Research Papers
Light-Driven Changes in Energy Metabolism Directly Entrain the Cyanobacterial Circadian Oscillator
Circadian clocks are self-sustained biological oscillators that can be entrained by environmental cues. Although this phenomenon has been studied in many organisms, the molecular mechanisms of entrainment remain unclear. Three cyanobacterial proteins and adenosine triphosphate (ATP) are sufficient to generate oscillations in phosphorylation in vitro. We show that changes in illumination that induce a phase shift in cultured cyanobacteria also cause changes in the ratio of ATP to adenosine diphosphate (ADP). When these nucleotide changes are simulated in the in vitro oscillator, they cause phase shifts similar to those observed in vivo. Physiological concentrations of ADP inhibit kinase activity in the oscillator, and a mathematical model constrained by data shows that this effect is sufficient to quantitatively explain entrainment of the cyanobacterial circadian clock.
Rust MJ, Golden SS, O’Shea EK. (Jan 2011) Light-driven changes in energy metabolism directly entrain the cyanobacterial circadian oscillator. Science. 14;331(6014):220-3. PubMed
A Vision for a Biomedical Cloud (J Intern Med. 2011 Dec 5. doi: 10.1111/j.1365-2796.2011.02491)
and Kevin White
A Vision for a Biomedical Cloud
By: Grossman RL, White KP.
Institute for Genomics and Systems Biology Departments of Medicine Human Genetics KCBD 10100 The University of Chicago Chicago, IL 60637
Abstract:
We present a vision for a Biomedical Cloud that draws on progress in the fields of Genomics, Systems Biology, and Biomedical Data Mining. The successful fusion of these areas will merge biomarkers, genetic variants, and environmental variables to build predictive models that will drastically increase the specificity and timeliness of diagnosis for a wide range of common diseases, while delivering accurate predictions about the efficacy of treatment options. However, the amount of data being generated by each of these areas is staggering, as is the task of managing and analyzing it. Adequate computing infrastructure needs to be developed in order to assemble, manage, and mine the enormous and rapidly growing corpus of ‘omics’ data along with clinical information. We have now arrived at an intersection point between genome technology, cloud computing and biological data mining. This intersection point provides a launch pad for developing a globally applicable cloud computing platform capable of supporting a new paradigm of data intensive, cloud-enabled Systems Medicine.
Copyright © 2011 The Association for the Publication of the Journal of Internal Medicine
Consequences of eukaryotic enhancer architecture for gene expression dynamics, development, and fitness (Department of Ecology and Evolution, University of Chicago, Chicago, Illinois, United States of America)
Ludwig MZ, Manu, Kittler R, White KP, Kreitman M.
Source: Department of Ecology and Evolution, University of Chicago, Chicago, Illinois, United States of America.
Abstract
The regulatory logic of time- and tissue-specific gene expression has mostly been dissected in the context of the smallest DNA fragments that, when isolated, recapitulate native expression in reporter assays. It is not known if the genomic sequences surrounding such fragments, often evolutionarily conserved, have any biological function or not. Using an enhancer of the even-skipped gene of Drosophila as a model, we investigate the functional significance of the genomic sequences surrounding empirically identified enhancers. A 480 bp long “minimal stripe element” is able to drive even-skipped expression in the second of seven stripes but is embedded in a larger region of 800 bp containing evolutionarily conserved binding sites for required transcription factors. To assess the overall fitness contribution made by these binding sites in the native genomic context, we employed a gene-replacement strategy in which whole-locus transgenes, capable of rescuing even-skipped(-) lethality to adulthood, were substituted for the native gene. The molecular phenotypes were characterized by tagging Even-skipped with a fluorescent protein and monitoring gene expression dynamics in living embryos. We used recombineering to excise the sequences surrounding the minimal enhancer and site-specific transgenesis to create co-isogenic strains differing only in their stripe 2 sequences. Remarkably, the flanking sequences were dispensable for viability, proving the sufficiency of the minimal element for biological function under normal conditions. These sequences are required for robustness to genetic and environmental perturbation instead. The mutant enhancers had measurable sex- and dose-dependent effects on viability. At the molecular level, the mutants showed a destabilization of stripe placement and improper activation of downstream genes. Finally, we demonstrate through live measurements that the peripheral sequences are required for temperature compensation. These results imply that seemingly redundant regulatory sequences beyond the minimal enhancer are necessary for robust gene expression and that “robustness” itself must be an evolved characteristic of the wild-type enhancer.
Genome-wide meta-analysis identifies variants associated with platinating agent susceptibility across populations.
Wheeler HE, Gamazon ER, Stark AL, O’Donnell PH, Gorsic LK, Huang RS, Cox NJ, Dolan ME.
Source: Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, IL, USA
Abstract:
Platinating agents are used in the treatment of many cancers, yet they can induce toxicities and resistance that limit their utility. Using previously published and additional world population panels of diverse ancestry totaling 608 lymphoblastoid cell lines (LCLs), we performed meta-analyses of over 3 million single-nucleotide polymorphisms (SNPs) for both carboplatin- and cisplatin-induced cytotoxicity. The most significant SNP in the carboplatin meta-analysis is located in an intron of NBAS (neuroblastoma amplified sequence; P=5.1 × 10(-7)). The most significant SNP in the cisplatin meta-analysis is upstream of KRT16P2 (P=5.8 × 10(-7)). We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Most of the variants that associate with platinum-induced cytotoxicity are polymorphic across multiple world populations; therefore, they could be tested in follow-up studies in diverse clinical populations. Seven genes previously implicated in platinating agent response, including BCL2 (B-cell CLL/lymphoma 2), GSTM1 (glutathione S-transferase mu 1), GSTT1, ERCC2 and ERCC6, were also implicated in our meta-analyses.
The Pharmacogenomics Journal advance online publication, 16 August 2011; doi:10.1038/tpj.2011.38.
Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data (Proc Natl Acad Sci U S A. 2011 Jul 5.)
Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data.
Tao Y, Ruan J, Yeh SH, Lu X, Wang Y, Zhai W, Cai J, Ling S, Gong Q, Chong Z, Qu Z, Li Q, Liu J, Yang J, Zheng C, Zeng C, Wang HY, Zhang J, Wang SH, Hao L, Dong L, Li W, Sun M, Zou W, Yu C, Li C, Liu G, Jiang L, Xu J, Huang H, Li C, Mi S, Zhang B, Chen B, Zhao W, Hu S, Zhuang SM, Shen Y, Shi S, Brown C, White KP, Chen DS, Chen PJ, Wu CI.
SourceLaboratory of Disease Genomics and Individualized Medicine, and China Academy of Sciences Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, People’s Republic of China.
Abstract
We present the analysis of the evolution of tumors in a case of hepatocellular carcinoma. This case is particularly informative about cancer growth dynamics and the underlying driving mutations. We sampled nine different sections from three tumors and seven more sections from the adjacent nontumor tissues. Selected sections were subjected to exon as well as whole-genome sequencing. Putative somatic mutations were then individually validated across all 9 tumor and 7 nontumor sections. Among the mutations validated, 24 were amino acid changes; in addition, 22 large indels/copy number variants (>1 Mb) were detected. These somatic mutations define four evolutionary lineages among tumor cells. Separate evolution and expansion of these lineages were recent and rapid, each apparently having only one lineage-specific protein-coding mutation. Hence, by using a cell-population genetic definition, this approach identified three coding changes (CCNG1, P62, and an indel/fusion gene) as tumor driver mutations. These three mutations, affecting cell cycle control and apoptosis, are functionally distinct from mutations that accumulated earlier, many of which are involved in inflammation/immunity or cell anchoring. These distinct functions of mutations at different stages may reflect the genetic interactions underlying tumor growth.
A user's guide to the encyclopedia of DNA elements (ENCODE). (PLoS Biol. 2011 Apr;9(4):e1001046. Epub 2011 Apr 19)
A user’s guide to the encyclopedia of DNA elements (ENCODE).
ENCODE Project Consortium, Myers RM, Stamatoyannopoulos J, Snyder M, Dunham I, Hardison RC, Bernstein BE, Gingeras TR, Kent WJ, Birney E, Wold B, Crawford GE.
Collaborators (329)Bernstein BE, Epstein CB, Shoresh N, Ernst J, Mikkelsen TS, Kheradpour P, Zhang X, Wang L, Issner R, Coyne MJ, Durham T, Ku M, Truong T, Ward LD, Altshuler RC, Lin MF, Kellis M, Gingeras TR, Davis CA, Kapranov P, Dobin A, Zaleski C, Schlesinger F, Chakrabortty S, Jha S, Lin W, Drenkow J, Wang H, Bell K, Gao H, Bell I, Dumais E, Dumais J, Antonarakis SE, Borel C, Guigo R, Djebali S, Ribeca P, Sammeth M, Alioto T, Merkel A, Tilgner H, Carninci P, Hayashizaki Y, Lassmann T, Takahashi H, Abdelhamid RF, Hannon G, Fejes-Toth K, Preall J, Gordon A, Sotirova V, Reymond A, Howald C, Graison EA, Chrast J, Ruan Y, Ruan X, Shahab A, Ting Poh W, Wei CL, Crawford GE, Furey TS, Boyle AP, Sheffield NC, Song L, Shibata Y, Vales T, Winter D, Zhang Z, London D, Wang T, Birney E, Iyer VR, Lee BK, McDaniell RM, Liu Z, Battenhouse A, Bhinge AA, Lieb JD, Grasfeder LL, Showers KA, Giresi PG, Kim SK, Shestak C, Myers RM, Pauli F, Reddy TE, Gertz J, Christopher E, Jain P, Sprouse RO, Bansal A, Pusey B, Muratet MA, Varley KE, Bowling KM, Newberry KM, Nesmith AS, Dilocker JA, Parker SL, Waite LL, Thibeault K, Roberts K, Absher DM, Mortazavi A, Williams B, Marinov G, Pepke S, King B, McCue K, Kirilusha A, DeSalvo G, Fisher-Aylor K, Amrhein H, Vielmetter J, Sherlock G, Sidow A, Batzoglou S, Rauch R, Kundaje A, Libbrecht M, Margulies EH, Parker SC, Elnitski L, Green ED, Kokocinski F, Frankish A, Hunt T, Despacio G, Kay M, Mukherjee G, Bignell A, Saunders G, Boychenko V, Van Baren MJ, Brown RH, Khurana E, Balasubramanian S, Zhang Z, Lam H, Cayting P, Robilotto R, Lu Z, Guigo R, Tanzer A, Knowles DG, Mariotti M, James Kent W, Haussler D, Harte R, Diekhans M, Kellis M, Lin M, Kheradpour P, Ernst J, Reymond A, Howald C, Graison EA, Chrast J, Tress M, Manuel J, Snyder M, Landt SG, Raha D, Shi M, Euskirchen G, Grubert F, Kasowski M, Lian J, Cayting P, Lacroute P, Monahan H, Patacsil D, Slifer T, Yang X, Charos A, Reed B, Wu L, Auerbach RK, Habegger L, Hariharan M, Rozowsky J, Abyzov A, Weissman SM, Struhl K, Lamarre N, Lindahl M, Miotto B, Moqtaderi Z, Fleming JD, Newburger P, Farnham PJ, Frietze S, O’Geen H, Xu X, Blahnik KR, Cao AR, Iyengar S, Stamatoyannopoulos JA, Kaul R, Thurman RE, Wang H, Navas PA, Sandstrom R, Sabo PJ, Weaver M, Canfield T, Lee K, Neph S, Reynolds A, Johnson A, Rynes E, Giste E, Neri J, Frum T, Johnson EM, Nguyen ED, Ebersol AK, Sanchez ME, Sheffer HH, Lotakis D, Haugen E, Humbert R, Kutyavin T, Shafer T, Dekker J, Lajoie BR, Sanyal A, James Kent W, Rosenbloom KR, Dreszer TR, Raney BJ, Barber GP, Meyer LR, Sloan CA, Malladi VS, Cline MS, Learned K, Swing VK, Zweig AS, Rhead B, Fujita PA, Roskin K, Karolchik D, Kuhn RM, Haussler D, Birney E, Dunham I, Wilder SP, Keefe D, Sobral D, Herrero J, Beal K, Lukk M, Brazma A, Vaquerizas JM, Luscombe NM, Bickel PJ, Boley N, Brown JB, Li Q, Huang H, Habegger L, Sboner A, Rozowsky J, Auerbach RK, Yip KY, Cheng C, Yan KK, Bhardwaj N, Wang J, Lochovsky L, Jee J, Gibson T, Leng J, Du J, Hardison RC, Harris RS, Miller W, Haussler D, Roskin K, Suh B, Wang T, Paten B, Noble WS, Hoffman MM, Buske OJ, Weng Z, Dong X, Wang J, Xi H, Tenenbaum SA, Doyle F, Penalva LO, Chittur S, Tullius TD, Parker SC, White KP, Karmakar S, Victorsen A, Jameel N, Grossman RL, Snyder M, Landt SG, Yang X, Patacsil D, Slifer T, Dekker J, Lajoie BR, Sanyal A, Weng Z, Whitfield TW, Wang J, Collins PJ, Trinklein ND, Partridge EC, Myers RM, Giddings MC, Chen X, Khatun J, Maier C, Yu Y, Gunawardena H, Risk B, Feingold EA, Lowdon RF, Dillon LA, Good PJ.
SourceHudsonAlpha Institute for Biotechnology, Huntsville, Alabama, United States of America. rmyers@hudsonalpha.org
Abstract
The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome.
Determinants of Antigenicity and Specificity in immune response for Protein Sequences (BMC Bioinformatics. 2011 Jun 21;12(1):251)
Determinants of Antigenicity and Specificity in immune response for Protein Sequences.
Wang Y, Wu W, Negre NN, White KP, Li C, Shah PK.
BACKGROUND: Target specific antibodies are pivotal for the design of vaccines, immunodiagnostic tests, studies on proteomics for cancer biomarker discovery, identification of protein-DNA and other interactions, and small and large biochemical assays. Therefore, it is important to understand the properties of protein sequences that are important for antigenicity and to identify small peptide epitopes and large regions in the linear sequence of the proteins whose utilization result in specific antibodies.
RESULTS: Our analysis using protein properties suggested that sequence composition combined with evolutionary information and predicted secondary structure, as well as solvent accessibility is sufficient to predict successful peptide epitopes. The antigenicity and the specificity in immune response were also found to depend on the epitope length. We trained the B-Cell Epitope Oracle (BEOracle), a support vector machine (SVM) classifier, for the identification of continuous B-Cell epitopes with these protein properties as learning features. The BEOracle achieved an F1-measure of 81.37% on a large validation set. The BEOracle classifier outperformed the classical methods based on propensity and sophisticated methods like BCPred and Bepipred for B-Cell epitope prediction. The BEOracle classifier also identified peptides for the ChIP-grade antibodies from the modENCODE/ENCODE projects with 96.88% accuracy. High BEOracle score for peptides showed some correlation with the antibody intensity on Immunofluorescence studies done on fly embryos. Finally, a second SVM classifier, the B-Cell Region Oracle (BROracle) was trained with the BEOracle scores as features to predict the performance of antibodies generated with large protein regions with high accuracy. The BROracle classifier achieved accuracies of 75.26-63.88% on a validation set with immunofluorescence, immunohistochemistry, protein arrays and western blot results from Protein Atlas database.
CONCLUSIONS: Together our results suggest that antigenicity is a local property of the protein sequences and that protein sequence properties of composition, secondary structure, solvent accessibility and evolutionary conservation are the determinants of antigenicity and specificity in immune response. Moreover, specificity in immune response could also be accurately predicted for large protein regions without the knowledge of the protein tertiary structure or the presence of discontinuous epitopes. The dataset prepared in this work and the classifier models are available for download at https://sites.google.com/site/oracleclassifiers/.
ChIP-chip versus ChIP-seq: lessons for experimental design and data analysis (BMC Genomics. 2011 Feb 28;12:134)
ChIP-chip versus ChIP-seq: lessons for experimental design and data analysis.
Ho JW, Bishop E, Karchenko PV, Nègre N, White KP, Park PJ.
SourceDepartment of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA, USA.
BACKGROUND: Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq) allows genome-wide discovery of protein-DNA interactions such as transcription factor bindings and histone modifications. Previous reports only compared a small number of profiles, and little has been done to compare histone modification profiles generated by the two technologies or to assess the impact of input DNA libraries in ChIP-seq analysis. Here, we performed a systematic analysis of a modENCODE dataset consisting of 31 pairs of ChIP-chip/ChIP-seq profiles of the coactivator CBP, RNA polymerase II (RNA PolII), and six histone modifications across four developmental stages of Drosophila melanogaster.
RESULTS: Both technologies produce highly reproducible profiles within each platform, ChIP-seq generally produces profiles with a better signal-to-noise ratio, and allows detection of more peaks and narrower peaks. The set of peaks identified by the two technologies can be significantly different, but the extent to which they differ varies depending on the factor and the analysis algorithm. Importantly, we found that there is a significant variation among multiple sequencing profiles of input DNA libraries and that this variation most likely arises from both differences in experimental condition and sequencing depth. We further show that using an inappropriate input DNA profile can impact the average signal profiles around genomic features and peak calling results, highlighting the importance of having high quality input DNA data for normalization in ChIP-seq analysis.
CONCLUSIONS: Our findings highlight the biases present in each of the platforms, show the variability that can arise from both technology and analysis methods, and emphasize the importance of obtaining high quality and deeply sequenced input DNA libraries for ChIP-seq analysis.
Large-scale genetic epistasis networks using RNAi (Nat Methods. 2011 Apr;8(4):341-6)
Wang X, White KP.
Pairwise quantitative genetic interactions are mapped by combinatorial RNA interference in metazoan cells
An epistatic interaction between two genes is observed if the combined effects of mutations in both genes deviate from the sum of each mutation’s individual effects. We need to understand epistatic genetic interactions if we want to solve the puzzle of genotype-phenotype relationships in complex human diseases. Advances in genetic tools and automated technology have enabled investigators to construct genome-scale genetic interaction maps in yeast and bacteria1. However, it has been a challenge to develop comparable methodologies in metazoan cells. In this issue of Nature Methods, groups led by Michael Boutros and Wolfgang Huber report their pilot experiments in systematically mapping pairwise genetic interactions in cultured Drosophila melanogaster cells by RNA interference (RNAi)2.
Genetic interactions underlie the robustness of biological systems and the complexity of common genetic disorders. So far, genetic interaction networks are mostly constructed from experimental data in yeast, taking advantage of the large collection of deletion strains, and the automated technology to sort and score double mutants3, 4, 5. In higher organisms and tissue culture cells, such large deletion collections do not exist, and the process to generate double mutants is much more labor-intensive and time-consuming. RNAi is the most practical way to simultaneously perturb multiple genes in these systems. Several groups have used RNAi to map genetic interactions based on semiquantitative readouts such as lethality and sterility1, 6, 7. But concerns about inherent noise in RNAi assays, caused by off-target effects and variable knockdown efficiencies, delayed the use of RNAi in large-scale mapping of genetic interactions based on more quantitative readouts.
Huber, Boutros and colleagues set out to overcome the limitations of using RNAi for quantitative genetic interaction mapping by combining a rigorous experimental design with robust statistical modeling2. To assay pairwise synthetic interactions between 93 genes, they designed two independent ds-RNAs to each gene and did reciprocal RNAi knockdowns with all four combinations in Drosophila Schneider S2 cells (Fig. 1). Therefore, they assayed each pair of genes in eight biological replicates. And within each biological replicate, they repeated the phenotypic measurements eight times. The measurements in both biological and technical replicates were highly reproducible, allowing calculation of reliable interaction scores for gene pairs from a multipli-cative model.
Figure 1: Workflow of a combinatorial RNAi screen for genetic interactions developed by Horn et al.2.
shRNA, short hairpin RNA.
Full size image (92 KB)
An intriguing aspect of this study is its multiparametric design. Instead of focusing on a specific pathway or a defined biological process, they assayed three phenotypes that are easily measured: cell number, nuclear area and fluorescence intensity of the nuclear area after Hoechst (DNA) staining. This is the first effort to quantify multiple complex phenotypic features at a large scale for genetic epistasis in metazoan cells. These readouts provided rich and nonredundant information on genetic interactions. Only 20% of all the 637 interactions discovered were common among phenotypes, and about 50% of the interactions were specific to a single phenotypic readout.
Although individual interactions are context dependent, the global interaction profiles can provide important aggregate information that reveals biologically relevant interactions. Horn et al.2 constructed the interaction profiles for each gene as the vector of its interaction scores with all other genes. Without any prior assumptions, genes that have similar functions were clustered robustly by their global interaction profiles, independent of the phenotypes from which these profiles were generated. This property was consistent with the finding in yeast studies that genes in the same biological pathway tend to have similar profiles of interactions4, 5.
Horn et al.2 use these global interaction profiles to predict functions of previously uncharacterized genes. They first trained a classifier on the combined multiparametric interaction profiles of known genes in two signaling pathways, Ras-MAPK and JNK pathways. The classifier correctly classified both positive and negative regulators of the Ras-MAPK pathway and also led to the discovery of a new positive regulator of the Ras-MAPK pathway, whose function is conserved between fruit flies and humans. Although the number of genes in each pathway studied is relatively small, the predictive power of the classifier is very strong, which bodes well for the application of the method to larger datasets as they become available.
This elegant study illustrated how combinatorial RNAi can be used to systematically assess pairwise genetic interactions of metazoan genes. Although the current study accessed signaling molecules at a medium scale, this method could be readily applied to different gene sets at variable scales by other investigators with the appropriate resources, such as small interfering RNA libraries, liquid-handling robotics and automated imaging systems. These epistasis mapping approaches also pave the way for studies in mammalian cells, which will require improvements in RNAi knockdown efficiencies and avoidance of off-target effects. But for now it will be interesting to see this approach expanded in Drosophila cells. For example, larger-scale mapping may reveal the extent to which interactions among the cellular networks derived from yeast studies are conserved in higher organisms. Additionally, chemical-gene interaction networks can be mapped, as has been demonstrated in yeast5, to allow discovery of targets for small-molecule drugs. A future challenge will be to develop complementary computational methods to extract more information from the interaction profiles, to integrate data from different experiments, and to guide experimental design using existing data. As all-by-all genetic interaction matrix mapping is still a daunting prospect, it will be very useful to select gene sets that are likely to provide the richest information based on prior information8. Horn et al.2 have cleared the way for large-scale studies of genetic interactions in metazoan cells.
Beta-Blocker Use Is Associated With Improved Relapse-Free Survival in Patients With Triple-Negative Breast Cancer (J Clin Oncol. 2011 May 31)
(J Clin Oncol. 2011 May 31.)
Melhem-Bertrandt A, Chavez-Macgregor M, Lei X, Brown EN, Lee RT, Meric-Bernstam F, Sood AK, Conzen SD, Hortobagyi GN, Gonzalez-Angulo AM.
SourceAmal Melhem-Bertrandt, Mariana Chavez-MacGregor, Xiudong Lei, Erika N. Brown, Richard T. Lee, Funda Meric-Bernstam, Anil K. Sood, Gabriel N. Hortobagyi, Ana-Maria Gonzalez-Angulo, The University of Texas MD Anderson Cancer Center, Houston, TX; and Suzanne D. Conzen, the University of Chicago, Chicago, IL.
PURPOSE To examine the association between beta-blocker (BB) intake, pathologic complete response (pCR) rates, and survival outcomes in patients with breast cancer treated with neoadjuvant chemotherapy. PATIENTS AND METHODS We retrospectively reviewed 1,413 patients with breast cancer who received neoadjuvant chemotherapy between 1995 and 2007. Patients taking BBs at the start of neoadjuvant therapy were compared with patients with no BB intake. Rates of pCR between the groups were compared using a χ(2) test. Cox proportional hazards models were fitted to determine the association between BB intake, relapse-free survival (RFS), and overall survival (OS). Results Patients who used BBs (n = 102) were compared with patients (n = 1,311) who did not. Patients receiving BBs tended to be older and obese (P < .001). The proportion of pCR was not significantly different between the groups (P = .48). After adjustment for age, race, stage, grade, receptor status, lymphovascular invasion, body mass index, diabetes, hypertension, and angiotensin-converting enzyme inhibitor use, BB intake was associated with a significantly better RFS (hazard ratio [HR], 0.52; 95% CI, 0.31 to 0.88) but not OS (P = .09). Among patients with triple-negative breast cancer (TNBC; n = 377), BB intake was associated with improved RFS (HR, 0.30; 95% CI, 0.10 to 0.87; P = .027) but not OS (HR, 0.35; 95% CI, 0.12 to 1.00; P = .05). CONCLUSION In this study, BB intake was associated with improved RFS in all patients with breast cancer and in patients with TNBC. Additional studies evaluating the potential benefits of beta-adrenergic blockade on breast cancer recurrence with a focus on TNBC are warranted. (J Clin Oncol. 2011 May 31)
Genetic pathways leading to therapy-related myeloid neoplasms (Mediterr J Hematol Infect Dis. 2011;3(1):e2011019. Epub 2011 May 16.)
Genetic pathways leading to therapy-related myeloid neoplasms.
Stoddart A, McNerney ME, Bartom E, Bergerson R, Young DJ, Qian Z, Wang J, Fernald AA, Davis EM, Larson RA, White KP, Le Beau MM.
SourceUniversity of Chicago, Chicago, IL, USA.
Abstract
Therapy-related myeloid neoplasm (t-MN) is a distinctive clinical syndrome occurring after exposure to chemotherapy or radiotherapy. t-MN arises in most cases from a multipotential hematopoietic stem cell or, less commonly, in a lineage committed progenitor cell. The prognosis for patients with t-MN is poor, as current forms of therapy are largely ineffective. Cytogenetic analysis, molecular analysis and gene expression profiling analysis of t-MN has revealed that there are distinct subtypes of the disease; however, our understanding of the genetic basis of t-MN is incomplete. Elucidating the genetic pathways and molecular networks that are perturbed in t-MNs, may facilitate the identification of therapeutic targets that can be exploited for the development of urgently-needed targeted therapies.
Genome-wide tissue-specific occupancy of the hox protein ultrabithorax and hox cofactor homothorax in Drosophila (PLoS One, 2011 Apr 5)
PLoS One. 2011 Apr 5;6(4):e14686.
Genome-wide tissue-specific occupancy of the hox protein ultrabithorax and hox cofactor homothorax in Drosophila.
Slattery M, Ma L, Négre N, White KP, Mann RS.
SourceDepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, United States of America.
Abstract
The Hox genes are responsible for generating morphological diversity along the anterior-posterior axis during animal development. The Drosophila Hox gene Ultrabithorax (Ubx), for example, is required for specifying the identity of the third thoracic (T3) segment of the adult, which includes the dorsal haltere, an appendage required for flight, and the ventral T3 leg. Ubx mutants show homeotic transformations of the T3 leg towards the identity of the T2 leg and the haltere towards the wing. All Hox genes, including Ubx, encode homeodomain containing transcription factors, raising the question of what target genes Ubx regulates to generate these adult structures. To address this question, we carried out whole genome ChIP-chip studies to identify all of the Ubx bound regions in the haltere and T3 leg imaginal discs, which are the precursors to these adult structures. In addition, we used ChIP-chip to identify the sites bound by the Hox cofactor, Homothorax (Hth). In contrast to previous ChIP-chip studies carried out in Drosophila embryos, these binding studies reveal that there is a remarkable amount of tissue- and transcription factor-specific binding. Analyses of the putative target genes bound and regulated by these factors suggest that Ubx regulates many downstream transcription factors and developmental pathways in the haltere and T3 leg. Finally, we discovered additional DNA sequence motifs that in some cases are specific for individual data sets, arguing that Ubx and/or Hth work together with many regionally expressed transcription factors to execute their functions. Together, these data provide the first whole-genome analysis of the binding sites and target genes regulated by Ubx to specify the morphologies of the adult T3 segment of the fly.
Large-scale genetic epistasis networks using RNAi. (Nat Methods, 2011 Apr 8)
Nat Methods. 2011 Apr;8(4):299-301.
Large-scale genetic epistasis networks using RNAi.
Wang X, White KP.
SourceInstitute for Genomics and Systems Biology and Department of Human Genetics, University of Chicago, Chicago, Illinois, USA.
Abstract
Pairwise quantitative genetic interactions are mapped by combinatorial RNA interference in metazoan cells.
PMID: 21451517 [PubMed - in process]
A cis-regulatory map of the Drosophila genome (Nature, 2011 March 24)
Nature. 2011 Mar 24;471(7339):527-31.
A cis-regulatory map of the Drosophila genome.
Nègre N, Brown CD, Ma L, Bristow CA, Miller SW, Wagner U, Kheradpour P, Eaton ML, Loriaux P, Sealfon R, Li Z, Ishii H, Spokony RF, Chen J, Hwang L, Cheng C, Auburn RP, Davis MB, Domanus M, Shah PK, Morrison CA, Zieba J, Suchy S, Senderowicz L, Victorsen A, Bild NA, Grundstad AJ, Hanley D, MacAlpine DM, Mannervik M, Venken K, Bellen H, White R, Gerstein M, Russell S, Grossman RL, Ren B, Posakony JW, Kellis M, White KP.
Institute for Genomics and Systems Biology, Department of Human Genetics, The University of Chicago, 900 East 57th Street, Chicago, Illinois 60637, USA.
Comment in:
* Nature. 2011 Mar 24;471(7339):458-9.
Abstract
Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.
Light-driven changes in energy metabolism directly entrain the cyanobacterial circadian oscillator (Science, 2011 Jan 14)
Science. 2011 Jan 14;331(6014):220-3.
Light-driven changes in energy metabolism directly entrain the cyanobacterial circadian oscillator.
Rust MJ, Golden SS, O’Shea EK.
Howard Hughes Medical Institute, Center for Systems Biology, Department of Molecular and Cellular Biology, and Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Abstract
Circadian clocks are self-sustained biological oscillators that can be entrained by environmental cues. Although this phenomenon has been studied in many organisms, the molecular mechanisms of entrainment remain unclear. Three cyanobacterial proteins and adenosine triphosphate (ATP) are sufficient to generate oscillations in phosphorylation in vitro. We show that changes in illumination that induce a phase shift in cultured cyanobacteria also cause changes in the ratio of ATP to adenosine diphosphate (ADP). When these nucleotide changes are simulated in the in vitro oscillator, they cause phase shifts similar to those observed in vivo. Physiological concentrations of ADP inhibit kinase activity in the oscillator, and a mathematical model constrained by data shows that this effect is sufficient to quantitatively explain entrainment of the cyanobacterial circadian clock.
PMID: 21233390 [PubMed - in process]
Identification of Functional Elements and Regulatory Circuits by Drosophila modENCODE (Science, 2010 Dec 22)
Science. 2010 Dec 22. [Epub ahead of print]
Identification of Functional Elements and Regulatory Circuits by Drosophila modENCODE.
The modENCODE Consortium, Roy S, Ernst J, Kharchenko PV, Kheradpour P, Negre N, Eaton ML, Landolin JM, Bristow CA, Ma L, Lin MF, Washietl S, Arshinoff BI, Ay F, Meyer PE, Robine N, Washington NL, Di Stefano L, Berezikov E, Brown CD, Candeias R, Carlson JW, Carr A, Jungreis I, Marbach D, Sealfon R, Tolstorukov MY, Will S, Alekseyenko AA, Artieri C, Booth BW, Brooks AN, Dai Q, Davis CA, Duff MO, Feng X, Gorchakov AA, Gu T, Henikoff JG, Kapranov P, Li R, Macalpine HK, Malone J, Minoda A, Nordman J, Okamura K, Perry M, Powell SK, Riddle NC, Sakai A, Samsonova A, Sandler JE, Schwartz YB, Sher N, Spokony R, Sturgill D, van Baren M, Wan KH, Yang L, Yu C, Feingold E, Good P, Guyer M, Lowdon R, Ahmad K, Andrews J, Berger B, Brenner SE, Brent MR, Cherbas L, Elgin SC, Gingeras TR, Grossman R, Hoskins RA, Kaufman TC, Kent W, Kuroda MI, Orr-Weaver T, Perrimon N, Pirrotta V, Posakony JW, Ren B, Russell S, Cherbas P, Graveley BR, Lewis S, Micklem G, Oliver B, Park PJ, Celniker SE, Henikoff S, Karpen GH, Lai EC, Macalpine DM, Stein LD, White KP, Kellis M.
Collaborators (76)
Acevedo D, Auburn R, Barber G, Bellen HJ, Bishop EP, Bryson TD, Chateigner A, Chen J, Clawson H, Comstock CL, Contrino S, Denapoli LC, Ding Q, Dobin A, Domanus MH, Drenkow J, Dudoit S, Dumais J, Eng T, Fagegaltier D, Gadel SE, Ghosh S, Guillier F, Hanley D, Hannon GJ, Hansen KD, Heinz E, Hinrichs AS, Hirst M, Jha S, Jiang L, Jung YL, Kashevsky H, Kennedy CD, Kephart ET, Langton L, Lee OK, Li S, Li Z, Lin W, Linder-Basso D, Lloyd P, Lyne R, Marchetti SE, Marra M, Mattiuzzo NR, McKay S, Meyer F, Miller D, Miller SW, Moore RA, Morrison CA, Prinz JA, Rooks M, Moore R, Rutherford KM, Ruzanov P, Scheftner DA, Senderowicz L, Shah PK, Shanower G, Smith R, Stinson EO, Suchy S, Tenney AE, Tian F, Venken KJ, Wang H, White R, Wilkening J, Willingham AT, Zaleski C, Zha Z, Zhang D, Zhao Y, Zieba J.
Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
Abstract
To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.
Evolution and inheritance of early embryonic patterning in D. Simulans and D. Sechellia (Evolution, Dec 1 2010)
Evolution. 2010 Dec 1. doi: 10.1111/j.1558-5646.2010.01206.x. [Epub ahead of print]
EVOLUTION AND INHERITANCE OF EARLY EMBRYONIC PATTERNING IN D. SIMULANS AND D. SECHELLIA.
Lott SE, Ludwig MZ, Kreitman M.
Committee on Genetics and the Department of Ecology and Evolution, University of Chicago, 1101 E 57th St., Chicago, IL, 60637, USA Present Address: Department of Molecular and Cell Biology, 387 Stanley Hall #3220, University of California, Berkeley, CA 94720, USA E-mail: slott@uchicago.edu.
Abstract
Pattern formation in Drosophila is a widely studied example of a robust developmental system. Such robust systems pose a challenge to adaptive evolution, as they mask variation which selection may otherwise act upon. Yet we find variation in the localization of expression domains (henceforth ‘stripe allometry’) in the pattern formation pathway. Specifically, we characterize differences in the gap genes giant and Kruppel, and the pair-rule gene even-skipped, which differ between the sibling species D. simulans and D. sechellia. In a double-backcross experiment, stripe allometry is consistent with maternal inheritance of stripe positioning and multiple genetic factors, with a distinct genetic basis from embryo length. Embryos produced by F1 and F2 backcross mothers exhibit novel spatial patterns of gene expression relative to the parental species, with no measurable increase in positional variance among individuals. Buffering of novel spatial patterns in the backcross genotypes suggests that robustness need not be disrupted in order for the trait to evolve, and perhaps the system is incapable of evolving to prevent the expression of all genetic variation. This limitation, and the ability of natural selection to act on minute genetic differences that are within the “margin of error” for the buffering mechanism, indicates that developmentally buffered traits can evolve without disruption of robustness.
Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase (Nature. 2010 Nov 11)
Nature. 2010 Nov 11;468(7321):330-3.
Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase.
Yi C, Jia G, Hou G, Dai Q, Zhang W, Zheng G, Jian X, Yang CG, Cui Q, He C.
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Abstract
Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology, including the oxidative demethylation of methylated nucleic acids and histones. Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes. Here we present structural characterization of oxidation intermediates for these demethylases. Using a chemical cross-linking strategy, complexes of AlkB-double stranded DNA (dsDNA) containing 1,N(6)-etheno adenine (εA), N(3)-methyl thymine (3-meT) and N(3)-methyl cytosine (3-meC) are stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo. Glycol (from εA) and hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.
PMID: 21068844 [PubMed - in process]
Modeling bistable cell-fate choices in the Drosophila eye: qualitative and quantitative perspectives (Development July 15, 2010)
Development. 2010 Jul;137(14):2265-78.
Modeling bistable cell-fate choices in the Drosophila eye: qualitative and quantitative perspectives.
Graham TG, Tabei SM, Dinner AR, Rebay I.
Ben May Department for Cancer Research, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
Abstract
A major goal of developmental biology is to understand the molecular mechanisms whereby genetic signaling networks establish and maintain distinct cell types within multicellular organisms. Here, we review cell-fate decisions in the developing eye of Drosophila melanogaster and the experimental results that have revealed the topology of the underlying signaling circuitries. We then propose that switch-like network motifs based on positive feedback play a central role in cell-fate choice, and discuss how mathematical modeling can be used to understand and predict the bistable or multistable behavior of such networks.
Sterile alpha motif domain-mediated self-association plays an essential role in modulating the activity of the Drosophila ETS family transcriptional repressor Yan (Molecular and Cellular Biology, March 2010)
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. Jan 2010)
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. April 2010)
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.
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.