Pengyu Hong

Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays

To evaluate the specificity of long dsRNAs used in high-throughput RNA interference (RNAi) screens performed at the Drosophila RNAi Screening Center (DRSC), we performed a global analysis of their activity in 30 genome-wide screens completed at our facility. Notably, our analysis predicts that dsRNAs containing ≥19-nucleotide perfect matches identified in silico to unintended targets may contribute to a significant false positive error rate arising from off-target effects. We confirmed experimentally that such sequences in dsRNAs lead to false positives and to efficient knockdown of a cross-hybridizing transcript, raising a cautionary note about interpreting results based on the use of a single dsRNA per gene. Although a full appreciation of all causes of false positive errors remains to be determined, we suggest simple guidelines to help ensure high-quality information from RNAi high-throughput screens.

Incorporate support vector machines to content-based image retrieval with relevance feedback

By using relevance feedback, content-based image retrieval (CBIR) allows the user to retrieve images interactively. Beginning with a coarse query, the user can select the most relevant images and provide a weight of preference for each relevant image to refine the query. The high level concept borne by the user and perception subjectivity of the user can be automatically captured by the system to some degree. This paper proposes an approach to utilize both positive and negative feedbacks for image retrieval. Support vector machines (SVM) is applied to classifying the positive and negative images. The SVM learning results are used to update the preference weights for the relevant images. This approach releases the user from manually providing preference weight for each positive example. Experimental results show that the proposed approach has improvement over the previous approach (Rui et al. 1997) that uses positive examples only.

Comparative Analysis of Argonaute-Dependent Small RNA Pathways in Drosophila

The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA, and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes.

Real-time speech-driven face animation with expressions using neural networks

A real-time speech-driven synthetic talking face provides an effective multimodal communication interface in distributed collaboration environments. Nonverbal gestures such as facial expressions are important to human communication and should be considered by speech-driven face animation systems. In this paper, we present a framework that systematically addresses facial deformation modeling, automatic facial motion analysis, and real-time speech-driven face animation with expression using neural networks. Based on this framework, we learn a quantitative visual representation of the facial deformations, called the motion units (MUs). A facial deformation can be approximated by a linear combination of the MUs weighted by MU parameters (MUPs). We develop an MU-based facial motion tracking algorithm which is used to collect an audio-visual training database. Then, we construct a real-time audio-to-MUP mapping by training a set of neural networks using the collected audio-visual training database. The quantitative evaluation of the mapping shows the effectiveness of the proposed approach. Using the proposed method, we develop the functionality of real-time speech-driven face animation with expressions for the iFACE system. Experimental results show that the synthetic expressive talking face of the iFACE system is comparable with a real face in terms of the effectiveness of their influences on bimodal human emotion perception.

The Hippo Signaling Pathway Interactome

Dissecting Hippo Interactions The Hippo signaling pathway plays key roles in many processes, from cell proliferation and cell death to regulation of stem cells and cancer cells. Kwon et al. (p. 737, published 10 October) attempted to systematically identify all components of the pathway. A protein-protein interaction screen identified more than 200 interactions among approximately 150 proteins. A protein identified in the screen, Leash, restrained the activity of the transcriptional coactivator Yorkie, which regulates gene expression in response to Hippo signaling. A proteomics approach for protein-protein interactions reveals new components of a conserved cell signaling pathway. The Hippo pathway controls metazoan organ growth by regulating cell proliferation and apoptosis. Many components have been identified, but our knowledge of the composition and structure of this pathway is still incomplete. Using existing pathway components as baits, we generated by mass spectrometry a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67% of the proteins by RNA interference regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively. We selected for further characterization a new member of the alpha-arrestin family, Leash, and show that it promotes degradation of Yki through the lysosomal pathway. Given the importance of the Hippo pathway in tumor development, the Hippo-PPIN will contribute to our understanding of this network in both normal growth and cancer.

Identification of neural outgrowth genes using genome-wide RNAi.

While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system.

Imaging analysis of clock neurons reveals light buffers the wake-promoting effect of dopamine

How animals maintain proper amounts of sleep yet remain flexible to changes in environmental conditions remains unknown. We found that environmental light suppressed the wake-promoting effects of dopamine in fly brains. The ten large lateral-ventral neurons (l-LNvs), a subset of clock neurons, are wake-promoting and respond to dopamine, octopamine and light. Behavioral and imaging analyses suggested that dopamine is a stronger arousal signal than octopamine. Notably, light exposure not only suppressed l-LNv responses, but also synchronized responses of neighboring l-LNvs. This regulation occurred by distinct mechanisms: light-mediated suppression of octopamine responses was regulated by the circadian clock, whereas light regulation of dopamine responses occurred by upregulation of inhibitory dopamine receptors. Plasticity therefore alters the relative importance of diverse cues on the basis of the environmental mix of stimuli. The regulatory mechanisms described here may contribute to the control of sleep stability while still allowing behavioral flexibility.

Sensitive and Specific Identification of Wild Type and Variant Proteins from 8 to 669 kDa Using Top-down Mass Spectrometry

Top-down and bottom-up mass spectrometry methods can generate gas phase fragments and use these to identify proteins. Top-down methods, in addition, can provide the mass of the protein itself and therefore additional structural information. Despite the conceptual advantage of top-down methods, the market share advantage belongs to bottom-up methods as a result of their more robust sample preparation, fragmentation, and data processing methods. Here we report improved fragmentation and data processing methods for top-down mass spectrometry. Specifically we report the use of funnel-skimmer dissociation, a variation of nozzle-skimmer dissociation, and compare its performance with electron capture dissociation. We also debut BIG Mascot, an extended version of Mascot with incorporated top-down MS2 search ability and the first search engine that can perform both bottom-up and top-down searches. Using BIG Mascot, we demonstrated the ability to identify proteins 1) using only intact protein MS1, 2) using only MS2, and 3) using the combination of MS1 and MS2. We correctly identified proteins with a wide range of masses, including 13 amyotrophic lateral sclerosis-associated variants of the protein Cu/Zn-superoxide dismutase, and extended the upper mass limit of top-down protein identification to 669 kDa by identifying thyroglobulin.

Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling.

Interactome mapping and functional genomics in Drosophila reveal common and specific components of a mitogen-activated protein kinase pathway. Finding the Shared and the Specific Components Regulating MAPK Signals Even in extensively studied pathways, such as the extracellular signal–regulated kinase (ERK) pathway that is activated by receptor tyrosine kinases, there remain gaps in our knowledge. Friedman et al. combined protein-protein interaction screens with RNAi functional genomic screens in Drosophila cell lines to identify components of the ERK pathway downstream of two receptor tyrosine kinases. Their analysis suggested that these receptors may compete for some common components, in addition to using receptor-specific and cell-specific signal transduction pathways. Knockdown of several newly identified pathway regulators resulted in wing phenotypes in vivo, confirming these as components in the pathway. Detailed understanding of this pathway has clinical relevance because of its importance in both physiological and pathophysiological contexts, such as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)–Ras–extracellular signal–regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification–mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type– and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine–protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

A boosting approach for motif modeling using ChIP-chip data

MOTIVATION Building an accurate binding model for a transcription factor (TF) is essential to differentiate its true binding targets from those spurious ones. This is an important step toward understanding gene regulation. RESULTS This paper describes a boosting approach to modeling TF-DNA binding. Different from the widely used weight matrix model, which predicts TF-DNA binding based on a linear combination of position-specific contributions, our approach builds a TF binding classifier by combining a set of weight matrix based classifiers, thus yielding a non-linear binding decision rule. The proposed approach was applied to the ChIP-chip data of Saccharomyces cerevisiae. When compared with the weight matrix method, our new approach showed significant improvements on the specificity in a majority of cases.