Modeling of cis-elements in the upstream regions of genes is a challenging computational problem. A set of regulatory motifs present in the promoters of a set of genes can be modeled by a biclique. Combinations of cis-elements play a vital role in ascertaining that the correct co-action of transcription factors binding to the gene promoter, results in appropriate gene expression in response to various stimuli. Geometrical and spatial constraints in transcription factor binding also impose restrictions on order and separation of cis-elements. Not all regulatory elements that coexist are biologically significant. If the set of genes in which a set of regulatory elements co-occur, are tightly correlated with respect to gene expression data over a set of treatments, the regulatory element combination can be biologically directed. The system developed in this work, XcisClique, consists of a comprehensive infrastructure for annotated genome and gene expression data for Arabidopsis thaliana. XcisClique models cis-regulatory elements as regular expressions and detects maximal bicliques of genes and motifs, called itemsets. An itemset consists of a set of genes (called a geneset) and a set of motifs (called a motifset) such that every motif in the motifset occurs in the promoter of every gene in the geneset. XcisClique differs from existing tools of the same kind in that, it offers a common platform for the integration of sequence and gene expression data. Itemsets identified by XcisClique are not only evaluated for statistical over-representation in sequence data, but are also examined with respect to the expression patterns of the corresponding geneset. Thus, the results produced are biologically directed. XcisClique is also the only tool of its kind for Arabidopsis thaliana, and can also be used for other organisms in the presence of appropriate sequence, expression, and regulatory element data. The web-interface to a subset of functionalities, source code and supplemental material are available online at http://bioinformatics.cs.vt.edu/xcisclique. / Master of Science

La régulation transcriptionnelle des gènes est un processus indispensable sans lequel la diversité phénotypique des cellules ainsi que l’adaptation à leur environnement serait inexistant. L’identification des éléments de régulation dans le génome est d’une importance capitale afin de comprendre les mécanismes gouvernant l’expression des gènes spécifiques à un type cellulaire donné. Ainsi, suite au pic de LH, le follicule ovarien entre dans un programme intensif de différentiation cellulaire, orchestré par des modifications majeures du profile transcriptionnel des cellules de granulosa, déclenchant ultimement l’ovulation et la lutéinisation, processus indispensables à la fertilité femelle. L’hypothèse supportée par cette étude stipule qu’une réorganisation de la structure chromatinienne survient aux régions régulatrices d’une panoplie de gènes dans les heures suivant le pic de LH et qu’en isolant et identifiant ces régions, il serait possible de retrouver des éléments essentiels aux processus d’ovulation et de lutéinisation. Ainsi, en utilisant un protocole standard de superovulation chez la souris, les éléments de régulation se modifiant 4h suivant l’administration de hCG ont été isolés et identifiés dans les cellules de granulosa en utilisant la méthode FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combinée à un séquençage haut débit. Cette étude a démontré que suite au stimulus ovulatoire, les cellules de granulosa subissent une reprogrammation majeure des éléments de régulation, qui est corrélée avec une modification drastique de leurs fonctions biologiques. De plus, cette étude a mis en évidence une association majoritaire des éléments de régulation à des régions intergéniques distales et à des introns, indiquant que ces régions ont une importance capitale dans la régulation transcriptionnelle dans les cellules de granulosa. Cette étude a également permis d’identifier une panoplie de régulateurs transcriptionnels reconnus pour être essentiels à la fonction ovarienne, ainsi que leur sites de liaison dans le génome, démontrant que la méthode FAIRE est une méthode assez puissante pour permettre la prédiction d’événements moléculaires précis ayant un sens physiologique réel. / Identification of regulatory elements in the genome is of paramount importance to understanding the mechanisms governing the expression of specific genes in a given cell type. Following the LH surge, the ovarian peri-ovulatory follicle enters an intensive program of cellular differentiation, orchestrated by major changes in the transcriptional profile of granulosa cells, ultimately triggering ovulation and luteinization, processes essentials for fertility in females. In the mouse, several genes essential to the success of this program are induced 2 to 6 hours after the ovulatory stimulus. Using a standard protocol for superovulation in mice, the regulatory elements were isolated and identified in granulosa cells 4h after administration of hCG using the method FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combined with next generation sequencing. The results of this analysis demonstrate that after the ovulatory stimulus, granulosa cells undergo a major reprogramming of regulatory elements, which is correlated with the extensive changes in their biological functions. In addition, this study showed that most regulatory elements were associated with distal intergenic regions and introns, indicating that these regions are important in transcriptional regulation in granulosa cells. A variety of transcriptional regulators known to be essential for ovarian function, and their binding sites were also identified in this analysis, demonstrating that the FAIRE method has the power to predict molecular events that have correlates in the known physiology of ovarian processes.

La régulation transcriptionnelle des gènes est un processus indispensable sans lequel la diversité phénotypique des cellules ainsi que l’adaptation à leur environnement serait inexistant. L’identification des éléments de régulation dans le génome est d’une importance capitale afin de comprendre les mécanismes gouvernant l’expression des gènes spécifiques à un type cellulaire donné. Ainsi, suite au pic de LH, le follicule ovarien entre dans un programme intensif de différentiation cellulaire, orchestré par des modifications majeures du profile transcriptionnel des cellules de granulosa, déclenchant ultimement l’ovulation et la lutéinisation, processus indispensables à la fertilité femelle. L’hypothèse supportée par cette étude stipule qu’une réorganisation de la structure chromatinienne survient aux régions régulatrices d’une panoplie de gènes dans les heures suivant le pic de LH et qu’en isolant et identifiant ces régions, il serait possible de retrouver des éléments essentiels aux processus d’ovulation et de lutéinisation. Ainsi, en utilisant un protocole standard de superovulation chez la souris, les éléments de régulation se modifiant 4h suivant l’administration de hCG ont été isolés et identifiés dans les cellules de granulosa en utilisant la méthode FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combinée à un séquençage haut débit. Cette étude a démontré que suite au stimulus ovulatoire, les cellules de granulosa subissent une reprogrammation majeure des éléments de régulation, qui est corrélée avec une modification drastique de leurs fonctions biologiques. De plus, cette étude a mis en évidence une association majoritaire des éléments de régulation à des régions intergéniques distales et à des introns, indiquant que ces régions ont une importance capitale dans la régulation transcriptionnelle dans les cellules de granulosa. Cette étude a également permis d’identifier une panoplie de régulateurs transcriptionnels reconnus pour être essentiels à la fonction ovarienne, ainsi que leur sites de liaison dans le génome, démontrant que la méthode FAIRE est une méthode assez puissante pour permettre la prédiction d’événements moléculaires précis ayant un sens physiologique réel. / Identification of regulatory elements in the genome is of paramount importance to understanding the mechanisms governing the expression of specific genes in a given cell type. Following the LH surge, the ovarian peri-ovulatory follicle enters an intensive program of cellular differentiation, orchestrated by major changes in the transcriptional profile of granulosa cells, ultimately triggering ovulation and luteinization, processes essentials for fertility in females. In the mouse, several genes essential to the success of this program are induced 2 to 6 hours after the ovulatory stimulus. Using a standard protocol for superovulation in mice, the regulatory elements were isolated and identified in granulosa cells 4h after administration of hCG using the method FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combined with next generation sequencing. The results of this analysis demonstrate that after the ovulatory stimulus, granulosa cells undergo a major reprogramming of regulatory elements, which is correlated with the extensive changes in their biological functions. In addition, this study showed that most regulatory elements were associated with distal intergenic regions and introns, indicating that these regions are important in transcriptional regulation in granulosa cells. A variety of transcriptional regulators known to be essential for ovarian function, and their binding sites were also identified in this analysis, demonstrating that the FAIRE method has the power to predict molecular events that have correlates in the known physiology of ovarian processes.

Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders. Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease. Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.

A transformação genética é uma alternativa ao melhoramento convencional de citros que permite a modificação de genótipos pela introdução de um ou mais genes oriundos de organismos semelhantes ou filogeneticamente distantes do hospedeiro. Os genes transferidos para espécies de interesse devem ser controlados por promotores, os quais regulam a expressão gênica de forma temporal, espacial e na magnitude desejada. Na maioria dos casos, os genes são expressos de forma constitutiva utilizando o promotor CaMV35S isolado do Vírus do Mosaico da Couve Flor. No entanto, o desenvolvimento de novas abordagens de transformação de plantas (cisgenia e intragenia), que fazem o uso de genes e sequências regulatórias derivadas da mesma espécie ou espécies relacionadas, requer a disponibilidade de elementos genéticos, incluindo promotores constitutivos, isolados de citros. Assim, o objetivo do trabalho foi clonar e caracterizar promotores constitutivos de Citrus sinensis. Para isso, a região promotora dos genes Fator de elongação 1-α (CsEF1), Gliceraldeido-3-fosfato-desidrogenase C2 (CsGAPC2) e Cyclofilina (CsCYC) foi isolada e avaliados pela fusão ao gene repórter uidA. A funcionalidade dos três promotores foi confirmada por ensaio histoquímico da atividade GUS em folhas, caules e raízes de plantas transgênicas de citros cv. \'Hamlin\'. A análise de RT-qPCR mostra que a expressão do gene uidA sob controle dos promotores CsCYC, CsGAPC2 e CsEF1 correspondeu a uma atividade aproximada de 64%, 58% e 47%, respectivamente em comparação com o promotor CaMV35S. A análise in silico dos promotores CsGAPC2, CsCYC e CsEF1 mostra que a atividade de cada um é controlada por uma série de putativos elementos cis-regulatórios. A sequência completa e versões truncadas originadas a partir de deleções em cada promotor foram fundidas ao gene uidA e analisadas em plantas transgênicas de Nicotiana benthamiana pelo ensaio histoquímico e fluorimétrico da atividade GUS. As análises de deleções não causaram perda de função dos promotores em estudo, mas afetaram a expressão gênica nos promotores CsGAPC2 e CsEF1. Os promotores isolados representam bons candidatos a serem utilizados em trabalhos de transformação genética de citros. / Genetic transformation is an alternative to citros conventional breeding that allows the modification of genotypes by the introduction of one or more genes derived from different organisms that can not be crossed by natural means. The transferred genes to the species of interest are controlled by promoters, which regulate a gene expression temporally, spatially and in the desired magnitude. In most cases, the introduced genes have been constitutively expressed using the CaMV35S promoter obtained from the cauliflower mosaic virus. However, the development of novel plant transformation approaches (cisgenesis and intragenesis) imply the use of genetic material from the same species or from closely related species capable of sexual hybridization, which requires the isolation of genetic elements, including citros constitutive promoters. The objective of this study was clone and characterize Citrus sinensis constitutive promoters. For this, the promoter region of the genes Elongation Factor 1-α (CsEF1), Glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2) and Cyclofiline (CsCYC) was isolated and evaluated by fusion to the uidA reporter gene. The functionality of three promoter was confirmed by histochemical GUS assay in leaves, stems and roots of transgenic citrus plants cv. \'Hamlin\'. RT-qPCR analysis revealed that uidA gene expression under control of the CsCYC, CsGAPC2 and CsEF1 promoters was approximately 64%, 58% and 47% expression compared with the CaMV35S promoter. In silico analysis of the CsGAPC2, CsCYC and CsEF1 promoters displays their activity is controlled by a series of putative cis-regulatory elements. The full length promoter and truncated versions originated from deletions in promoters sequences were fused to the uidA gene and analyzed in Nicotiana benthamiana transgenic plants by histochemical and fluorimetric GUS assay. Deletion analysis did not cause loss of function on any of the promoters, but affected the gene expression on CsGAPC2 and CsEF1 truncated versions. The isolated promoters represent good candidates to be used in citros genetic transformation.

AN ABSTRACT OF THE DISSERTATION OF BELAN M. KHALIL, for the Doctor of Philosophy degree in Plant Biology, presented July 11, 2018, at Southern Illinois University Carbondale. TITLE: ANALYSIS OF THE CIS-REGULATORY ELEMENT LEXICON IN UPSTREAM GENE PROMOTERS OF ARABIDOPSIS THALIANA AND ORYZA SATIVA. MAJOR PROFESSOR: Dr Matt Geisler Gene expression in plants is partly regulated through an interaction of trans-acting factors with the promoter regions of the gene. Trans-acting factor binding sites consist of short nucleotide sequences most often present in the upstream promoter region. These binding sites, the cis-regulatory elements (CREs), vary in structure, complexity and function. In binding to trans-acting factors, CREs connect genes to signalling and regulatory pathways that affect plant growth, development, and response to the environment. As words in a language, CREs and thus promoters can be analyzed by looking for spelling (patterns of nucleotides) associated with meaning (functions). Considering CREs as words in a language, this kind of analysis provides a great opportunity for comprehensive understanding of promoter language. Identification and characterization of CREs are challenging either experimentally or bioinformatically, and has previously been accomplished by discovering degenerate words, with ambiguous nucleotides. This kind of result implicitly makes a hypothesis that binding of a specific trans-acting factor is somewhat promiscuous (or sloppy) and that all words represented by a degenerate pattern are equally good at binding. In this study, we unpack the “degeneracy hypothesis” by systematically considering each combination of letters independently for CRE function. Our results demonstrate that not all degenerate combinations of published CREs have the same effect on gene expression. A systematic search and comparison of all 65,536 possible 8 bp CRE words were searched in the 500 bp and 1000 bp upstream promoters of all genes in Arabidopsis thaliana and Oryza sativa, respectively. The function of each CRE was evaluated by statistically comparing the presence or absence of the element in the promoter with that genes response (induction or suppression) to stimuli in 1691 public availability transcriptomes of differential gene expression data. Arabidopsis, a model dicot plant had a much larger number of such data sets, than rice, however rice was chosen as a comparison as it had the largest number of datasets for a monocot, the most distantly related plant group with sufficient data available. A comprehensive list of 8 bp words associated with differential gene expression, linguistically known as lexicon, was retrieved for both species by establishing that the presence of a CRE significantly increased the likelihood for differential expression by at least one stimulus. The lexicons were composed of 641 and 856 CREs respectively in Arabidopsis and rice, and there were only 78 shared CREs between the two lexicons. The CRE lexicon was then characterized for their strength and breadth of response, occurrence frequency, sequence complexity, and sequence conservation between two species. In Arabidopsis, evening element (EE) showed the strongest response to a cold stress transcriptome (p-value 10-99). In rice, the element AAACCCTA showed strongest response to a tissue specific transcriptome (p-value 10-79). The breadth of response varied between the two species due to number of transcriptomes used in the study. The element AAACCCTA and GCGGCGGA significantly correlated to 197 and 58 transcriptomes in both Arabidopsis and rice, respectively. On the other side of the breadth scale there were also many CREs with very restricted response. There were 291 and 258 CREs in Arabidopsis and rice, respectively, significantly correlated to a single stimulus. Occurrence frequency revealed that the most abundant CREs in Arabidopsis and rice genes were TATA box and TATA box like CREs. The structure of the CREs in the lexicon was also varied. CREs were distributed on seven levels of complexity. Level one comprised CREs having 8 copies of the same nucleotide, level seven comprised CREs having two copies of the same nucleotide. In Arabidopsis, out of 641 CREs, 314 were of level 6 complexity, which means having 3 copies of the same nucleotide. In rice, the majority of the lexicon, 263 CREs were of level 5 complexity, which means having 4 copies of the same nucleotide. Each CRE of the lexicon was correlated to at least one experimental condition in the differential gene expression data, but many were correlated to multiple and often related conditions such as drought, temperature and salinity. Therefore, each CRE was assigned a “meaning”, i.e. the associated stimuli, thus providing a sort of CRE function dictionary in addition to the lexicon itself. Many CREs possessed different meanings (termed homographs in language), and in many cases the meanings of different CREs overlapped like language synonyms. Sharing meanings (synonyms) was often among CREs with strong sequence similarity (homonyms or homophones), however, not in all cases. Analyzed as a linguistic aspect, CRE homonymity and synonymity was applied to explore the hypothesis “all CRE synonyms are also homonyms and all CRE homonyms are also synonyms.” To the end a single CRE was compared to all possible CREs with only one letter mismatch in their sequences are considered as homonyms. The CREs meaning was converted to a matrix of stimuli to generate clusters of synonyms that were analyzed for similarity of spelling (sequence). This analysis showed that not all homonyms are synonyms, however most synonyms are homonyms. Furthermore, despite a search of all one letter mismatches among homonyms, many of the functional homonyms shared smaller 4-5bp core sequence and only varied at the flanks. Synonyms being homonyms in the language of promoters raises a question, how did this evolve? Duplication of transcription factors in the genome generated transcription factor families where each family member shares the same core domain, usually a DNA recognition site. We here propose that CREs also duplicate during gene duplication process building CRE families in parallel. Members of CRE families may show different connectivity and affinity to individual members of transcription factors in a transcription factor family. In environmental sensors and developmental decision panel, this association of two families of interaction factors is called dense overlapping region (or DOR) and is a highly overrepresented network topology in biological systems. This also explains the degeneracy of initially discovered CREs. The fact is only a portion of nucleotide combinations implied by a degenerate CRE is bioactive, it represents an overlap of different members of a CRE family which is part of the process of family expansion and diversification and done as compensatory mutations as the family of transcription factors expanded and diversified. We also extensively studied CREs involved abiotic stress and identifies shared elements among abiotic stresses as well as abiotic stress specific CREs. Furthermore, CREs follow a time-sensitive response rule, which means some CREs participates in gene expression regulation only at a certain period during the course of exposure to the abiotic stress.

A transformação genética é uma alternativa ao melhoramento convencional de citros que permite a modificação de genótipos pela introdução de um ou mais genes oriundos de organismos semelhantes ou filogeneticamente distantes do hospedeiro. Os genes transferidos para espécies de interesse devem ser controlados por promotores, os quais regulam a expressão gênica de forma temporal, espacial e na magnitude desejada. Na maioria dos casos, os genes são expressos de forma constitutiva utilizando o promotor CaMV35S isolado do Vírus do Mosaico da Couve Flor. No entanto, o desenvolvimento de novas abordagens de transformação de plantas (cisgenia e intragenia), que fazem o uso de genes e sequências regulatórias derivadas da mesma espécie ou espécies relacionadas, requer a disponibilidade de elementos genéticos, incluindo promotores constitutivos, isolados de citros. Assim, o objetivo do trabalho foi clonar e caracterizar promotores constitutivos de Citrus sinensis. Para isso, a região promotora dos genes Fator de elongação 1-α (CsEF1), Gliceraldeido-3-fosfato-desidrogenase C2 (CsGAPC2) e Cyclofilina (CsCYC) foi isolada e avaliados pela fusão ao gene repórter uidA. A funcionalidade dos três promotores foi confirmada por ensaio histoquímico da atividade GUS em folhas, caules e raízes de plantas transgênicas de citros cv. \'Hamlin\'. A análise de RT-qPCR mostra que a expressão do gene uidA sob controle dos promotores CsCYC, CsGAPC2 e CsEF1 correspondeu a uma atividade aproximada de 64%, 58% e 47%, respectivamente em comparação com o promotor CaMV35S. A análise in silico dos promotores CsGAPC2, CsCYC e CsEF1 mostra que a atividade de cada um é controlada por uma série de putativos elementos cis-regulatórios. A sequência completa e versões truncadas originadas a partir de deleções em cada promotor foram fundidas ao gene uidA e analisadas em plantas transgênicas de Nicotiana benthamiana pelo ensaio histoquímico e fluorimétrico da atividade GUS. As análises de deleções não causaram perda de função dos promotores em estudo, mas afetaram a expressão gênica nos promotores CsGAPC2 e CsEF1. Os promotores isolados representam bons candidatos a serem utilizados em trabalhos de transformação genética de citros. / Genetic transformation is an alternative to citros conventional breeding that allows the modification of genotypes by the introduction of one or more genes derived from different organisms that can not be crossed by natural means. The transferred genes to the species of interest are controlled by promoters, which regulate a gene expression temporally, spatially and in the desired magnitude. In most cases, the introduced genes have been constitutively expressed using the CaMV35S promoter obtained from the cauliflower mosaic virus. However, the development of novel plant transformation approaches (cisgenesis and intragenesis) imply the use of genetic material from the same species or from closely related species capable of sexual hybridization, which requires the isolation of genetic elements, including citros constitutive promoters. The objective of this study was clone and characterize Citrus sinensis constitutive promoters. For this, the promoter region of the genes Elongation Factor 1-α (CsEF1), Glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2) and Cyclofiline (CsCYC) was isolated and evaluated by fusion to the uidA reporter gene. The functionality of three promoter was confirmed by histochemical GUS assay in leaves, stems and roots of transgenic citrus plants cv. \'Hamlin\'. RT-qPCR analysis revealed that uidA gene expression under control of the CsCYC, CsGAPC2 and CsEF1 promoters was approximately 64%, 58% and 47% expression compared with the CaMV35S promoter. In silico analysis of the CsGAPC2, CsCYC and CsEF1 promoters displays their activity is controlled by a series of putative cis-regulatory elements. The full length promoter and truncated versions originated from deletions in promoters sequences were fused to the uidA gene and analyzed in Nicotiana benthamiana transgenic plants by histochemical and fluorimetric GUS assay. Deletion analysis did not cause loss of function on any of the promoters, but affected the gene expression on CsGAPC2 and CsEF1 truncated versions. The isolated promoters represent good candidates to be used in citros genetic transformation.

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O Coup¬TF, αποτελεί ορφανό μέλος της υπεροικογένειας των υποδοχέων των στεροειδών/θυρεοειδών ορμονών και κατέχει κυρίαρχο ρόλο στην ανάπτυξη των εμβρύων όλων των μεταζώων. Στην παρούσα Διατριβή μελετήθηκε η cis¬ ρυθμιστική περιοχή του γονιδίου του, με σκοπό την ένταξή του στο γονιδιακό ρυθμιστικό δίκτυο του εμβρύου του αχινού. Με πειράματα in situ υβριδοποίησης βρέθηκε ότι το γονίδιο PlCoup¬TF εκφράζεται στο στοματικό εξώδερμα του γαστριδίου και στη βλεφαριδωτή ζώνη στον πλουτέα, στο είδος Paracentrotus lividus. Από παλαιότερα πειράματα είχε βρεθεί ότι το τμήμα της ανοδικής περιοχής που εκτείνεται από το -232 ως το ¬532 (τμήμα a), είναι απαραίτητο και επαρκές για την έκφραση του γονιδίου αναφοράς (gfp) στη βλεφαριδωτή ζώνη του πλουτέα. Εντός της περιοχής a ανευρέθησαν τρία πιθανά ρυθμιστικά στοιχεία (¬ 453, ¬432 και ¬377) του γονιδίου PlCoup¬TF, τα οποία αναγνωρίζονται από πρωτεΐνες εμβρυικού πυρηνικού εκχυλίσματος. Στοχευμένες μεταλλάξεις των στοιχείων αυτών, οδήγησαν σε μείωση της έκφρασης του γονιδίου αναφοράς (στοιχείο ¬453) και στην εκτοπική έκφρασή του (στοιχεία ¬432 και ¬377). Περαιτέρω μελέτη των παραγόντων που αναγνωρίζουν τα στοιχεία αυτά, οδήγησε στο συμπέρασμα ότι ο μεταγραφικός παράγοντας PlElk αναγνωρίζει το στοιχείο ¬ 453 και ρυθμίζει θετικά το γονιδίο του PlCoup¬TF και ο μεταγραφικός παράγοντας PlOtx αναγνωρίζει το στοιχείο -377 και καταστέλλει την έκφραση του PlCoup¬TF στο αντιστοματικό εξώδερμα. Τα αποτελέσματα της παρούσης εργασίας οδήγησαν στην ένταξη του γονιδίου PlCoup¬TF και των δύο ρυθμιστών του στο γονιδιακό ρυθμιστικό δίκτυο που καθορίζει τη διαφοροποίηση της βλεφαριδωτής ζώνης εντός του εμβρυικού εξωδέρματος. / Coup­TF, an orphan member of the nuclear receptor super family, has a fundamental role in the development of metazoan embryos. The study of the gene's regulatory circuit in the sea urchin embryo will facilitate the placement of this transcription factor in the well­studied embryonic Gene Regulatory Network (GRN). The Paracentrotus lividus Coup­TF gene (PlCoup­TF) is expressed throughout embryonic development preferentially in the oral ectoderm of the gastrula and the ciliary band of the pluteus stage. Two overlapping λ genomic clones, containing three exons and upstream sequences of PlCoup­TF, were isolated from a genomic library. The transcription initiation site was determined and 5′ deletions and individual segments of a 1930 bp upstream region were placed ahead of a GFP reporter cassette and injected into fertilized P.lividus eggs. Module a (−532 to −232), was necessary and sufficient to confer ciliary band expression to the reporter. Comparison of P.lividus and Strongylocentrotus purpuratusupstream Coup­TF sequences, revealed considerable conservation, but none within module a. 5′ and internal deletions into module a, defined a smaller region that confers ciliary band specific expression. Putative regulatory cis­acting elements (RE1, RE2 and RE3) within module a, were specifically bound by proteins in sea urchin embryonic nuclear extracts. Site­specific mutagenesis of these elements resulted in loss of reporter activity (RE1) or ectopic expression (RE2, RE3). It is proposed that sea urchin transcription factors, which bind these three regulatory sites, are necessary for spatial and quantitative regulation of the PlCoup­TF gene at pluteus stage sea urchin embryos. Additional experiments led us to the conclusion that transcription factor PlElk binds to the ­453 regulatory element and positively regulates PlCoup­TF gene in the ciliary band. Furthermore, PlOtx binds to the ­377 regulatory element and negatively regulates PlCoup­TF gene in the aboral ectoderm. These findings lead to the hierarchical positioning of PlCoup­TF within the embryonic GRN

A major challenge in life sciences is the understanding of mechanisms that regulate the expression of genes. An important step towards this goal is the ability to identify transcriptional regulatory elements like binding sites for transcription factors. In computational biology, a popular approach for this task is comparative sequence analysis using both distantly as well as closely related species. Although this method has successfully identified conserved regulatory regions, the majority of binding sites can change rapidly even between closely related species. This makes it difficult to detect them using DNA sequences alone. In this thesis, we introduce two new approaches for the detection and evolutionary analysis of transcriptional elements that consider the challenges of binding site turnover. In the first part, we develop a method for detecting homologous motifs in a given set of sequences in order to obtain evidence for evolutionary events and turnover. Based on a detailed theoretical scaffold, we develop a simple, but effective and efficient heuristic for assembling local pairwise sequence alignments into a local multiple sequence alignment. This kind of multiple alignment only contains conserved motifs represented in columns which satisfy the order implied by the underlying sequences. By favoring motifs that are contained in a great range of sequences, our method is additionally able to detect even small conserved motifs. Furthermore, the calculation of the initial local pairwise alignments is generic. This allows the use of fast heuristic methods in case of large data sets while exact alignment programs can be used for small data sets where detailed information is needed. Application to artificial as well as biological data sets demonstrate the capabilities of our algorithm. In the second part, we propose a conceptually simple, but mathematically non-trivial, phenomenological model for the binding site turnover at a genomic locus. The model is based on the assumption that binding sites have a constant rate of origination and a constant decay rate per binding site. The elementary derivation of the transient probability distribution is affirmed by simulations of sequence evolution as well as biological data. Based on the derived distribution, we develop a phenomenological model of binding site number dynamics in order to detect changes in selective constraints acting on transcription factor binding sites. Using a maximum likelihood implementation as well as exploratory data analysis, we show the functionality of the model by identifying functionally important changes in the evolutionary turnover rates on biological data. Each part of this thesis leads to the development of a new program. While Tracker allows the computation of conserved homologous motifs and their representation in a local multiple alignment, Creto determines the evolutionary turnover rates for arbitrary clades of a phylogenetic tree with given binding site numbers at the final taxa. Both software tools are freely available to the scientific community for further research in this important and exciting field.