京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24947号 / 生博第509号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 原田 浩, 教授 松本 智裕, 教授 鈴木 淳 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Mosquito reproduction is tightly regulated by the endocrine system. The sesquiterpenoid insect hormone, Juvenile hormone (JH), plays a crucial role in mosquito reproductive maturation. JH signaling pathways consist of a hierarchy of transcriptional regulators that modulate the transcriptional responses to this hormone. Genomic action of JH is mediated through the intracellular receptor Methoprene tolerant (Met) and Krüppel homolog 1 (Kr-h1), an intermediate effector that acts downstream of Met. Kr-h1 is an essential transcription factor for proper oogenesis and egg production in several insects. However, the regulatory mechanism of Kr-h1 in mosquito reproduction has not been well studied. In the current study, we performed global analyses of the Kr-h1 binding sites at multiple time points before and after a blood meal. In addition to known JH-regulated genes, we identified the binding of Kr-h1 to several genes that are controlled by the insect steroid hormone 20-hydroxyecdysone (20E). Kr-h1 seemed to have different roles in regulating the 20E-responsive genes before and after the blood-feeding. RNAi mediated Kr-h1 silencing demonstrated the activator role of Kr-h1 on several 20E-regulated genes in the previtellogenic stage, while Kr-h1 mostly repressed those genes after blood feeding. On the genes that were activated by Kr-h1 in the previtellogenic stage, the binding of Kr-h1 was associated with an increase of the histone marker H3K27ac. For the first time, we demonstrated that the regulatory action of Kr-h1 involves histone modification on the 20E-responsive genes. This study significantly extends our understanding of the regulatory mechanism of Kr-h1, and the cross-talk of JH and 20E in coordinating mosquito reproduction. JH analogs are commonly used as mosquito larvicides. Recent studies reported that the application of a JH analog, pyriproxyfen (PPF), on adult female mosquitoes substantially reduces their reproduction. A big knowledge gap was the poor understanding of the mechanism underlying this sterilizing effect of PPF. Here, with our customized laboratory setup that mimics the bed net intervention, we established a dose-dependent effect of PPF in compromising mosquito fecundity and fertility. We carefully assessed the effects of PPF exposure on mosquito physiology and follicular development. PPF induced excessive growth of primary follicles during the previtellogenic stage. However, the follicular development in the PPF-treated mosquitoes was severely impaired after blood feeding. The primary follicles were much smaller than their counterparts in the control groups and their development stopped at Christopher's stage III. Moreover, PPF triggered the atypical premature growth of secondary follicles at ~36 h PBM. In addition to the follicular developmental reprogramming, PPF also altered the levels of storage metabolites, enhancing the accumulation of glycogen and triglyceride (TAG) before a blood meal and speeding their depletion after blood-feeding. Consistent with the observed phenotypical changes and relevant metabolic genes, several 20E-responsive genes were significantly altered in their expression as a result of PPF exposure. Furthermore, RNAi experiments demonstrated that the JH receptor Met is required in the PPF-induced sterilization. In summary, we evaluated the sterilizing effects of PPF on mosquito reproduction, investigated the molecular action of PPF in regulating mosquito gene expression, and determined the signaling pathway involved in the PPF-induced sterilization of female mosquitoes. / Doctor of Philosophy / Among different insect-borne diseases, mosquito causes the highest disease burden with almost 700 million infections and over a million deaths every year. Aedes aegypti mosquitoes are the major vehicle to transmit several viral diseases including dengue, yellow fever, chikungunya, and Zika fever. They pose a global threat to public health and economic sectors. Different mosquito control strategies are used, and a very quick, powerful, and popular strategy is using chemical insecticides to decrease mosquito populations. However, insecticide resistance in mosquitoes and non-specific toxicity to other animals are great challenges associated with the commonly used insecticides. To resolve this problem, new insecticides are urgently needed. If we can broaden our understanding of mosquito reproductive biology, new targets will be identified and can be exploited to develop new insecticides. In our study, we investigated an insect-specific hormone, Juvenile hormone (JH), to understand its regulatory action in mosquito reproduction. Also, this study improved our knowledge of the molecular understanding of the insecticide (synthetic JH-like compound) in decreasing mosquito egg numbers and reducing the hatching rate. Overall, we gained a significant understanding of the hormonal regulation of mosquito reproduction. This knowledge can be used in the future to develop new insecticides with better efficiency to decrease the mosquito population and mosquito-borne disease burden.

The quorum sensing signaling system based on intercellular exchange of N-acyl-homoserine lactones is used by many proteobacteria to regulate the transcription of essential genes in a signal density-dependent manner. It is involved in a number of processes including the development of highly organized bacterial communities, e.g., biofilms, the regulation of expression of virulence factors, production of antibiotics, and bioluminescence. The extensive genetic and biochemical data available on the quorum sensing system in Vibrio fischeri allows the development of a systems biology approach to undertake a spatial and dynamical analysis of the regulation throughout the population. The quorum sensing regulated lux genes are organized in two divergent transcriptional units: luxR and luxICDABEG. The latter contains the genes required for luminescence and the luxI gene necessary for synthesis of an N-acyl-homoserine lactone commonly called autoinducer (AI). The luxR gene codes for a transcriptional regulatory protein that activates the transcription of both operons at a threshold concentration of AI. The positive feedback loop induces a rapid increase of transcription level of the lux genes when a critical population density is reached (reflected by the concentration of AI in the environment). With a combination of molecular biology tools, physiological analysis, and mathematical modeling we identified critical characteristics of the system and expect to assign parameter values in order to achieve a comprehensive understanding of the dynamics. An ordinary differential equation mathematical model is used to investigate the dynamics of the system and derive parameter values. In parallel a novel microfluidic cell culture experimental set-up is used to carefully control environmental parameters as well as to achieve chemostatic conditions for high-density cell populations. An unstable variant of the green fluorescent protein was used as a reporter to follow the time response at a single cell level. Thus spatial organization and noise across the population can be analyzed. Plasmids carrying different genetic constructs were transformed in a recombinant Escherichia coli strain to specifically identify genetic and biochemical elements involved in the regulation of the lux genes under diverse conditions. Then the quantitative data extracted from batch culture and microfluidic assays were used to assign parameter values in the models. The particular question being investigated first is the nature of the regulation to increasing concentration of the signal. The hypothesis tested is that the regulation of the production of the signal by individual cells is biphasic and, therefore, quorum sensing should be robust to global and local variations in cell density. / Master of Science

Juvenile hormone (JH) is one of the principal hormones that regulate insect development and reproduction. Accumulating evidence suggests that Methoprene-tolerant (Met) protein is a nuclear receptor of JH. Work by others has shown that Met is capable of binding JH at physiological concentration. An RNAi knockdown of Met causes down-regulated expression of JH-responsive genes and a phenotype similar to that observed in JH-deficient insects, suggesting that Met is required for mediating physiological and molecular responses to JH. The work in this report aims to understand the mechanisms underlying gene regulation by JH via Met. Met is a bHLH-PAS (basic-helix-loop-helix Per-ARNT-Sim) family protein. Many proteins in this family function as heterodimers formed with other proteins of this family. In a yeast two-hybrid screening, we discovered that another bHLH-PAS family protein, FISC, interacts with Met in the presence of JH. FISC is also required for JH functions as an RNAi knockdown of FISC down-regulated JH-responsive genes. To elucidate how Met and FISC mediate JH functions in gene regulation, we employed molecular biology techniques and characterized the formation of a JH-Met-FISC complex and its actions in activating gene expression using mosquito Aedes aegypti as a model. My results demonstrated that Met and FISC forms a complex when JH is present via their conserved N-terminal domains. The complex then binds to E box-like sequences presented in the promoter of JH-responsive genes to activate gene expression. This mechanism also applies to the fruit fly Drosophila melanogaster, suggesting that it is a conserved action of JH in insects. Further studies showed that DNA-binding by Met and FISC requires the basic regions of the bHLH domains of both proteins. Lastly we identified a consensus binding-site of Met and FISC. / Ph. D.

The transcription factor, Pit-1, is involved in the transcriptional regulation of the mammalian prolactin (Prl), growth hormone (GH) and thyroid stimulating hormone b-subunit genes (TSHb) as well as its own gene. The role of Pit-1 in avian species is unknown. Three turkey (t) Pit-1 isoforms have been identified that arise from alternative transcription initiation and alternative splicing. Splicing of exon 1 to an alternative acceptor splice site in exon 2 results in a 28 amino acid insertion in tPit-1β* relative to tPit-1*. Both isoforms initiate transcription at exon 1. A tPit-1 transcript unique to the turkey has been identified and arises following transcription initiation upstream of the alternative acceptor splice site in exon 2. Western blot analysis of pituitary extracts has revealed two isoforms of 37 and 40 kDa. The ability of Pit-1 to transactivate the Prl, GH, and Pit-1 promoters was determined with cotransfection assays. The tPrl, tGH, tPit-1 and rat (r) Prl promoters were cloned upstream of the luciferase gene in a reporter construct. Turkey Pit-1 isoforms and rPit-1 were expressed under the control of the Avian Sarcoma Virus Long Terminal Repeat (ASVLTR) promoter. Cotransfection analyses in mouse L cells indicate that tPit-1* activates the tPrl, tGH, tPit-1 and rPrl promoters 4.6-, 3.8-, 1.7-, and 29.0-fold, respectively. Similar results were observed when cotransfection assays were performed in a turkey pituitary-derived cell line and in primary turkey pituitary cells. These results indicate that tPit-1 is not a strong activator of the tPrl, tGH, or tPit-1 genes, whereas Pit-1 does activate these genes in mammals. A point mutation at amino acid position 176 (ser ⟹ leu) in the POU-homeodomain results in a mutant tPit-1 that shows decreased activity on all promoters tested. Turkey Pit-1* (ser-176) activates the rPrl promoter 14-fold lower than the wild type tPit-1* (leu-176). / Master of Science

Clostridium perfringens is a Gram-positive (Gr+) anaerobic pathogen that was found to contain Type IV pilus (T4P) system genes within the genomes of all its sequenced strains. T4P are widely used in Gram-negative organisms for aggregation, biofilm formation, adherence, and DNA uptake. Because few examples of T4P-utilizing Gram-positive bacteria are studied to date, we wanted to characterize the T4P system in this Gr+ bacterium. To understand the regulation of T4P genes and therefore better understand their expression, we employed the highly powerful next-generation sequencing tool RNA-seq in a variety of conditions. RNA-seq uncovered previously unknown regulatory mechanisms surrounding T4P genes as well as provided transcriptional information for most of the genes in the C. perfringens strain 13 genome. We also utilized reporter gene assays to look at post-transcriptional regulation of T4P promoters. The wealth of RNA-seq data acted as a jumping-off point for many smaller projects involving transcriptional regulators that may influence T4P expression. We investigated a novel small RNA in close proximity to the major T4P operon, as well as two little-characterized transcriptional regulators that function in the same conditions as T4P genes. RNA-seq also provided data to develop a method for protein purification from C. perfringens without induction. / Master of Science

Doctor of Philosophy / Genetics Interdepartmental Program, Plant Pathology / Xiaoyan Tang / Pseudomonas syringae bacteria utilize the type III secretion system (T3SS) to deliver effector proteins into host cells. The T3SS and effector genes (together called the T3 genes hereafter) are repressed in nutrient rich medium but are rapidly induced after the bacteria are transferred into minimal medium (MM) or infiltrated into the plant. The induction of the T3 genes is mediated by HrpL, an alternative sigma factor that recognizes the conserved hrp box motif in the T3 gene promoters. The induction of hrpL is mediated by HrpR and HrpS, two homologous proteins that bind the hrpL promoter. To identify additional genes involved in regulation of the T3 genes, P. s. pv. phaseolicola (Psph) NPS3121 transposon insertion mutants were screened for reduced induction of avrPto-luc and hrpL-luc, reporter genes for promoters of effector gene avrPto and hrpL, respectively. Determination of the transposon-insertion sites led to the identification of genes with putative functions in signal transduction and transcriptional regulation, protein synthesis, and basic metabolism. A transcriptional regulator (AefRNPS3121) identified in the screen is homologous to AefR, a regulator of the quorum sensing signal and epiphytic (plant-associated) traits that was not known previously to regulate the T3 genes in P. s. pv. syringae (Psy) B728a. AefRNPS3121 in Psph NPS3121 and AefR in Psy B728a are similar in regulating the quorum sensing signal in liquid medium but different in regulating epiphytic traits such as swarming motility, entry into leaves, and survival on the leaf surface. The two component system RhpRS was identified in Pseudomonas syringae as a regulator of the T3 genes (Xiao et al. 2007). In the rhpS- mutant, the response regulator RhpR represses the induction of the T3 gene regulatory cascade, but induces its own promoter in a phosphorylation-dependent manner. Deletion and mutagenesis analyses revealed an inverted repeat (IR) element GTATC-N6-GATAC in the rhpR promoter that confers the RhpR-dependent induction. Computational search of the P. syringae genomes for the putative IR elements and Northern blot analysis of the genes with a putative IR element in the promoter region uncovered five genes that were upregulated (PSPTO2036, PSPTO2767, PSPTO3477, PSPTO3574, and PSPTO3660) and two genes that were down-regulated (PSPTO0536 and PSPTO0897) in an RhpR-dependent manner. ChIP assays indicated that RhpR binds the promoters containing a putative IR element but not the hrpR and hrpL promoters that do not have an IR element, suggesting that RhpR indirectly regulates the transcriptional cascade of hrpRS, hrpL, and the T3 genes. To identify additional genes involved in the rhpRS pathway, suppressor mutants were screened that restored the induction of the avrPto-luc reporter gene in the rhpS- mutant. Determination of the transposon-insertion sites led to the identification of rhpR, an ATP-dependent Lon protease, a sigma 70 family protein (PSPPH1909), and other metabolic genes. A lon- rhpS- double mutant exhibited phenotypes typical of a lon- mutant, suggesting that rhpS acts with or through lon. The expression of lon was elevated in rhpS- and other T3-deficient mutants, indicating a negative feedback mechanism. Both the lon- rhpS- and the PSPPH1909- rhpS- double mutant displayed enhanced transcription of hrpL in MM than did the rhpS- mutant.

<p>Next generation sequencing technologies have provided us with a wealth of data profiling a diverse range of biological processes. In an effort to better understand the process of gene regulation, two predictive machine learning models specifically tailored for analyzing gene transcription and polyadenylation are presented.</p><p>Transcriptional enhancers are specific DNA sequences that act as ``information integration hubs" to confer regulatory requirements on a given cell. These non-coding DNA sequences can regulate genes from long distances, or across chromosomes, and their relationships with their target genes are not limited to one-to-one. With thousands of putative enhancers and less than 14,000 protein-coding genes, detecting enhancer-gene pairs becomes a very complex machine learning and data analysis challenge. </p><p>In order to predict these specific-sequences and link them to genes they regulate, we developed McEnhancer. Using DNAseI sensitivity data and annotated in-situ hybridization gene expression clusters, McEnhancer builds interpolated Markov models to learn enriched sequence content of known enhancer-gene pairs and predicts unknown interactions in a semi-supervised learning algorithm. Classification of predicted relationships were 73-98% accurate for gene sets with varying levels of initial known examples. Predicted interactions showed a great overlap when compared to Hi-C identified interactions. Enrichment of known functionally related TF binding motifs, enhancer-associated histone modification marks, along with corresponding developmental time point was highly evident.</p><p>On the other hand, pre-mRNA cleavage and polyadenylation is an essential step for 3'-end maturation and subsequent stability and degradation of mRNAs. This process is highly controlled by cis-regulatory elements surrounding the cleavage site (polyA site), which are frequently constrained by sequence content and position. More than 50\% of human transcripts have multiple functional polyA sites, and the specific use of alternative polyA sites (APA) results in isoforms with variable 3'-UTRs, thus potentially affecting gene regulation. Elucidating the regulatory mechanisms underlying differential polyA preferences in multiple cell types has been hindered by the lack of appropriate tests for determining APAs with significant differences across multiple libraries. </p><p>We specified a linear effects regression model to identify tissue-specific biases indicating regulated APA; the significance of differences between tissue types was assessed by an appropriately designed permutation test. This combination allowed us to identify highly specific subsets of APA events in the individual tissue types. Predictive kernel-based SVM models successfully classified constitutive polyA sites from a biologically relevant background (auROC = 99.6%), as well as tissue-specific regulated sets from each other. The main cis-regulatory elements described for polyadenylation were found to be a strong, and highly informative, hallmark for constitutive sites only. Tissue-specific regulated sites were found to contain other regulatory motifs, with the canonical PAS signal being nearly absent at brain-specific sites. We applied this model on SRp20 data, an RNA binding protein that might be involved in oncogene activation and obtained interesting insights. </p><p>Together, these two models contribute to the understanding of enhancers and the key role they play in regulating tissue-specific expression patterns during development, as well as provide a better understanding of the diversity of post-transcriptional gene regulation in multiple tissue types.</p> / Dissertation

<p>Understanding transcriptional regulation in development and disease is one of the central questions in modern biology. The current working model is that Transcription Factors (TFs) combinatorially bind to specific regions of the genome and drive the expression of groups of genes in a cell-type specific fashion. In organisms with large genomes, particularly mammals, TFs bind to enhancer regions that are often several kilobases away from the genes they regulate, which makes identifying the regulators of gene expression difficult. In order to overcome these obstacles and uncover transcriptional regulatory networks, we used an approach combining expression profiling and genome-wide identification of enhancers followed by motif analysis. Further, we applied these approaches to uncover the TFs important in mammalian sex determination.</p><p>Using expression data from a panel of 19 human cell lines we identified genes showing patterns of cell-type specific up-regulation, down-regulation and constitutive expression. We then utilized matched DNase-seq data to assign DNase Hypersensitivity Sites (DHSs) to each gene based on proximity. These DHSs were scanned for matches to motifs and compiled to generate scores reflecting the presence of TF binding sites (TFBSs) in each gene's putative regulatory regions. We used a sparse logistic regression classifier to classify differentially regulated groups of genes. Comparing our approach to proximal promoter regions, we discovered that using sequence features in regions of open chromatin provided significant performance improvement. Crucially, we discovered both known and novel regulators of gene expression in different cell types. For some of these TFs, we found cell-type specific footprints indicating direct binding to their cognate motifs.</p><p>The mammalian gonad is an excellent system to study cell fate determination processes and the dynamic regulation orchestrated by TFs in development. At embryonic day (E) 10.5, the bipotential gonad initiates either testis development in XY embryos, or ovarian development in XX embryos. Genetic studies over the last 3 decades have revealed about 30 genes important in this process, but there are still significant gaps in our understanding. Specifically, we do not know the network of TFs and their specific combinations that cause the rapid changes in gene expression observed during gonadal fate commitment. Further, more than half the cases of human sex reversal are as yet unexplained. </p><p>To apply the methods we developed to identify regulators of gene expression to the gonad, we took two approaches. First, we carried out a careful dissection of the transcriptional dynamics during gonad differentiation in the critical window between E11.0 and E12.0. We profiled the transcriptome at 6 equally spaced time points and developed a Hidden Markov Model to reveal the cascades of transcription that drive the differentiation of the gonad. Further, we discovered that while the ovary maintains its transcriptional state at this early stage, concurrent up- and down-regulation of hundreds of genes are orchestrated by the testis pathway. Further, we compared two different strains of mice with differential susceptibility to XY male-to-female sex reversal. This analysis revealed that in the C57BL/6J strain, the male pathway is delayed by ~5 hours, likely explaining the increased susceptibility to sex reversal in this strain. Finally, we validated the function of Lmo4, a transcriptional co-factor up-regulated in XY gonads at E11.6 in both strains. RNAi mediated knockdown of Lmo4 in primary gonadal cells led to the down-regulation of male pathway genes including key regulators such as Sox9 and Fgf9. </p><p>To find the enhancers in the XY gonad, we conducted DNase-seq in E13.5 XY supporting cells. In addition, we conducted ChIP-seq for H3K27ac, a mark correlated with active enhancer activity. Further, we conducted motif analysis to reveal novel regulators of sex determination. Our work is an important step towards combining expression and chromatin profiling data to assemble transcriptional networks and is applicable to several systems.</p> / Dissertation

Statistical mechanics has been applied to a wide range of systems in physics, biology, medicine and even anthropology. This theory has been recently used to model the complex biochemical processes of gene expression and regulation. In particular, genetic networks offer a large number of interesting phenomena, such as multistability and oscillatory behaviour, that can be modelled with statistical mechanics tools. In the first part of this thesis we introduce gene regulation, genetic switches, and the colonization of a spatially structured media. We also introduce statistical mechanics and some of its useful tools, such as the master equation and mean- field theories. We present simple examples that are both pedagogical and also set the basis for the study of more complicated scenarios. In the second part we consider the exclusive genetic switch, a fundamental example of genetic networks. In this system, two proteins compete to regulate each other's dynamics. We characterize the switch by solving the stationary state in different limits of the protein binding and unbinding rates. We perform a study of the bistability of the system by examining its probability distribution, and by applying information theory techniques. We then present several versions of a mean field theory that offers further information about the switch. Finally, we compute the stationary probability distribution with an exact perturbative approach in the unbinding parameter, obtaining a valid result for a wide range of parameters values. The techniques used for this calculation are successfully applied to other switches. The topic studied in the third part of the thesis is the propagation of a trait inside an expanding population. This trait may represent resistance to an antibiotic or being infected with a certain virus. Although our model accounts for different examples in the genetic context, it is also very useful for the general study of a trait propagating in a population. We compute the speed of expansion and the stationary population densities for the invasion of an established and an expanding population, finding non-trivial criteria for speed selection and interesting speed transitions. The obtained formulae for the different wave speeds show excellent agreement with the results provided by simulations. Moreover, we are able to obtain the value of the speeds through a detailed analysis of the populations, and establish the requirements for our equations to present speed transitions. We finally apply our model to the propagation in a position-dependent fitness landscape. In this situation, the growth rate or the maximum concentration depends on the position. The amplitudes and speeds of the waves are again successfully predicted in every case.