Gene regulation lies at the heart of most biological processes and transcription factors are the key molecules that control tissues specific gene expression. In higher eukaryotes transcription factors control gene expression by binding regulatory DNA segments called cis-regulatory modules (CRMs). The increasing number of sequenced genomes of multicellular eukaryotes along with high-throughput methods such as whole genome microarray expression data allows for systematic characterization of the CRMs that control gene expression. A first step towards understanding gene regulation is the identification of the regulatory elements present in the genome. We take advantage of the large database of spatio-temporal patterns of gene expression in D. melanogaster embryogenesis to identify sets of developmentally co-expressed genes. We developed a computational method that identifies DNA binding sites for transcription factors from families of co-regulated genes that are expressed during Drosophila embryo development. This method discovers over-represented motifs in a set of co-regulated genes using the exhaustive motif enumeration technique. Clustering the predicted motifs identifies the CRMs, which assist in translating a combinatorial code of TF inputs into a specific gene expression output. The predicted CRMs were verified experimentally by searching the whole genome for the predicted CRMs and establishing expression pattern of the genes that are associated with these CRMs. It is well know that the gene expression is substantially controlled through CRMs and those key regulatory sequences are conserved in related species. The conservation of CRMs can be studied by comparing the related genomes and alignment methods are widely used computational tools for comparing the sequences. However, in distantly related species the CRM sequences are simply not align able. To identify the similar CRMs in distantly related species we developed a non-alignment based method for discovering similar CRMs in related species. This method is based on word frequencies where the given sequences are compared using Poisson based metric. When starting with a set of CRMs involved in Drosophila early embryo development, we show here that our non-alignment method successfully detects similar CRMs in distantly related species ( D. ananassae, D. pseudoobscura, D. willisoni, D. mojavensis, D. virilis, D. grimshawi ). This method proved efficient in discriminating the functional CRMs from the non-functional ones.

Gene regulation lies at the heart of most biological processes and transcription factors are the key molecules that control tissues specific gene expression. In higher eukaryotes transcription factors control gene expression by binding regulatory DNA segments called cis-regulatory modules (CRMs). The increasing number of sequenced genomes of multicellular eukaryotes along with high-throughput methods such as whole genome microarray expression data allows for systematic characterization of the CRMs that control gene expression. A first step towards understanding gene regulation is the identification of the regulatory elements present in the genome. We take advantage of the large database of spatio-temporal patterns of gene expression in D. melanogaster embryogenesis to identify sets of developmentally co-expressed genes. We developed a computational method that identifies DNA binding sites for transcription factors from families of co-regulated genes that are expressed during Drosophila embryo development. This method discovers over-represented motifs in a set of co-regulated genes using the exhaustive motif enumeration technique. Clustering the predicted motifs identifies the CRMs, which assist in translating a combinatorial code of TF inputs into a specific gene expression output. The predicted CRMs were verified experimentally by searching the whole genome for the predicted CRMs and establishing expression pattern of the genes that are associated with these CRMs. It is well know that the gene expression is substantially controlled through CRMs and those key regulatory sequences are conserved in related species. The conservation of CRMs can be studied by comparing the related genomes and alignment methods are widely used computational tools for comparing the sequences. However, in distantly related species the CRM sequences are simply not align able. To identify the similar CRMs in distantly related species we developed a non-alignment based method for discovering similar CRMs in related species. This method is based on word frequencies where the given sequences are compared using Poisson based metric. When starting with a set of CRMs involved in Drosophila early embryo development, we show here that our non-alignment method successfully detects similar CRMs in distantly related species ( D. ananassae, D. pseudoobscura, D. willisoni, D. mojavensis, D. virilis, D. grimshawi ). This method proved efficient in discriminating the functional CRMs from the non-functional ones.

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.

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

Bioinformatics is a valuable tool to understand gene regulatory networks. Cis-regulatory elements (CREs) previously found in promoter regions are known to recruit transcription in signaling pathways. In this work it has been hypothesized to consider CREs as a family of related words that interact/bind to a family of related transcription factors, and thus have similar but distinct regulation patterns. A 1460 microarray gene expression collection was obtained via online databases to create a transcriptomic meta-dataset. A novel bioinformatic algorithm was applied to annotate all 65536 (64k) potential 8-letter CREs in the 500 bp upstream promoter region of all A. thaliana genes across the transcriptomic meta-dataset. Of the possible words, only 2,498 were significantly associated with a pattern of regulation in any of the 1,460 microarrays tested whereas the remaining motifs appeared not to be regulatory. Unique CREs were categorized into 4 regulatory types: inducer, suppressor, biregulator and insulator. A predicted protein protein interactome was created for an economically important plant Coffea canephora. Here, it has been hypothesized that evolutionary conservation of many core biological processes enable generation of predicted protein interactome for species with few resources other than sequenced genome. Of over 12,000 genes identified, 939 were predicted to have 4,587 interactions. Gene Ontology analysis revealed enrichment of processes conserved in all eukaryotes but depletion in unique plant processes. A third study was conducted to determine if homology modeling, evolutionary analysis, and structural evolution could determine key factors involved in function, and interaction specificity in Pus10 (EC 5.4.99.25) found in Archaea and Eukaryotes. Redundancy of Pus10 and the bacterial TrmA and TruB orthologs appear to have resulted in significant molecular evolution of Pus10 function. Neofunctionalization was identified in animal kingdom where thiouridine synthase, methylases and PSUSs (THUMP)-domain modification in early animal evolution coincides with appearance of TNF-related apoptosis-inducing ligand (TRAIL) apoptosis components. Subfunctionalization was identified for Thermococcales lineage of Archaea where a shorter forefinger-loop coincides with the loss of Ψ54 specificity as experimentally verified in P. furiosus. Absence of Pus10 was observed in Sulfolobus and higher fungi whereas in plant kingdom Pus10 function remains unknown with possible pseudogene in some lineages

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