Predição de promotores de Bacillus subtilis usando técnicas de aprendizado de máquina

One of the most important goals of bioinformatics is the ability to identify genes in uncharacterized DNA sequences on world wide database. Gene expression on prokaryotes initiates when the RNA-polymerase enzyme interacts with DNA regions called promoters. In these regions are located the main regulatory elements of the transcription process. Despite the improvement of in vitro techniques for molecular biology analysis, characterizing and identifying a great number of promoters on a genome is a complex task. Nevertheless, the main drawback is the absence of a large set of promoters to identify conserved patterns among the species. Hence, a in silico method to predict them on any species is a challenge. Improved promoter prediction methods can be one step towards developing more reliable ab initio gene prediction methods. In this work, we present an empirical comparison of Machine Learning (ML) techniques such as Na¨ýve Bayes, Decision Trees, Support Vector Machines and Neural Networks, Voted Perceptron, PART, k-NN and and ensemble approaches (Bagging and Boosting) to the task of predicting Bacillus subtilis. In order to do so, we first built two data set of promoter and nonpromoter sequences for B. subtilis and a hybrid one. In order to evaluate of ML methods a cross-validation procedure is applied. Good results were obtained with methods of ML like SVM and Naïve Bayes using B. subtilis. However, we have not reached good results on hybrid database

Identification of two steroid-responsive promoters of different strength controlled by the same estrogen-responsive element in the 5'-end region of the Xenopus laevis vitellogenin gene A1.

A structural and functional analysis of the 5'-end region of the Xenopus laevis vitellogenin gene A1 revealed two transcription initiation sites located 1.8 kilobases apart. A RNA polymerase II binding assay indicates that both promoters form initiation complexes efficiently. In vitro, using a transcription assay derived from a HeLa whole-cell extract, the upstream promoter is more than 10-fold stronger than the downstream one. In contrast, both promoters have a similar strength in a HeLa nuclear extract. In vivo, that is in estrogen-stimulated hepatocytes, it is the downstream promoter homologous to the one used by the other members of the vitellogenin gene family, which is 50-fold stronger than the upstream promoter. Thus, if functional vitellogenin mRNA results from this latter activity, it would contribute less than 1% to the synthesis of vitellogenin by fully induced Xenopus hepatocytes expressing the four vitellogenin genes. In contrast, both gene A1 promoters are silent in uninduced hepatocytes. Transfection experiments using the Xenopus cell line B3.2 in which estrogen-responsiveness has been introduced reveal that the strong downstream promoter is controlled by an estrogen responsive element (ERE) located 330 bp upstream of it. The upstream promoter can also be controlled by the same ERE. Since the region comprising the upstream promoter is flanked by a 200 base pair long inverted repeat with stretches of homology to other regions of the X. laevis genome, we speculate that it might have been inserted upstream of the vitellogenin gene A1 by a recombination event and consequently brought under control of the ERE lying 1.5 kilobases downstream.

Identification and isolation of plant promoters induced by thiocyanate

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Identification of Novel Cellular Transcription Factors that Regulate Early Promoters of Human Papillomavirus Types 18 and 16

Background. The long control region (LCR) of human papillomavirus (HPV) regulates early gene transcription by interaction with several viral and cellular transcription factors (TFs). Methods. To identify novel TFs that could influence early expression of HPV type 18 (HPV-18) and HPV type 16 (HPV-16), a high-throughput transfection array was used. Results. Among the 704 TFs tested, 28 activated and 36 inhibited the LCR of HPV-18 by more than 2-fold. For validation, C33 cells were cotransfected with increasing amounts of selected TF expression plasmids in addition to LCR-luciferase vectors of different molecular variants of HPV-18 and HPV-16. Among the TFs identified, only GATA3, FOXA1, and MYC have putative binding sites within the LCR sequence, as indicated using the TRANSFAC database. Furthermore, we demonstrated FOXA1 and MYC in vivo binding to the LCR of both HPV types using chromatin immunoprecipitation assay. Conclusions. We identified new TFs implicated in the regulation of the LCR of HPV-18 and HPV-16. Many of these factors are mutated in cancer or are putative cancer biomarkers and could potentially be involved in the regulation of HPV early gene expression.

IDENTIFICATION OF FACTORS INVOLVED IN DNA METHYLATION OF CPG-ISLAND-PROMOTERS

Repression of many tumor suppressor genes (TSGs) in cancer is mediated by aberrantly increased DNA methylation levels at promoter CpG islands (CGI). About one-fourth of empirically defined human promoters are surrounded by or contain clustered repetitive elements. It was previously observed that a sharp transition of methylation occurs between highly methylated repetitive elements (SINE or LINE) and unmethylated CGI-promoters (e.g. P16, VHL, CDH and RIL) in normal tissues. The functions that lead to increased CGI methylation in cancer remain poorly understood. We propose that CGI-promoters contain cis-elements for triggering de novo DNA methylation. In the first part of our project, we established a site-specific integration system with enforced local transcriptional repression in colorectal cancer cells and monitored the occurrence of de novo DNA methylation in exogenous fragments containing a CGI-promoter and repetitive elements. Initial de novo methylation was seeded at specific CG sites in a repetitive element, and accelerated by persistent binding of a KRAB-containing transcriptional repressor. Furthermore, additional repetitive elements (LINE and SINE) located adjacent to the promoter could confer DNA methylation spreading into the CGI particularly in the setting of KRAB-factor binding. However, a repressive chromatin alone was not sufficient to initiate DNA methylation, which required specific DNA sequences and was integration-site (and/or cell-line) specific. In addition, all the methylation observed showed slow and gradual accumulation over several months of culture. Overall, these results demonstrate a requirement for specific DNA sequences to trigger de novo DNA methylation, and repetitive elements as cis-regulatory factors to cooperate with strengthened transcriptional repression in promoting methylation spreading. In the second part, we re-introduced disrupted DNMT3B or DNMT1 into HCT116 DKO cells and mapped the remethylation pattern through a profiling method (DREAM). Moderate remethylation occurred when DNMT3B was re-expressed with a preference toward non-CGI and non-promoter regions. Hence, there exists a set of genomic regions with priority to be targets for DNMT3B in somatic cells.

Identification of a minimal promoter sequence for the human N-acetyltransferase Type I gene that binds AP-1 (activator protein 1) and YY-1 (Yin and Yang 1)

Human N-acetyltransferase Type I (NAT1) catalyses the acetylation of many aromatic amine and hydrazine compounds and it has been implicated in the catabolism of folic acid. The enzyme is widely expressed in the body, although there are considerable differences in the level of activity between tissues. A search of the mRNA databases revealed the presence of several NAT1 transcripts in human tissue that appear to be derived from different promoters. Because little is known about NAT1 gene regulation, the present study was undertaken to characterize one of the putative promoter sequences of the NAT1 gene located just upstream of the coding region. We show with reverse-transcriptase PCR that mRNA transcribed from this promoter (Promoter 1) is present in a variety of human cell-lines, but not in quiescent peripheral blood mononuclear cells. Using deletion mutant constructs, we identified a 20 bp sequence located 245 bases upstream of the translation start site which was sufficient for basal NAT1 expression. It comprised an AP-1 (activator protein 1)-binding site, flanked on either side by a TCATT motif. Mutational analysis showed that the AP-1 site and the 3' TCATT sequence were necessary for gene expression, whereas the 5' TCATT appeared to attenuate promoter activity. Electromobility shift assays revealed two specific bands made up by complexes of c-Fos/Fra, c-Jun, YY-1 (Yin and Yang 1) and possibly Oct-1. PMA treatment enhanced expression from the NAT1 promoter via the AP-1-binding site. Furthermore, in peripheral blood mononuclear cells, PMA increased endogenous NAT1 activity and induced mRNA expression from Promoter I, suggesting that it is functional in vivo.

Experimental procedures for the identification of material properties of intervertebal disc - a contribution for the development of biomimetic substitutes for the nucleus pulposus

Tese de Doutoramento em Engenharia Mecânica.

I. Identification and characterisation of epigenetically altered tumor supressor genes by proteomic and genomic approches / II. Studies of genes regualted by MeCP2 union to its promoter regions by proteomic and genmomic approches

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.

Comparative study and identification of potent eukaryotic transcriptional repressors in gene switch systems.

In mammalian cells, proper gene regulation is achieved by the complex interplay of transcription factors that activate or repress gene expression by binding to the regulatory regions of target promoters. While transcriptional activators have been extensively characterised and classified into functional groups, relatively little is known about the comparative strength and cell type-specificity of transcriptional repressors. Here, we have compared the ability of a series of eukaryotic repression domains to silence basal and activated transcription. A series of the most potent repression domains was further tested in the context of a gene therapy gene-switch system in various cell types. The results indicate that the analysed repression domains exert varying silencing activities in different promoter contexts. Furthermore, their potential for gene silencing varies also depending on the cellular context. When multimerised within one chimeric repressor protein, particular combinations of repressor domains were found to display synergistic repressing effects and efficient repression in a panel of cell lines. This approach thus allowed the identification of transcriptional repressors that are both potent and versatile in terms of cellular specificity as a basis for gene switch systems.

Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line.

The expression of a hybrid gene formed by the promoter region of the Xenopus laevis vitellogenin gene B1 and the CAT coding region is regulated by estrogen when the gene is transfected into hormone-responsive MCF-7 cells. Furthermore, the 5' flanking region of the gene B1 alone can confer inducibility to heterologous promoters, although to a varying extent depending on the promoter used. Deletion mapping of he vitellogenin hormone-responsive sequences revealed that a 13 bp element 5'-AGTCACTGTGACC-3' at position -334 is essential for estrogen inducibility. We have shown previously that this 13 bp element is present upstream of several liver-specific estrogen-inducible genes.

Identification of novel functional TBP-binding sites and general factor repertoires.

Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)-on-chip analysis for 28 transcription factors on a large set of known and novel TATA-binding protein (TBP)-binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP-binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP-on-chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)-specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG and non-CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP-binding sites in the human genome.

Identification of HMX1 target genes: a predictive promoter model approach.

PURPOSE: A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes. METHODS: We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. RESULTS: The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. CONCLUSIONS: Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.

Identification in silico d’éléments de réponse de récepteurs nucléaires impliqués dans le cancer du sein

La croissance de deux tiers des tumeurs mammaires dépend des œstrogènes. Le réseau de gènes responsable de propager les signaux prolifératifs des œstrogènes est encore mal connu. Des micropuces d’ADN de cellules de carcinome mammaire MCF7 traitées à l’œstradiol (E2) avec ou sans l’inhibiteur de synthèse protéique cycloheximide (CHX) ont permis d’identifier de nombreux gènes cibles primaires et secondaires. La séquence des promoteurs des gènes cibles a été criblée à l’aide d’une banque de 300 matrices modélisant les sites reconnus par divers facteurs de transcription. Les éléments de réponse aux œstrogènes (ERE) sont enrichis dans les promoteurs des gènes primaires. Les sites E2F sont enrichis dans les promoteurs des gènes cible secondaires. Un enrichissement similaire a été observé avec les régions liées par ERα et E2F1 en ChIP-on-chip pour chacune des catégories de gènes. La croissance des cellules de carcinome mammaire est inhibée par des traitements à l’acide rétinoïque (RA). L’analyse de micropuces d’ADN de MCF7 traitées avec RA a permis d’identifier de nombreux gènes cibles potentiels. Un enrichissement d’éléments de réponse à l’acide rétinoïque (RARE) est observable dans les promoteurs de ces gènes après avoir exclus les RARE se trouvant à l’intérieur d’éléments transposables. Des RARE présents dans des éléments transposables spécifiques aux primates sont aussi fixés in vivo dans les promoteurs de cibles connues de RA : BTG2, CASP9 et GPRC5A. Certains gènes cibles de RA dans les MCF7 sont aussi des cibles de E2, suggérant que le contrôle que ces molécules exercent sur la prolifération est en partie attribuable à des effets opposés sur un ensemble commun de gènes.

Searching for novel gene functions in yeast : identification of thousands of novel molecular interactions by protein-fragment complementation assay followed by automated gene function prediction and high-throughput lipidomics

La compréhension de processus biologiques complexes requiert des approches expérimentales et informatiques sophistiquées. Les récents progrès dans le domaine des stratégies génomiques fonctionnelles mettent dorénavant à notre disposition de puissants outils de collecte de données sur l’interconnectivité des gènes, des protéines et des petites molécules, dans le but d’étudier les principes organisationnels de leurs réseaux cellulaires. L’intégration de ces connaissances au sein d’un cadre de référence en biologie systémique permettrait la prédiction de nouvelles fonctions de gènes qui demeurent non caractérisées à ce jour. Afin de réaliser de telles prédictions à l’échelle génomique chez la levure Saccharomyces cerevisiae, nous avons développé une stratégie innovatrice qui combine le criblage interactomique à haut débit des interactions protéines-protéines, la prédiction de la fonction des gènes in silico ainsi que la validation de ces prédictions avec la lipidomique à haut débit. D’abord, nous avons exécuté un dépistage à grande échelle des interactions protéines-protéines à l’aide de la complémentation de fragments protéiques. Cette méthode a permis de déceler des interactions in vivo entre les protéines exprimées par leurs promoteurs naturels. De plus, aucun biais lié aux interactions des membranes n’a pu être mis en évidence avec cette méthode, comparativement aux autres techniques existantes qui décèlent les interactions protéines-protéines. Conséquemment, nous avons découvert plusieurs nouvelles interactions et nous avons augmenté la couverture d’un interactome d’homéostasie lipidique dont la compréhension demeure encore incomplète à ce jour. Par la suite, nous avons appliqué un algorithme d’apprentissage afin d’identifier huit gènes non caractérisés ayant un rôle potentiel dans le métabolisme des lipides. Finalement, nous avons étudié si ces gènes et un groupe de régulateurs transcriptionnels distincts, non préalablement impliqués avec les lipides, avaient un rôle dans l’homéostasie des lipides. Dans ce but, nous avons analysé les lipidomes des délétions mutantes de gènes sélectionnés. Afin d’examiner une grande quantité de souches, nous avons développé une plateforme à haut débit pour le criblage lipidomique à contenu élevé des bibliothèques de levures mutantes. Cette plateforme consiste en la spectrométrie de masse à haute resolution Orbitrap et en un cadre de traitement des données dédié et supportant le phénotypage des lipides de centaines de mutations de Saccharomyces cerevisiae. Les méthodes expérimentales en lipidomiques ont confirmé les prédictions fonctionnelles en démontrant certaines différences au sein des phénotypes métaboliques lipidiques des délétions mutantes ayant une absence des gènes YBR141C et YJR015W, connus pour leur implication dans le métabolisme des lipides. Une altération du phénotype lipidique a également été observé pour une délétion mutante du facteur de transcription KAR4 qui n’avait pas été auparavant lié au métabolisme lipidique. Tous ces résultats démontrent qu’un processus qui intègre l’acquisition de nouvelles interactions moléculaires, la prédiction informatique des fonctions des gènes et une plateforme lipidomique innovatrice à haut débit , constitue un ajout important aux méthodologies existantes en biologie systémique. Les développements en méthodologies génomiques fonctionnelles et en technologies lipidomiques fournissent donc de nouveaux moyens pour étudier les réseaux biologiques des eucaryotes supérieurs, incluant les mammifères. Par conséquent, le stratégie présenté ici détient un potentiel d’application au sein d’organismes plus complexes.