Submitted to the faculty of Indiana University in partial fulfillment of the requirements for the degree Master of Science in the department of Bioinformatics in School of Informatics of Indiana University 29 January, 2004 / Background: Toll and Toll-related proteins play an important role in antibacterial innate immunity and are widespread in insects, plants, and mammals. The completion of new genomes such as Anopheles gambiae has provided an avenue for a deeper understanding of Toll evolution. While most evolutionary analyses are performed on protein sequences, here, we present a unique phylogenetic analysis of Toll genes from the perspective of upstream regulatory regions so as to study the importance of evolutionary information inherited in such sequences. Results: In a comparative study, phylogeny on the protein products of Toll like genes showed consistency with earlier literature except for the single point of divergence between insects and mammals. On the other hand, the phylogeny based on upstream regulatory sequences (-3000 to +10) showed a broader distinction between the plants and the rest, though the tree was not well resolved probably due to poor alignment of these sequences. The phylogeny based on TFBs necessitated the development of a supervised statistical approach to determine their “evolutionary informativeness”. Employing the frequency of evolutionarily informative TFBs, a phylogeny was derived using pair-wise distances. It suggested a closer relationship between Anopheles and plants than to Drosophila and a significant homology among mammalian TLRs. Conclusions: A unique approach of using TFBs in studying evolution of Toll genes has been developed. Broadly, this approach showed results similar to the protein phylogeny. The inclusion of the evolutionary information from TFBs may be relevant to such analyses due to the selective pressure of conservation in upstream sequences.

Promoters, neighboring regulatory regions and those extending further upstream of the 5’end of genes, are considered one of the main components affecting the expression status of genes in a specific phenotype. More recently research by Chen et al. (2006, 2012) and Mapendano et al. (2010) demonstrated that the 3’end regulatory regions of genes also influence gene expression. However, the association between the regulatory regions surrounding 3’end of genes and their over- or under-expression status in a particular phenotype has not been systematically studied. The aim of this study is to ascertain if regulatory regions surrounding the 3’end of genes contain sufficient regulatory information to correlate genes with their expression status in a particular phenotype. Over- and under-expressed ovarian cancer (OC) genes were used as a model. Exploratory analysis of the 3’end regions were performed by transforming the annotated regions using principal component analysis (PCA), followed by clustering the transformed data thereby achieving a clear separation of genes with different expression status. Additionally, several classification algorithms such as Naïve Bayes, Random Forest and Support Vector Machine (SVM) were tested with different parameter settings to analyze the discriminatory capacity of the 3’end regions of genes related to their gene expression status. The best performance was achieved using the SVM classification model with 10-fold cross-validation that yielded an accuracy of 98.4%, sensitivity of 99.5% and specificity of 92.5%. For gene expression status for newly available instances, based on information derived from the 3’end regions, an SVM predictive model was developed with 10-fold cross-validation that yielded an accuracy of 67.0%, sensitivity of 73.2% and specificity of 61.0%. Moreover, building an SVM with polynomial kernel model to PCA transformed data yielded an accuracy of 83.1%, sensitivity of 92.5% and specificity of 74.8% using 10-fold cross-validation for evaluation. These clustering and classification analyses strongly suggest that the regions surrounding the 3’end of genes contain sufficiently rich regulatory information to discriminate between over- and under-expressed genes; at least in the case of genes implicated in OC.

L’expression des immunoglobulines (Ig) par les cellules B est dictée par de multiples remaniements géniques appelées recombinaisons V(D)J, commutation de classe (CSR) et hypermutation somatique (SHM). Tous ces événements sont contrôlés par des éléments cis-régulateurs notamment au locus IgH. Les mieux décrits sont la région intronique Eµ et ses régions d’attachement à la matrice nucléaire MARsEµ et la région régulatrice en 3’ du locus IgH (3’RR), constituée de 4 éléments activateurs (hs3a ; hs1,2 ; hs3b ; hs4) et possédant une structure quasi-palindromique très conservée. Afin d’étudier ces régions régulatrices, nous avons analysé plusieurs modèles murins présentant la délétion de la totalité de la région Eµ, des MARsEµ, et de diverses parties de la 3’RR. Ces modèles ont mis au jour des fonctions insoupçonnées de ces régions régulatrices, à divers stades du développement B. Au delà de son rôle régulateur des réarrangements précoces de DH vers JH, nous avons précisé la fenêtre d’action de région Eµ et son rôle sur la transcription et l’expression de la chaîne lourde µ du stade pré-B jusqu’au stade B transitionnel. L’activateur Eµ module en effet l’expression du pré-BCR et du BCR et par conséquent conditionne l’orientation des cellules B matures vers les compartiments de la zone marginale et folliculaire. Notre étude indique également que Eµ n’influence pas le choix des segments VH au cours de la recombinaison VDJ. Nous montrons que les régions MARsEµ, sont impliquées dans le ciblage des gènes d’Ig par l’hypermutation somatique. Le mode d’action de ces régions MARsEµ reste à définir mais nous décrivons qu’il est découplé de la transcription et qu’il affecte également des loci situés sur des chromosomes différents : loci codant les chaînes légères d’Ig (Ig) et loci des cibles illégitimes de AID (Bcl6 et Cd83).Une étude comparant un nouveau modèle murin déficient pour la région quasi-palindromique 3’IgH (de hs3a à hs3b) à deux autres modèles de notre laboratoire (3’RR KO et hs3b-hs4 KO) nous a permis d’identifier deux modules fonctionnels complémentaires dans la région 3’RR. Le module distal (hs4) impliqué dans la régulation de l’expression de la chaîne lourde du stade B immature au stade B naïf et le module proximal (de hs3a à hs3b) nécessaire aux stades matures pour la SHM et la CSR vers la majorité des isotypes après activation, mais également pour la synthèse des anticorps par les plasmocytes. / Immunoglobulin (Ig) gene expression in B cells depends on multiple genetic rearrangements named V(D)J recombination, class switch recombination (CSR) and somatic hypermutation (SHM). These events are controlled by cis-regulatory elements which are, especially numerous within the IgH locus. Major IgH regulatory elements are Eµ composed of a core enhancer flanked by nuclear matrix attachment regions MARsEµ and the regulatory region located at the 3’end of the locus (3’RR), composed of 4 enhancers (hs3a; hs1-2; hs3b; hs4) harbouring a highly conserved quasi-palindromic structure. To study these regulatory regions, we analyzed multiple mice models carrying endogenous deletions of either the entire Eµ region, MARsEµ only or various parts of the 3’RR. These models revealed unsuspected functions for all IgH regulatory regions, at various stages of B cell development. Beyond its role to control accessibility prior DH to JH rearrangements, we identified the window of activity for Eµ region and its particular role on transcription and expression of the µ heavy chain from pre-B to the transitional stages. Indeed, the Eµ enhancer modulates pre-BCR and BCR expression and consequently modulates the mature B cell fate toward marginal zone and follicular compartments. Our study also indicated that the absence of Eµ has no effect on VH segment usage during V to DJ recombination. We also showed that MARsEµ are implicated in the targeting of Ig genes by SHM. The mechanism by which MARsEµ enhances SHM remains to be clarified but we showed that the process does not depend on transcription. Surprisingly MARsEµ also modulates SHM occuring on genes located on different chromosomes: the Ig light chain loci (Igκ) and AID off-targets loci (Bcl6 and Cd83).In a study comparing a new mouse model devoid of the 3’ IgH quasi-palindromic region (hs3a to hs3b) to previous relevant models available in our laboratory (3’RR KO and hs3b-hs4 KO), we identified two complementary functional modules within the 3’RR. The distal module (hs4), which regulates Ig heavy chain expression (and therefore BCR expression) from the immature to mature naïve B cell stages and the proximal module (hs3a to hs3b and its quasi-palindromic structure) required at mature stages for SHM, CSR to most isotypes in activated B cells, and antibody production in plasma cells.

The tumour suppressor p53 protein plays a central role in the DNA damage response/checkpoint pathways leading to DNA repair, cell cycle arrest, apoptosis and senescence. The activation of p53-mediated pathways is primarily facilitated by the binding of tetrameric p53 to two 'half-sites', each consisting of a decameric p53 response element (RE). Functional REs are directly adjacent or separated by a small number of 1-13 'spacer' base pairs (bp). The p53 RE is detected by exact or inexact matches to the palindromic sequence represented by the regular expression [AG][AG][AG]C[AT][TA]G[TC][TC][TC] or a position weight matrix (PWM). The use of matrix-based and regular expression pattern-matching techniques, however, leads to an overwhelming number of false positives. A more specific model, which combines multiple factors known to influence p53-dependent transcription, is required for accurate detection of the binding sites. In this thesis, we present a logistic regression based model which integrates sequence information and epigenetic information to predict human p53 binding sites. Sequence information includes the PWM score and the spacer length between the two half-sites of the observed binding site. To integrate epigenetic information, we analyzed the surrounding region of the binding site for the presence of mono- and trimethylation patterns of histone H3 lysine 4 (H3K4). Our model showed a high level of performance on both a high-resolution data set of functional p53 binding sites from the experimental literature (ChIP data) and the whole human genome. Comparing our model with a simpler sequence-only model, we demonstrated that the prediction accuracy of the sequence-only model could be improved by incorporating epigenetic information, such as the two histone modification marks H3K4me1 and H3K4me3.

Understanding the mechanisms of transcriptional regulation and the constraints that operate in gene promoter sequences is the key step in understanding the evolutionary conservation of transcriptional regulation. It is well known that regulatory regions with the same expression outputs do not have to share the sequence similarity. The most important elements in regulatory sequences are transcription factor binding sites and their position relocation does not usually influence the expression output. The least complex transcriptional regulation is characteristic for housekeeping genes. For their expression they require only basal core promoter elements (sometimes only CpG islands are sufficient) and general transcription factors, so they can be transcribed easily and immediately whenever they are needed. In this study we focused on transcriptional regulation of invertebrate amphioxus (Branchiostoma floridae) housekeeping genes in vertebrate systems. We prepared a set of constructs with amphioxus regulatory regions for testing their activity in different mammalian cell lines and a set of constructs with the same amphioxus regulatory regions for observing their spatial recognition in developing medaka fish embryo. We found that half of investigated amphioxus regulatory regions are recognized by...