Hangnoh Lee

DNA copy number evolution in Drosophila cell lines

BackgroundStructural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles.ResultsOur work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167).ConclusionOur study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

Effects of Gene Dose, Chromatin, and Network Topology on Expression in Drosophila melanogaster.

Deletions, commonly referred to as deficiencies by Drosophila geneticists, are valuable tools for mapping genes and for genetic pathway discovery via dose-dependent suppressor and enhancer screens. More recently, it has become clear that deviations from normal gene dosage are associated with multiple disorders in a range of species including humans. While we are beginning to understand some of the transcriptional effects brought about by gene dosage changes and the chromosome rearrangement breakpoints associated with them, much of this work relies on isolated examples. We have systematically examined deficiencies of the left arm of chromosome 2 and characterize gene-by-gene dosage responses that vary from collapsed expression through modest partial dosage compensation to full or even over compensation. We found negligible long-range effects of creating novel chromosome domains at deletion breakpoints, suggesting that cases of gene regulation due to altered nuclear architecture are rare. These rare cases include trans de-repression when deficiencies delete chromatin characterized as repressive in other studies. Generally, effects of breakpoints on expression are promoter proximal (~100bp) or in the gene body. Effects of deficiencies genome-wide are in genes with regulatory relationships to genes within the deleted segments, highlighting the subtle expression network defects in these sensitized genetic backgrounds.

NetREX: Network Rewiring using EXpression - Towards Context Specific Regulatory Networks

Understanding gene regulation is a fundamental step towards understanding of how cells function and respond to environmental cues and perturbations. An important step in this direction is the ability to infer the transcription factor (TF)-gene regulatory network (GRN). However gene regulatory networks are typically constructed disregarding the fact that regulatory programs are conditioned on tissue type, developmental stage, sex, and other factors. Due to lack of the biological context specificity, these context-agnostic networks may not provide insight for revealing the precise actions of genes for a specific biological system under concern. Collecting multitude of features required for a reliable construction of GRNs such as physical features (TF binding, chromatin accessibility) and functional features (correlation of expression or chromatin patterns) for every context of interest is costly. Therefore we need methods that is able to utilize the knowledge about a context-agnostic network (or a network constructed in a related context) for construction of a context specific regulatory network. To address this challenge we developed a computational approach that utilizes expression data obtained in a specific biological context such as a particular development stage, sex, tissue type and a GRN constructed in a different but related context (alternatively an incomplete or a noisy network for the same context) to construct a context specific GRN. Our method, NetREX, is inspired by network component analysis (NCA) that estimates TF activities and their influences on target genes given predetermined topology of a TF-gene network. To predict a network under a different condition, NetREX removes the restriction that the topology of the TF-gene network is fixed and allows for adding and removing edges to that network. To solve the corresponding optimization problem, which is non-convex and non-smooth, we provide general mathematical framework allowing use of the recently proposed Proximal Alternative Linearized Maximization technique and prove that our formulation has the properties required for convergence. We tested our NetREX on simulated data and subsequently applied it to gene expression data in adult females from 99 hemizygotic lines of the Drosophila deletion (DrosDel) panel. The networks predicted by NetREX showed higher biological consistency than alternative approaches. In addition, we used the list of recently identified targets of the Doublesex (DSX) transcription factor to demonstrate the predictive power of our method.

External RNA Controls Consortium Beta Version Update.

Spike-in RNAs are valuable controls for a variety of gene expression measurements. The External RNA Controls Consortium developed test sets that were used in a number of published reports. Here we provide an authoritative table that summarizes, updates, and corrects errors in the test version that ultimately resulted in the certified Standard Reference Material 2374. We have noted existence of anti-sense RNA controls in the material, corrected sub-pool memberships, and commented on control RNAs that displayed inconsistent behavior.

Drosophila Cell Lines to Model Selection for Aneuploid States

Abnormal numbers of chromosomes, or aneuploid segments of chromosomes, are associated with multiple genetic disorders and cancers. In many chromosomal abnormalities, it is thought that genic balance of protein complexes or pathways are disrupted. In cancers and immortal cell lines, it is thought that aneuploidy confers a growth and senescence advantage. The karyotype and gene expression profiles of 19 Drosophila modENCODE cell lines highlight the evolution of advantageous gene copy numbers while maintaining genic balance. These highly aneuploidcells show coherent changes in copy number among genes encoding components of multiprotein complexes, which may reflect strong selection for genic balance. They also show copy number increases in genes that positively regulate cell cycle progression or decreases in copy number of genes that negatively regulate cell cycle progression, highlighting multiple evolutionary paths to increased growth. Some copy number changes, both increases and decreases, are recurrent. This suggests that there are some critical primary drivers of evolving the ability to grow in vitro. The small, highly rearranged genome, of Drosophila cell lines provides a powerful model system for studying numerical changes in genome, their effect, and dosage compensation against the effect.

Whole genome screen reveals a novel relationship between Wolbachia and Drosophila host translation

Wolbachia is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. To investigate Wolbachia-insect interactions we used the Wolbachia/Drosophila model that provides a genetically tractable system for studying host-pathogen interactions. We coupled genome-wide RNAi screening with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. A diverse set of host gene networks was identified to regulate Wolbachia levels and unexpectedly revealed that perturbations of host translation components such as the ribosome and translation initiation factors results in increased Wolbachia levels both in vitro using RNAi and in vivo using mutants and a chemical-based translation inhibition assay. This work provides evidence for Wolbachia-host translation interaction and strengthens our general understanding of the Wolbachia-host intracellular relationship. Author summary Insects such as mosquitos act as vectors to spread devastating human diseases such as Dengue, West Nile, and Zika. It is critical to develop control strategies to prevent the transmission of these diseases to human populations. A novel strategy takes advantage of an endosymbiotic bacterium Wolbachia pipientis. The presence of this bacterium in insect vectors prevents successful transmission of RNA viruses. The degree to which viruses are blocked by Wolbachia is dependent on the levels of the bacteria present in the host such that higher Wolbachia levels induce a stronger antiviral effect. In order to use Wolbachia as a tool against vector-borne virus transmission a better understanding of host influences on Wolbachia levels is needed. Here we performed a genome-wide RNAi screen in a model host system Drosophila melanogaster infected with Wolbachia to identify host systems that affect Wolbachia levels. We found that host translation can influence Wolbachia levels in the host.

Reprogramming of regulatory network using expression uncovers sex-specific gene regulation in Drosophila

Gene regulatory networks (GRNs) describe regulatory relationships between transcription factors (TFs) and their target genes. Computational methods to infer GRNs typically combine evidence across different conditions to infer context-agnostic networks. We develop a method, Network Reprogramming using EXpression (NetREX), that constructs a context-specific GRN given context-specific expression data and a context-agnostic prior network. NetREX remodels the prior network to obtain the topology that provides the best explanation for expression data. Because NetREX utilizes prior network topology, we also develop PriorBoost, a method that evaluates a prior network in terms of its consistency with the expression data. We validate NetREX and PriorBoost using the “gold standard” E. coli GRN from the DREAM5 network inference challenge and apply them to construct sex-specific Drosophila GRNs. NetREX constructed sex-specific Drosophila GRNs that, on all applied measures, outperform networks obtained from other methods indicating that NetREX is an important milestone toward building more accurate GRNs.For many applications knowledge of context-specific gene regulatory networks (GRNs) is desirable, but their inference remains a challenge. Here, the authors introduce a method for construction of context-specific GRNs, and apply it to construct sex-specific Drosophila GRNs.

Non-canonical Drosophila X chromosome dosage compensation and repressive topologically associated domains

BackgroundIn animals with XY sex chromosomes, X-linked genes from a single X chromosome in males are imbalanced relative to autosomal genes. To minimize the impact of genic imbalance in male Drosophila, there is a dosage compensation complex (MSL) that equilibrates X-linked gene expression with the autosomes. There are other potential contributions to dosage compensation. Hemizygous autosomal genes located in repressive chromatin domains are often derepressed. If this homolog-dependent repression occurs on the X, which has no pairing partner, then derepression could contribute to male dosage compensation.ResultsWe asked whether different chromatin states or topological associations correlate with X chromosome dosage compensation, especially in regions with little MSL occupancy. Our analyses demonstrated that male X chromosome genes that are located in repressive chromatin states are depleted of MSL occupancy; however, they show dosage compensation. The genes in these repressive regions were also less sensitive to knockdown of MSL components.ConclusionsOur results suggest that this non-canonical dosage compensation is due to the same transacting derepression that occurs on autosomes. This mechanism would facilitate immediate compensation during the evolution of sex chromosomes from autosomes. This mechanism is similar to that of C. elegans, where enhanced recruitment of X chromosomes to the nuclear lamina dampens X chromosome expression as part of the dosage compensation response in XX individuals.

Dosage-Dependent Expression Variation Suppressed on the Drosophila Male X Chromosome

DNA copy number variation is associated with many high phenotypic heterogeneity disorders. We systematically examined the impact of Drosophila melanogaster deletions on gene expression profiles to ask whether increased expression variability owing to reduced gene dose might underlie this phenotypic heterogeneity. Indeed, we found that one-dose genes have higher gene expression variability relative to two-dose genes. We then asked whether this increase in variability could be explained by intrinsic noise within cells due to stochastic biochemical events, or whether expression variability is due to extrinsic noise arising from more complex interactions. Our modeling showed that intrinsic gene expression noise averages at the organism level and thus cannot explain increased variation in one-dose gene expression. Interestingly, expression variability was related to the magnitude of expression compensation, suggesting that regulation, induced by gene dose reduction, is noisy. In a remarkable exception to this rule, the single X chromosome of males showed reduced expression variability, even compared with two-dose genes. Analysis of sex-transformed flies indicates that X expression variability is independent of the male differentiation program. Instead, we uncovered a correlation between occupancy of the chromatin-modifying protein encoded by males absent on the first (mof) and expression variability, linking noise suppression to the specialized X chromosome dosage compensation system. MOF occupancy on autosomes in both sexes also lowered transcriptional noise. Our results demonstrate that gene dose reduction can lead to heterogeneous responses, which are often noisy. This has implications for understanding gene network regulatory interactions and phenotypic heterogeneity. Additionally, chromatin modification appears to play a role in dampening transcriptional noise.

Interplay between copy number, dosage compensation and expression noise in Drosophila

Gene copy number variations are associated with many disorders characterized by high phenotypic heterogeneity. Disease penetrance differs even in genetically identical twins. Can such heterogeneity arise, in part, from increased expression variability of one dose genes? While increased variability in the context of single cell gene expression is well recognized, our computational simulations indicated that in a multicellular organism intrinsic single cell level noise should cancel out and thus the impact of gene copy reduction on organismal level expression variability must be due to something else. To systematically examine the impact of gene dose reduction on expression variability in a multi-cellular organism, we performed experimental gene expression measurements in Drosophila DrosDel autosomal deficiency lines. Genome-wide analysis revealed that autosomal one dose genes have higher gene expression variability relative to two dose genes. In flies, gene dose reduction is often accompanied by dosage compensation at the gene expression level. Surprisingly, expression noise was increased by compensation. This increased compensation-dependent variability was found to be a property of one dose autosomal genes but not X-liked genes in males despite the fact that they too are dosage compensated, suggesting that sex chromosome dosage compensation also results in noise reduction. Previous studies attributed autosomal dosage compensation to feedback loops in interaction networks. Our results suggest that these feedback loops are not optimized to deliver consistent responses to gene deletion events and thus gene deletions can lead to heterogeneous responses even in the context of an identical genetic background. Additionally, we show that expression variation associated with reduced dose of transcription factors propagate through the gene interaction network, impacting a large number of downstream genes. These properties of gene deletions could contribute to the phenotypic heterogeneity of diseases associated with haploinsufficiency. Author Summary Gene copy number variations are associated with many human disorders characterized by high phenotypic heterogeneity and understanding the effects of gene copy alterations is essential if we wish to understand factors influencing disease heterogeneity. While heterogeneous responses to reductions in gene dosage can be attributed in part to genetic background differences, we found that responses to dosage reduction vary, even in identical genetic backgrounds. Heterogeneities are not restricted to reduced dosage genes, but propagate through gene networks to affect downstream genes with normal copy number. We also found that reduction in gene dosage is associated with the introduction of expression variation or noise. Expression noise was also observed to propagate across the gene network, further contributing to the heterogeneous response to gene deletion. Through the use of computational simulation, we showed that the majority of the increased noise we observed is most likely due to extrinsic rather than intrinsic sources. Irrespective of the source of the observed noise, we propose that the presence of expression noise and its propagation through gene networks is likely to contribute to the heterogeneity of disease phenotypes observed in humans.