Isolation and characterization of strong gene regulatory sequences from apple, Malus x domestica

For the strong expression of genes in plant tissue, the availability of specific gene regulatory sequences is desired. We cloned promoter and terminator sequences of an apple (Malus x domestica) ribulose biphosphate carboxylase small subunit gene (MdRbcS), which is known for its high expression and used gus reporter gene expression to test the regulatory activity of the isolated promoter and terminator sequences in transgenic tobacco. The MdRbcS promoter itself seemed to be less strong than the CaMV35S promoter when both used in combination with the nos terminator. However, the combination of the promoter and terminator of MdRbcS was able to drive gus to similar expression levels as the reference construct with CaMV35S promoter and nos terminator. This observation indicates the importance of the terminator sequence for gene expression. It is concluded that the combination of the MdRbcS promoter and terminator is a suitable regulatory sequence set for the expression of transgenes to a high level in plants and for intragenesis in apple specificall

Parallel Genetics of Gene Regulatory Sequences in Caenorhabditis elegans

Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren.How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals

Evolution of gene expression and gene-regulatory sequences in Drosophila melanogaster

In this work, I investigate the role of gene regulatory changes in the evolution of Drosophila melanogaster. As a first step, I performed a survey of gene expression variation in the species using whole-genome microarrays. I surveyed eight strains from an ancestral African population and eight strains from a derived European population using an experimental design that allowed for the detection of expression differences within and between populations. Levels of gene expression variation were nearly equal within the two populations, but a higher amount of variation was detected in comparisons between the two populations. Most gene expression variation within populations appears to be limited by stabilizing selection. However, some genes that are differentially expressed between the two populations might be targets of positive selection. Some of these encode proteins associated with insecticide resistance, food choice, lipid metabolism, and flight. These genes are good candidates for studying adaptive regulatory evolution that accompanied the out-of-Africa migration of D. melanogaster. To verify the accuracy of the microarray experiments, I performed quantitative Real-Time PCR (qPCR), which is another method to measure gene expression, on a subset of genes. I compared the fold-changes in gene expression between pairs of strains determined by the two methods. I also compared the pattern of expression variation in male and female flies. The qPCR approach supported the general accuracy of the microarray experiments, as the fold-changes measured by the two techniques were highly correlated. Expression differences among the strains tended to be similar for male and females. However, exceptions to this general pattern could be found by looking at the pairwise fold-changes for individual genes, some of which differed in expression pattern between males and females. I also investigated the molecular evolution and population genetics of the protein-encoding and upstream regulatory regions of genes that have potentially undergone adaptive evolution at the gene-regulatory level. These genes represent a subset of the genes that showed a significant difference in gene expression between the African and European populations. A set of control genes, which showed no significant difference in expression between the two populations, was also included in the analysis. Overall, I found evidence for both positive and purifying selection in the coding and non-coding regions. However, patterns of polymorphism and divergence did not differ significantly between the candidate genes and the control genes. One of the genes that showed an interesting pattern of expression in the microarray and qPCR experiments was subjected to further, more detailed population genetic analysis. This gene, CG9509, has twofold higher expression in the European strains than in the African strains. The coding and the upstream regions of this gene show evidence of recurrent positive selection since the split of D. melanogaster and its close relative, D. sechellia. A polymorphism survey of the CG9509 region uncovered a 1.2-kb segment, which included the putative CG9509 promoter that showed no polymorphism in the European population. The European population also has several fixed or nearly-fixed derived mutations in this region. These observations, coupled with statistical analysis, provide evidence for a selective sweep in the European population. The selective sweep was likely driven by local adaptation at the level of gene expression

Use of H19 Gene Regulatory Sequences in DNA-Based Therapy for Pancreatic Cancer

Pancreatic cancer is the eighth most common cause of death from cancer in the world, for which palliative treatments are not effective and frequently accompanied by severe side effects. We propose a DNA-based therapy for pancreatic cancer using a nonviral vector, expressing the diphtheria toxin A chain under the control of the H19 gene regulatory sequences. The H19 gene is an oncofetal RNA expressed during embryo development and in several types of cancer. We tested the expression of H19 gene in patients, and found that 65% of human pancreatic tumors analyzed showed moderated to strong expression of the gene. In vitro experiments showed that the vector was effective in reducing Luciferase protein activity on pancreatic carcinoma cell lines. In vivo experiment results revealed tumor growth arrest in different animal models for pancreatic cancer. Differences in tumor size between control and treated groups reached a 75% in the heterotopic model (P = .037) and 50% in the orthotopic model (P = .007). In addition, no visible metastases were found in the treated group of the orthotopic model. These results indicate that the treatment with the vector DTA-H19 might be a viable new therapeutic option for patients with unresectable pancreatic cancer

motifDiverge: a model for assessing the statistical significance of gene regulatory motif divergence between two DNA sequences

Next-generation sequencing technology enables the identification of thousands of gene regulatory sequences in many cell types and organisms. We consider the problem of testing if two such sequences differ in their number of binding site motifs for a given transcription factor (TF) protein. Binding site motifs impart regulatory function by providing TFs the opportunity to bind to genomic elements and thereby affect the expression of nearby genes. Evolutionary changes to such functional DNA are hypothesized to be major contributors to phenotypic diversity within and between species; but despite the importance of TF motifs for gene expression, no method exists to test for motif loss or gain. Assuming that motif counts are Binomially distributed, and allowing for dependencies between motif instances in evolutionarily related sequences, we derive the probability mass function of the difference in motif counts between two nucleotide sequences. We provide a method to numerically estimate this distribution from genomic data and show through simulations that our estimator is accurate. Finally, we introduce the R package {\tt motifDiverge} that implements our methodology and illustrate its application to gene regulatory enhancers identified by a mouse developmental time course experiment. While this study was motivated by analysis of regulatory motifs, our results can be applied to any problem involving two correlated Bernoulli trials

Seamless Gene Tagging by Endonuclease-Driven Homologous Recombination

Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions

SOMIX: Motifs Discovery in Gene Regulatory Sequences Using Self-Organizing Maps

We present a clustering algorithm called Self-organizing Map Neural Network with mixed signals discrimination (SOMIX), to discover binding sites in a set of regulatory regions. Our framework integrates a novel intra-node soft competitive procedure in each node model to achieve maximum discrimination of motif from background signals. The intra-node competition is based on an adaptive weighting technique on two different signal models: position specific scoring matrix and markov chain. Simulations on real and artificial datasets showed that, SOMIX could achieve significant performance improvement in terms of sensitivity and specificity over SOMBRERO, which is a well-known SOM based motif discovery tool. SOMIX has also been found promising comparing against other popular motif discovery tools

Spatial re-organization of myogenic regulatory sequences temporally controls gene expression

During skeletal muscle differentiation, the activation of some tissue-specific genes occurs immediately while others are delayed. The molecular basis controlling temporal gene regulation is poorly understood. We show that the regulatory sequences, but not other regions of genes expressed at late times of myogenesis, are in close physical proximity in differentiating embryonic tissue and in differentiating culture cells, despite these genes being located on different chromosomes. Formation of these inter-chromosomal interactions requires the lineage-determinant MyoD and functional Brg1, the ATPase subunit of SWI/SNF chromatin remodeling enzymes. Ectopic expression of myogenin and a specific Mef2 isoform induced myogenic differentiation without activating endogenous MyoD expression. Under these conditions, the regulatory sequences of late gene loci were not in close proximity, and these genes were prematurely activated. The data indicate that the spatial organization of late genes contributes to temporal regulation of myogenic transcription by restricting late gene expression during the early stages of myogenesis. The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research