Predicting Transcript Production Rates in Yeast With Sparse Linear Models

To provide biological insights into transcriptional regulation, a couple of groups have recently presented models relating the promoter DNA-bound transcription factors (TFs) to downstream gene’s mean transcript level or transcript production rates over time. However, transcript production is dynamic in response to changes of TF concentrations over time. Also, TFs are not the only factors binding to promoters; other DNA binding factors (DBFs) bind as well, especially nucleosomes, resulting in competition between DBFs for binding at same genomic location. Additionally, not only TFs, but also some other elements regulate transcription. Within core promoter, various regulatory elements influence RNAPII recruitment, PIC formation, RNAPII searching for TSS, and RNAPII initiating transcription. Moreover, it is proposed that downstream from TSS, nucleosomes resist RNAPII elongation.

Here, we provide a machine learning framework to predict transcript production rates from DNA sequences. We applied this framework in the S. cerevisiae yeast for two scenarios: a) to predict the dynamic transcript production rate during the cell cycle for native promoters; b) to predict the mean transcript production rate over time for synthetic promoters. As far as we know, our framework is the first successful attempt to have a model that can predict dynamic transcript production rates from DNA sequences only: with cell cycle data set, we got Pearson correlation coefficient Cp = 0.751 and coefficient of determination r2 = 0.564 on test set for predicting dynamic transcript production rate over time. Also, for DREAM6 Gene Promoter Expression Prediction challenge, our fitted model outperformed all participant teams, best of all teams, and a model combining best team’s k-mer based sequence features and another paper’s biologically mechanistic features, in terms of all scoring metrics.

Moreover, our framework shows its capability of identifying generalizable fea- tures by interpreting the highly predictive models, and thereby provide support for associated hypothesized mechanisms about transcriptional regulation. With the learned sparse linear models, we got results supporting the following biological insights: a) TFs govern the probability of RNAPII recruitment and initiation possibly through interactions with PIC components and transcription cofactors; b) the core promoter amplifies the transcript production probably by influencing PIC formation, RNAPII recruitment, DNA melting, RNAPII searching for and selecting TSS, releasing RNAPII from general transcription factors, and thereby initiation; c) there is strong transcriptional synergy between TFs and core promoter elements; d) the regulatory elements within core promoter region are more than TATA box and nucleosome free region, suggesting the existence of still unidentified TAF-dependent and cofactor-dependent core promoter elements in yeast S. cerevisiae; e) nucleosome occupancy is helpful for representing +1 and -1 nucleosomes’ regulatory roles on transcription.

IL-10-producing Regulatory B Cell Development in Human Autoimmune Disease

B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of costimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and inflammation has been described and the concept of “regulatory B cells” has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported with interleukin-10 (IL-10) being the most studied. IL-10-producing regulatory B cells predominantly localize within a rare CD1dhiCD5+ B cell subset in mice and the CD24hiCD27+ B cell subset in adult humans. This specific IL-10-producing subset of regulatory B cells have been named “B10 cells” to highlight that the regulatory function of these rare B cells is primarily mediated by IL-10, and to distinguish them from other regulatory B cell subsets that regulate immune responses through different mechanisms. B10 cells have been studies in a variety of animal models with autoimmune disease and clinical settings of human autoimmunity. There are many unsolved questions related to B10 cells including their surface phenotype, their origin and development in vivo, and their role in autoimmunity.

In Chapter 3 of this dissertation, the role of the B cell receptor (BCR) in B10 cell development is highlighted. First, the BCR repertoire of mouse peritoneal cavity B10 cells is examined by single cell sequencing; peritoneal cavity B10 cells have clonally diverse germline BCRs that are predominantly unmutated. Second, mouse B10 cells are shown to have higher frequencies of λ+ BCRs compared to non-B10 cells which may indicate the involvement of BCR light chain editing early in the process of B10 cell development in vivo. Third, human peripheral blood B10 cells are examined and are also found to express higher frequencies of λ chains compared to non-b10 cells. Therefore, B10 cell BCRs are clonally diverse and enriched for unmutated germline sequences and λ light chains.

In Chapter 4 of this dissertation, B10 cells are examined in the healthy developing human across the entire age range of infancy, childhood and adolescence, and in a large cohort of children with autoimmunity. The study of B10 cells in the developing human documents a massive transient expansion during middle childhood when up to 30% of blood B cells were competent to produce IL-10. The surface phenotype of pediatric B10 cells was variable and reflective of overall B cell development. B10 cells down-regulated CD4+ T cell interferon-gamma (IFN-γ) production through IL-10-dependent pathways and IFN-γ inhibited whereas interleukin-21 (IL-21) promoted B cell IL-10 competency in vitro. Children with autoimmunity had a contracted B10 cell compartment, along with increased IFN-γ and decreased IL-21 serum levels compared to age-matched healthy controls. The decreased B10 cell frequencies and numbers in children with autoimmunity may be partially explained by the differential regulation of B10 cell development by IFN-γ and IL-21 and alterations in serum cytokine levels. The age-related changes of the B10 cell compartment during normal human development provide new insights into immune tolerance mechanisms involved in inflammation and autoimmunity.

These studies collectively demonstrate that BCR signals are the most important early determinant of B10 cell development in vivo, that human B10 cells are not a surface phenotype defined developmental B cell subset but a functionally defined regulatory B cell subset that regulates CD4+ T IFN-γ production through IL-10-dependent pathways and that human B10 cell development can be regulated by soluble factors in vivo such as the cytokine milieu. The findings of these studies provide new insights into immune tolerance mechanisms involved in human autoimmunity and the potent effects of IL-21 on human B cell IL-10 competence in vitro open new horizons in the development of autologous B10 cell-based therapies as an approach to treat human autoimmune disease in the future.

Recoding: reprogrammed genetic decoding with an emphasis on antizyme regulatory frameshifting

Recoding embraces mechanisms that augment the rules of standard genetic decoding. The deviations from standard decoding are often purposeful and their realisation provides diverse and flexible regulatory mechanisms. Recoding events such as programed ribosomal frameshifting are especially plentiful in viruses. In most organisms only a few cellular genes are known to employ programed ribosomal frameshifting in their expression. By far the most prominent and therefore well-studied case of cellular +1 frameshifting is in expression of antizyme mRNAs. The protein antizyme is a key regulator of polyamine levels in most eukaryotes with some exceptions such as plants. A +1 frameshifting event is required for the full length protein to be synthesized and this requirement is a conserved feature of antizyme mRNAs from yeast to mammals. The efficiency of the frameshifting event is dependent on the free polyamine levels in the cell. cis-acting elements in antizyme mRNAs such as specific RNA structures are required to stimulate the frameshifting efficiency. Here I describe a novel stimulator of antizyme +1 frameshifting in the Agaricomycotina class of Basidiomycete fungi. It is a nascent peptide that acts from within the ribosome exit tunnel to stimulate frameshifting efficiency in response to polyamines. The interactions of the nascent peptide with components of the peptidyl transferase centre and the protein exit tunnel emerge in our understanding as powerful means which the cell employs for monitoring and tuning the translational process. These interactions can modulate the rate of translation, protein cotranslational folding and localization. Some nascent peptides act in concert with small molecules such as polyamines or antibiotics to stall the ribosome. To these known nascent peptide effects we have added that of a stimulatory effect on the +1 frameshifting in antizyme mRNAs. It is becoming evident that nascent peptide involvement in regulation of translation is a much more general phenomenon than previously anticipated.

Combined analysis of variation in core, accessory and regulatory genome regions provides a super-resolution view into the evolution of bacterial populations

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug–resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.

Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use

Genetic decoding is not ‘frozen’ as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational ‘correction’ of problem or ‘savior’ indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5′ or 3′ of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3′ from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.

Diversity of luciferase sequences and bioluminescence production in Baltic Sea Alexandrium ostenfeldii

The toxic dinoflagellate Alexandrium ostenfeldii is the only bioluminescent bloom-forming phytoplankton in coastal waters of the Baltic Sea. We analysed partial luciferase gene (lcf) sequences and bioluminescence production in Baltic A. ostenfeldii bloom populations to assess the distribution and consistency of the trait in the Baltic Sea, and to evaluate applications for early detection of toxic blooms. Lcf was consistently present in 61 Baltic Sea A. ostenfeldii strains isolated from six separate bloom sites. All Baltic Sea strains except one produced bioluminescence. In contrast, the presence of lcf and the ability to produce bioluminescence did vary among strains from other parts of Europe. In phylogenetic analyses, lcf sequences of Baltic Sea strains clustered separately from North Sea strains, but variation between Baltic Sea strains was not sufficient to distinguish between bloom populations. Clustering of the lcf marker was similar to internal transcribed spacer (ITS) sequences with differences being minor and limited to the lowest hierarchical clusters, indicating a similar rate of evolution of the two genes. In relation to monitoring, the consistent presence of lcf and close coupling of lcf with bioluminescence suggests that bioluminescence can be used to reliably monitor toxic bloom-forming A. ostenfeldii in the Baltic Sea.

Diversity of luciferase sequences and bioluminescence production in Baltic Sea Alexandrium ostenfeldii

The toxic dinoflagellate Alexandrium ostenfeldii is the only bioluminescent bloom-forming phytoplankton in coastal waters of the Baltic Sea. We analysed partial luciferase gene (lcf) sequences and bioluminescence production in Baltic A. ostenfeldii bloom populations to assess the distribution and consistency of the trait in the Baltic Sea, and to evaluate applications for early detection of toxic blooms. Lcf was consistently present in 61 Baltic Sea A. ostenfeldii strains isolated from six separate bloom sites. All Baltic Sea strains except one produced bioluminescence. In contrast, the presence of lcf and the ability to produce bioluminescence did vary among strains from other parts of Europe. In phylogenetic analyses, lcf sequences of Baltic Sea strains clustered separately from North Sea strains, but variation between Baltic Sea strains was not sufficient to distinguish between bloom populations. Clustering of the lcf marker was similar to internal transcribed spacer (ITS) sequences with differences being minor and limited to the lowest hierarchical clusters, indicating a similar rate of evolution of the two genes. In relation to monitoring, the consistent presence of lcf and close coupling of lcf with bioluminescence suggests that bioluminescence can be used to reliably monitor toxic bloom-forming A. ostenfeldii in the Baltic Sea.

Comprehensive Transcriptome Analysis of Sex-Biased Expressed Genes Reveals Discrete Biological and Physiological Features of Male and Female Schistosoma japonicum

Schistosomiasis is a chronic and debilitating disease caused by blood flukes (digenetic trematodes) of the genus Schistosoma. Schistosomes are sexually dimorphic and exhibit dramatic morphological changes during a complex lifecycle which requires subtle gene regulatory mechanisms to fulfil these complex biological processes. In the current study, a 41,982 features custom DNA microarray, which represents the most comprehensive probe coverage for any schistosome transcriptome study, was designed based on public domain and local databases to explore differential gene expression in S. japonicum. We found that approximately 1/10 of the total annotated genes in the S. japonicum genome are differentially expressed between adult males and females. In general, genes associated with the cytoskeleton, and motor and neuronal activities were readily expressed in male adult worms, whereas genes involved in amino acid metabolism, nucleotide biosynthesis, gluconeogenesis, glycosylation, cell cycle processes, DNA synthesis and genome fidelity and stability were enriched in females. Further, miRNAs target sites within these gene sets were predicted, which provides a scenario whereby the miRNAs potentially regulate these sex-biased expressed genes. The study significantly expands the expressional and regulatory characteristics of gender-biased expressed genes in schistosomes with high accuracy. The data provide a better appreciation of the biological and physiological features of male and female schistosome parasites, which may lead to novel vaccine targets and the development of new therapeutic interventions.

The regulatory role of eNOS-derived nitric oxide on transcription in endothelial cells: Impact of S-nitrosylation on β-catenin signaling

Les cellules endothéliales forment une couche semi-perméable entre le sang et les organes. La prolifération, la migration et la polarisation des cellules endothéliales sont essentielles à la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, soit l’angiogenèse. Le facteur de croissance de l’endothélium vasculaire (VEGF) peut activer la synthase endothéliale du monoxyde d’azote (eNOS) et induire la production de monoxyde d’azote (NO) nécessaire pour la régulation de la perméabilité vasculaire et l’angiogenèse. β- caténine est une composante essentielle du complexe des jonctions d’ancrage ainsi qu’un régulateur majeur de la voie de signalisation de Wnt/β-caténine dans laquelle elle se joint au facteur de transcription TCF/LEF et module l’expression de nombreux gènes, dont certains sont impliqués dans l’angiogenèse. La S-nitrosylation (SNO) est un mécanisme de régulation posttraductionnel des protéines par l’ajout d’un groupement nitroso au niveau de résidus cystéines. Le NO produit par eNOS peut induire la S-nitrosylation de la β−caténine au niveau des jonctions intercellulaires et moduler la perméabilité de l’endothélium. Il a d’ailleurs été montré que le NO peut contrôler l’expression génique par la transcription. Le but de cette thèse est d’établir le rôle du NO au sein de la transcription des cellules endothéliales, spécifiquement au niveau de l’activité de β-caténine. Le premier objectif était de déterminer si la SNO de la β-caténine affecte son activité transcriptionnelle. Nous avons montré que le NO inhibe l’activité transcriptionnelle de β- caténine ainsi que la prolifération des cellules endothéliales induites par l’activation de la voie Wnt/β-caténine. Il est intéressant de constater que le VEGF, qui induit la production de NO via eNOS, réprime l’expression de AXIN2 qui est un gène cible de Wnt s’exprimant suite à la i i stimulation par Wnt3a et ce, dépendamment de eNOS. Nous avons identifié que la cystéine 466 de la β-caténine est un résidu essentiel à la modulation répressive de son activité transcriptionnelle par le NO. Lorsqu’il est nitrosylé, ce résidu est responsable de la perturbation du complexe de transcription formé de β-caténine et TCF-4 ce qui inhibe la prolifération des cellules endothéliales induite par la stimulation par Wnt3a. Puisque le NO affecte la transcription, nous avons réalisé l’analyse du transcriptome afin d’obtenir une vue d’ensemble du rôle du NO dans l’activité transcriptionnelle des cellules endothéliales. L’analyse différentielle de l’expression des gènes de cellules endothéliales montre que la répression de eNOS par siRNA augmente l’expression de gènes impliqués au niveau de la polarisation tels que : PARD3A, PARD3B, PKCZ, CRB1 et TJ3. Cette analyse suggère que le NO peut réguler la polarisation des cellules et a permis d’identifier des gènes responsables de l’intégrité des cellules endothéliales et de la réponse immunitaire. De plus, l’analyse de voies de signalisation par KEGG montre que certains gènes modulés par l’ablation de eNOS sont enrichis dans de nombreuses voies de signalisation, notamment Ras et Notch qui sont importantes lors de la migration cellulaire et la différenciation des cellules de têtes et de tronc (tip/stalk). Le regroupement des gènes exprimés chez les cellules traitées au VEGF (déplétées de eNOS ou non) révèle que le NO peut affecter l’expression de gènes contribuant au processus angiogénique, dont l’attraction chimiotactique. Notre étude montre que le NO module la transcription des cellules endothéliales et régule l’expression des gènes impliqués dans l’angiogenèse et la fonction endothéliale.

Bayesian Methods for Inferring Gene Regulatory Networks

Thesis (Ph.D.)--University of Washington, 2016-08

Analysis of Alpha CA Genes in Early Vertebrates and ; High-Invertebrates

Carbonic anhydrases are enzymes that are ubiquitously found in all organisms that are engaged in catalyzing the hydration of carbon dioxide to form bicarbonate and proton and vice versa. They are crucial in the process of respiration, bone resorption, pH regulation, ion transport, and photosynthesis in plants. Out of the five classes of carbonic anhydrase α, β, γ, δ, ζ this study focused in the α carbonic anhydrases. This class of CAs constitute of 16 subfamilies in mammals that include 3 non-active enzymes known as Carbonic Anhydrase Related Proteins. The inactiveness of these enzymes is due to the loss of one or more Histidine residues in the active site. This thesis was conducted based on the aim of studying evolutionary analysis of carbonic anhydrase sequences from organisms spanning from the Cambrian age. It was carried out in two phases. The first phase was the sequence collection, which involved many biological sequence databases as a source. The scope of this segment included sequence alignments and analysis of the sequence manually and in an automated form incorporating few analysis tools. The second Phase was phylogenetic analysis and exploring the subcellular location of the proteins, which was key for the evolutionary analysis. Through the medium of the methods conducted with respect to the phases mentioned above, it was possible to accomplish the desired result. Certain thought-provoking sequences were come across and analyzed thoroughly. Whereas, Phylogenetics showed interesting results to bolster previous findings and new findings as well which lay bedrock for future intensified studies.

Reorganisation ofHoxdregulatory landscapes during the evolution of a snake-like body plan

Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.

Identification and characterisation of telomere regulatory and signalling pathways after induction of telomere dysfunction

Telomeres are DNA-protein complexes which cap the ends of eukaryotic linear chromosomes. In normal somatic cells telomeres shorten and become dysfunctional during ageing due to the DNA end replication problem. This leads to activation of signalling pathways that lead to cellular senescence and apoptosis. However, cancer cells typically bypass this barrier to immortalisation in order to proliferate indefinitely. Therefore enhancing our understanding of telomere dysfunction and pathways involved in regulation of the process is essential. However, the pathways involved are highly complex and involve interaction between a wide range of biological processes. Therefore understanding how telomerase dysfunction is regulated is a challenging task and requires a systems biology approach. In this study I have developed a novel methodology for visualisation and analysis of gene lists focusing on the network level rather than individual or small lists of genes. Application of this methodology to an expression data set and a gene methylation data set allowed me to enhance my understanding of the biology underlying a senescence inducing drug and the process of immortalisation respectively. I then used the methodology to compare the effect of genetic background on induction of telomere uncapping. Telomere uncapping was induced in HCT116 WT, p21-/- and p53-/- cells using a viral vector expressing a mutant variant of hTR, the telomerase RNA template. p21-/- cells showed enhanced sensitivity to telomere uncapping. Analysis of a candidate pathway, Mismatch Repair, revealed a role for the process in response to telomere uncapping and that induction of the pathway was p21 dependent. The methodology was then applied to analysis of the telomerase inhibitor GRN163L and synergistic effects of hypoglycaemia with this drug. HCT116 cells were resistant to GRN163L treatment. However, under hypoglycaemic conditions the dose required for ablation of telomerase activity was reduced significantly and telomere shortening was enhanced. Overall this new methodology has allowed our group and collaborators to identify new biology and improve our understanding of processes regulating telomere dysfunction.

Real-time PCR quantification and diversity analysis of the functional genes aprA and dsrA of sulfate-reducing prokaryotes in marine sediments of the Peru continental margin and the Black Sea

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5'-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 10(8)/g sediment close to the sediment surface to less than 10(5)/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5-1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40-121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 10(4)/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere.

Transcriptional dynamics of the developing sweet cherry (Prunus avium L.) fruit: sequencing, annotation and expression profiling of exocarp-associated genes

The exocarp, or skin, of fleshy fruit is a specialized tissue that protects the fruit, attracts seed dispersing fruit eaters, and has large economical relevance for fruit quality. Development of the exocarp involves regulated activities of many genes. This research analyzed global gene expression in the exocarp of developing sweet cherry (Prunus avium L., 'Regina'), a fruit crop species with little public genomic resources. A catalog of transcript models (contigs) representing expressed genes was constructed from de novo assembled short complementary DNA (cDNA) sequences generated from developing fruit between flowering and maturity at 14 time points. Expression levels in each sample were estimated for 34 695 contigs from numbers of reads mapping to each contig. Contigs were annotated functionally based on BLAST, gene ontology and InterProScan analyses. Coregulated genes were detected using partitional clustering of expression patterns. The results are discussed with emphasis on genes putatively involved in cuticle deposition, cell wall metabolism and sugar transport. The high temporal resolution of the expression patterns presented here reveals finely tuned developmental specialization of individual members of gene families. Moreover, the de novo assembled sweet cherry fruit transcriptome with 7760 full-length protein coding sequences and over 20 000 other, annotated cDNA sequences together with their developmental expression patterns is expected to accelerate molecular research on this important tree fruit crop.