Research Papers
Sterile alpha motif domain-mediated self-association plays an essential role in modulating the activity of the Drosophila ETS family transcriptional repressor Yan
Mol Cell Biol. 2010 Mar;30(5):1158-70. Epub 2010 Jan 4.
Sterile alpha motif domain-mediated self-association plays an essential role in modulating the activity of the Drosophila ETS family transcriptional repressor Yan
Zhang J, Graham TG, Vivekanand P, Cote L, Cetera M, Rebay I.
University of Chicago, Ben May Department for Cancer Research, Chicago, IL 60637, USA.
Abstract
The ETS family transcriptional repressor Yan is an important downstream target and effector of the receptor tyrosine kinase (RTK) signaling pathway in Drosophila melanogaster. Structural and biochemical studies have shown that the N-terminal sterile alpha motif (SAM) of Yan is able to self associate to form a helical polymeric structure in vitro, although the extent and functional significance of self-association of full-length Yan remain unclear. In this study, we demonstrated that full-length Yan self associates via its SAM domain to form higher-order complexes in living cells. Introduction of SAM domain missense mutations that restrict Yan to a monomeric state reduces Yan’s transcriptional repression activity and impairs its function during embryonic and retinal development. Coexpression of combinations of SAM domain mutations that permit the formation of Yan dimers, but not higher-order oligomers, increases activity relative to that of monomeric Yan, but not to the level obtained with wild-type Yan. Mechanistically, self-association directly promotes transcriptional repression of target genes independent of its role in limiting mitogen-activated protein kinase (MAPK)-mediated phosphorylation and nuclear export of Yan. Thus, we propose that the formation of higher-order Yan oligomers contributes to proper repression of target gene expression and RTK signaling output in developing tissues.
Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila
Nat Struct Mol Biol. 2010 Jan;17(1):24-30. Epub 2009 Dec 27.
Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila.
Marques JT, Kim K, Wu PH, Alleyne TM, Jafari N, Carthew RW.
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois, USA.
Abstract
In Drosophila melanogaster, the small interfering RNA (siRNA) pathway is triggered by exogenous double-stranded RNA (dsRNA) or upon viral infection. This pathway requires Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) called R2D2. A potentially distinct siRNA pathway, which requires Dcr-2 in association with a different dsRBP, called Loquacious (Loqs), is activated by endogenous dsRNA derived from transposons, structured loci and overlapping transcripts. Here we show that different sources of dsRNA enter a common siRNA pathway that requires R2D2 and Loqs. R2D2 and loqs mutants show impaired silencing triggered by injection of exogenous dsRNA or by artificial and natural expression of endogenous dsRNA. In addition, we show that these dsRBPs function sequentially and nonredundantly in collaboration with Dcr-2. Loqs is primarily required for dsRNA processing, whereas R2D2 is essential for the subsequent loading of siRNAs into effector Ago-RISC complexes.
Novel opportunities for computational biology and sociology in drug discovery
Trends Biotechnol. 2010 Apr;28(4):161-70.
Novel opportunities for computational biology and sociology in drug discovery.
Yao L, Evans JA, Rzhetsky A.
Department of Biomedical Informatics, Center for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032, USA.
Corrected and republished from:
* Trends Biotechnol. 2009 Sep;27(9):531-40.
Abstract
Current drug discovery is impossible without sophisticated modeling and computation. In this review we outline previous advances in computational biology and, by tracing the steps involved in pharmaceutical development,explore a range of novel, high-value opportunities for computational innovation in modeling the biological process of disease and the social process of drug discovery.These opportunities include text mining for new drug leads, modeling molecular pathways and predicting the efficacy of drug cocktails, analyzing genetic overlap between diseases and predicting alternative drug use.Computation can also be used to model research teams and innovative regions and to estimate the value of academy-industry links for scientific and human benefit. Attention to these opportunities could promise punctuated advance and will complement the well-established computational work on which drug discovery currently relies.
Extracting physically intuitive reaction coordinates from transition networks of a beta-sheet minipr
J Phys Chem B. 2010 May 27;114(20):6979-89.
Extracting physically intuitive reaction coordinates from transition networks of a beta-sheet miniprotein.
Qi B, Muff S, Caflisch A, Dinner AR.
Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.
Abstract
Simulations are important for understanding complex reactions, but their interpretation is challenging owing to the large number of degrees of freedom typically involved. To address this issue, various means for relating the dynamics of a stochastic system to its structural and energetic features have been introduced. Here, we show how two leading approaches can be combined to advantage. We use the network of transitions observed in a reversible folding/unfolding simulation of a 20-residue three-stranded antiparallel beta-sheet peptide (beta3s) to estimate the probabilities of committing to stable states (the native state and major nonnative states), and these then serve as the basis for an efficient statistical procedure for identifying physical variables that describe the dynamics. We find that a single coordinate that jointly characterizes the formation of the two native turns of beta3s can adequately describe the overall folding process, despite its complex nature. Additional features associated with major pathways leading from individual nonnative states are resolved; indeed, a key result is an improved understanding of the unfolded state. Connections to other methods for analyzing complex reactions are discussed.
Mutational bias shaping fly copy number variation: implications for genome evolution
Trends Genet. 2010 Apr 21. [Epub ahead of print]
Mutational bias shaping fly copy number variation: implications for genome evolution.
Cardoso-Moreira MM, Long M.
Department of Ecology and Evolution, University of Chicago, 1101 E 57(th) St. 60637 Chicago, IL, USA; Graduate Program in Areas of Basic and Applied Biology, Universidade do Porto, Porto, Portugal; Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
Abstract
Copy number variants (CNVs) underlie several genomic disorders and are a major source of genetic innovation. Consequently, any bias affecting their placement in the genome will impact our understanding of human disease and genome evolution. Here we report a mutational bias affecting CNVs that generates different probabilities of duplication and deletion across the genome in association with DNA replication time. We show that this mutational bias has important consequences for genome evolution by leading to different probabilities of gene duplication for different classes of genes and by linking the probability of gene duplication with the transcriptional activity of genes. Copyright © 2010 Elsevier Ltd. All rights reserved.
Cleavable C-terminal His-tag vectors for structure determination.
J Struct Funct Genomics. 2010 Mar;11(1):31-9. Epub 2010 Mar 6.
Cleavable C-terminal His-tag vectors for structure determination.
Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B, Tan K, Kim Y, Joachimiak A.
Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Bldg. 202/Rm. BE111, 9700 South Cass Avenue, Argonne, IL 60439, USA.
Abstract
High-throughput structural genomics projects seek to delineate protein structure space by determining the structure of representatives of all major protein families. Generally this is accomplished by processing numerous proteins through standardized protocols, for the most part involving purification of N-terminally His-tagged proteins. Often proteins that fail this approach are abandoned, but in many cases further effort is warranted because of a protein’s intrinsic value. In addition, failure often occurs relatively far into the path to structure determination, and many failed proteins passed the first critical step, expression as a soluble protein. Salvage pathways seek to recoup the investment in this subset of failed proteins through alternative cloning, nested truncations, chemical modification, mutagenesis, screening buffers, ligands and modifying processing steps. To this end we have developed a series of ligation-independent cloning expression vectors that append various cleavable C-terminal tags instead of the conventional N-terminal tags. In an initial set of 16 proteins that failed with an N-terminal appendage, structures were obtained for C-terminally tagged derivatives of five proteins, including an example for which several alternative salvaging steps had failed. The new vectors allow appending C-terminal His(6)-tag and His(6)- and MBP-tags, and are cleavable with TEV or with both TEV and TVMV proteases.
Geometric cues for directing the differentiation of mesenchymal stem cells.
Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):4872-7. Epub 2010 Mar 1.
Geometric cues for directing the differentiation of mesenchymal stem cells.
Kilian KA, Bugarija B, Lahn BT, Mrksich M.
Department of Chemistry, and Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA.
Abstract
Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature-and with each occupying the same area-display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.
Understanding mechanisms underlying human gene expression variation with RNA sequencing.
Nature. 2010 Apr 1;464(7289):768-72. Epub 2010 Mar 10.
Understanding mechanisms underlying human gene expression variation with RNA sequencing.
Pickrell JK, Marioni JC, Pai AA, Degner JF, Engelhardt BE, Nkadori E, Veyrieras JB, Stephens M, Gilad Y, Pritchard JK.
Department of Human Genetics, The University of Chicago, Chicago 60637, USA. pickrell@uchicago.edu
Abstract
Understanding the genetic mechanisms underlying natural variation in gene expression is a central goal of both medical and evolutionary genetics, and studies of expression quantitative trait loci (eQTLs) have become an important tool for achieving this goal. Although all eQTL studies so far have assayed messenger RNA levels using expression microarrays, recent advances in RNA sequencing enable the analysis of transcript variation at unprecedented resolution. We sequenced RNA from 69 lymphoblastoid cell lines derived from unrelated Nigerian individuals that have been extensively genotyped by the International HapMap Project. By pooling data from all individuals, we generated a map of the transcriptional landscape of these cells, identifying extensive use of unannotated untranslated regions and more than 100 new putative protein-coding exons. Using the genotypes from the HapMap project, we identified more than a thousand genes at which genetic variation influences overall expression levels or splicing. We demonstrate that eQTLs near genes generally act by a mechanism involving allele-specific expression, and that variation that influences the inclusion of an exon is enriched within and near the consensus splice sites. Our results illustrate the power of high-throughput sequencing for the joint analysis of variation in transcription, splicing and allele-specific expression across individuals.
Chaperone networks: Tipping the balance in protein folding diseases
Chaperone networks: Tipping the balance in protein folding diseases.
Neurobiol Dis. 2010 May 13;
Authors: Voisine C, Pedersen JS, Morimoto RI
Adult-onset neurodegeneration and other protein conformational diseases are associated with the appearance, persistence, and accumulation of misfolded and aggregation prone proteins. To protect the proteome from long-term damage, the cell expresses a highly integrated protein homeostasis (proteostasis) machinery to ensure that proteins are properly expressed, folded, and cleared, and to recognize damaged proteins. Molecular chaperones have a central role in proteostasis as they have been shown to be essential to prevent the accumulation of alternate folded proteotoxic states as occurs in protein conformation diseases exemplified by neurodegeneration. Studies using invertebrate models expressing proteins associated with Huntington’s disease, Alzheimer’s disease, ALS, and Parkinson’s disease have provided insights into the genetic networks and stress signaling pathways that regulate the proteostasis machinery to prevent cellular dysfunction, tissue pathology, and organismal failure. These events appear to be further amplified by aging and provide evidence that age-related failures in proteostasis may be a common element in many diseases.
A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain
Nat Struct Mol Biol. 2010 Mar 28. [Epub ahead of print]
A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain.
Wojcik J, Hantschel O, Grebien F, Kaupe I, Bennett KL, Barkinge J, Jones RB, Koide A, Superti-Furga G, Koide S.
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
Interactions between Src homology 2 (SH2) domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody mimic (or ‘monobody’), termed HA4, that bound to the Abelson (Abl) kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and MS of intracellular HA4 interactors showed HA4’s specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.
A comprehensive map of insulator elements for the Drosophila genome
PLoS Genet. 2010 Jan 15;6(1):e1000814.
A comprehensive map of insulator elements for the Drosophila genome.
Nègre N, Brown CD, Shah PK, Kheradpour P, Morrison CA, Henikoff JG, Feng X, Ahmad K, Russell S, White RA, Stein L, Henikoff S, Kellis M, White KP.
Institute for Genomics and Systems Biology, Department of Human Genetics, and Department of Ecology and Evolution, University of Chicago, Chicago, Illinois, USA.
Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP-chip the genome-wide binding sites of 6 insulator-associated proteins-dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF-to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all classically defined insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila.
Systems analysis of EGF receptor signaling dynamics with microwestern arrays
Nature Methods
Systems analysis of EGF receptor signaling dynamics with microwestern arrays
Mark F Ciaccio, Joel P Wagner, Chih-Pin Chuu, Douglas A Lauffenburger & Richard B Jones
Published online: 24 January 2010 | doi:10.1038/nmeth.1418
Microwestern arrays combine the advantages of scalability of reverse phase protein arrays and the information content of western blotting for analyzing protein abundance and modification state with high sensitivity and throughput. The method is demonstrated for analyzing phosphorylation state changes in the EGF receptor signaling network using Bayesian network modeling.
Innate immune and chemically triggered oxidative stress modifies translational fidelity.
Nature. 2009 Nov 26;462(7272):522-6.
Innate immune and chemically triggered oxidative stress modifies translational fidelity.
Netzer N, Goodenbour JM, David A, Dittmar KA, Jones RB, Schneider JR, Boone D, Eves EM, Rosner MR, Gibbs JS, Embry A, Dolan B, Das S, Hickman HD, Berglund P, Bennink JR, Yewdell JW, Pan T.
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.
Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer
Science. 2009 Feb 27;323(5918):1218-22. Epub 2009 Jan 22.
Liu J, Ghanim M, Xue L, Brown CD, Iossifov I, Angeletti C, Hua S, Nègre N, Ludwig M, Stricker T, Al-Ahmadie HA, Tretiakova M, Camp RL, Perera-Alberto M, Rimm DL, Xu T, Rzhetsky A, White KP.
Institute for Genomics and Systems Biology, University of Chicago and Argonne National Laboratory, Chicago, IL 60637, USA.
We constructed a large-scale functional network model in Drosophila melanogaster built around two key transcription factors involved in the process of embryonic segmentation. Analysis of the model allowed the identification of a new role for the ubiquitin E3 ligase complex factor SPOP. In Drosophila, the gene encoding SPOP is a target of segmentation transcription factors. Drosophila SPOP mediates degradation of the Jun kinase phosphatase Puckered, thereby inducing tumor necrosis factor (TNF)/Eiger-dependent apoptosis. In humans, we found that SPOP plays a conserved role in TNF-mediated JNK signaling and was highly expressed in 99% of clear cell renal cell carcinomas (RCCs), the most prevalent form of kidney cancer. SPOP expression distinguished histological subtypes of RCC and facilitated identification of clear cell RCC as the primary tumor for metastatic lesions.
Flynet: a genomic resource for Drosophila melanogaster transcriptional regulatory networks
Bioinformatics. 2009 Nov 15;25(22):3001-4. Epub 2009 Aug 5.
Flynet: a genomic resource for Drosophila melanogaster transcriptional regulatory networks.
Tian F, Shah PK, Liu X, Negre N, Chen J, Karpenko O, White KP, Grossman RL.
School of Medicine, Tsinghua University, Beijing, China 100084.
MOTIVATION: The highly coordinated expression of thousands of genes in an organism is regulated by the concerted action of transcription factors, chromatin proteins and epigenetic mechanisms. High-throughput experimental data for genome wide in vivo protein-DNA interactions and epigenetic marks are becoming available from large projects, such as the model organism ENCyclopedia Of DNA Elements (modENCODE) and from individual labs. Dissemination and visualization of these datasets in an explorable form is an important challenge. RESULTS: To support research on Drosophila melanogaster transcription regulation and make the genome wide in vivo protein-DNA interactions data available to the scientific community as a whole, we have developed a system called Flynet. Currently, Flynet contains 101 datasets for 38 transcription factors and chromatin regulator proteins in different experimental conditions. These factors exhibit different types of binding profiles ranging from sharp localized peaks to broad binding regions. The protein-DNA interaction data in Flynet was obtained from the analysis of chromatin immunoprecipitation experiments on one color and two color genomic tiling arrays as well as chromatin immunoprecipitation followed by massively parallel sequencing. A web-based interface, integrated with an AJAX based genome browser, has been built for queries and presenting analysis results. Flynet also makes available the cis-regulatory modules reported in literature, known and de novo identified sequence motifs across the genome, and other resources to study gene regulation. AVAILABILITY: Flynet is available at https://www.cistrack.org/flynet/.
Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases
Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases.
Zhuang M, Calabrese MF, Liu J, Waddell MB, Nourse A, Hammel M, Miller DJ, Walden H, Duda DM, Seyedin SN, Hoggard T, Harper JW, White KP, Schulman BA.
Mol Cell. 2009 Oct 9;36(1):39-50.
Genomic antagonism between retinoic acid and estrogen signaling in breast cancer
Cell. 2009 Jun 26;137(7):1259-71.
Institute for Genomics and Systems Biology and Department of Human Genetics, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Retinoic acid (RA) triggers antiproliferative effects in tumor cells, and therefore RA and its synthetic analogs have great potential as anticarcinogenic agents. Retinoic acid receptors (RARs) mediate RA effects by directly regulating gene expression. To define the genetic network regulated by RARs in breast cancer, we identified RAR genomic targets using chromatin immunoprecipitation and expression analysis. We found that RAR binding throughout the genome is highly coincident with estrogen receptor alpha (ERalpha) binding, resulting in a widespread crosstalk of RA and estrogen signaling to antagonistically regulate breast cancer-associated genes. ERalpha- and RAR-binding sites appear to be coevolved on a large scale throughout the human genome, often resulting in competitive binding activity at nearby or overlapping cis-regulatory elements. The highly coordinated intersection between these two critical nuclear hormone receptor signaling pathways provides a global mechanism for balancing gene expression output via local regulatory interactions dispersed throughout the genome.
Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer
Mol Syst Biol. 2008;4:188. Epub 2008 Apr 15.
Hua S, Kallen CB, Dhar R, Baquero MT, Mason CE, Russell BA, Shah PK, Liu J, Khramtsov A, Tretiakova MS, Krausz TN, Olopade OI, Rimm DL, White KP.
Joint Institute for Genomics and Systems Biology, The University of Chicago and Argonne National Laboratory, Chicago, IL, USA.
We demonstrate an integrated approach to the study of a transcriptional regulatory cascade involved in the progression of breast cancer and we identify a protein associated with disease progression. Using chromatin immunoprecipitation and genome tiling arrays, whole genome mapping of transcription factor-binding sites was combined with gene expression profiling to identify genes involved in the proliferative response to estrogen (E2). Using RNA interference, selected ERalpha and c-MYC gene targets were knocked down to identify mediators of E2-stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the E2-inducible histone variant H2A.Z, is significantly associated with lymph node metastasis and decreased breast cancer survival. Detection of H2A.Z levels independently increased the prognostic power of biomarkers currently in clinical use. This integrated approach has accelerated the identification of a molecule linked to breast cancer progression, has implications for diagnostic and therapeutic interventions, and can be applied to a wide range of cancers.
