferentes. Análises de transcriptómica comparativa entre genes nativos e adquiridos nas três espécies foram realizadas a fim de fornecer uma primeira avaliação do impacto do conjunto dos genes adquiridos no metabolismo do hospedeiro. Níveis quantitativos de expressão génica e padrões de expressão diferencial foram estudados e analisados juntamente com a anotação funcional e o contexto evolutivo dos genes adquiridos. Os resultados indicam que, depen-dendo da espécie analisada, os níveis de expressão dos genes adquiridos podem ser seme-lhantes ou significativamente inferiores aos dos genes nativos. No entanto, em todos os casos, uma proporção importante destes genes é activamente regulada. Os genes adquiridos que são expressos frequentemente substituem genes pré-existentes nos genomas, frequentemente relacionados com a assimilação de fontes menos comuns de carbono e azoto. A análise do transcriptoma completo é uma ferramenta que complementa perfeitamente o conhecimento actual sobre a evolução do genoma no clado W/S, especialmente no que respeita à compreensão do impacto evolutivo de eventos de transferência horizontal de genes nestas leveduras

Transcription factor binding site prediction with multivariate gene expression data

Multi-sample microarray experiments have become a standard experimental method for studying biological systems. A frequent goal in such studies is to unravel the regulatory relationships between genes. During the last few years, regression models have been proposed for the de novo discovery of cis-acting regulatory sequences using gene expression data. However, when applied to multi-sample experiments, existing regression based methods model each individual sample separately. To better capture the dynamic relationships in multi-sample microarray experiments, we propose a flexible method for the joint modeling of promoter sequence and multivariate expression data. In higher order eukaryotic genomes expression regulation usually involves combinatorial interaction between several transcription factors. Experiments have shown that spacing between transcription factor binding sites can significantly affect their strength in activating gene expression. We propose an adaptive model building procedure to capture such spacing dependent cis-acting regulatory modules. We apply our methods to the analysis of microarray time-course experiments in yeast and in Arabidopsis. These experiments exhibit very different dynamic temporal relationships. For both data sets, we have found all of the well-known cis-acting regulatory elements in the related context, as well as being able to predict novel elements.Comment: Published in at http://dx.doi.org/10.1214/10.1214/07-AOAS142 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

Determining Physical Constraints in Transcriptional Initiation Complexes Using DNA Sequence Analysis

Eukaryotic gene expression is often under the control of cooperatively acting transcription factors whose binding is limited by structural constraints. By determining these structural constraints, we can understand the “rules” that define functional cooperativity. Conversely, by understanding the rules of binding, we can infer structural characteristics. We have developed an information theory based method for approximating the physical limitations of cooperative interactions by comparing sequence analysis to microarray expression data. When applied to the coordinated binding of the sulfur amino acid regulatory protein Met4 by Cbf1 and Met31, we were able to create a combinatorial model that can correctly identify Met4 regulated genes. Interestingly, we found that the major determinant of Met4 regulation was the sum of the strength of the Cbf1 and Met31 binding sites and that the energetic costs associated with spacing appeared to be minimal

Prediction of synergistic transcription factors by function conservation

A new strategy is proposed for identifying synergistic transcription factors by function conservation, leading to the identification of 51 homotypic transcription-factor combinations