Publications by authors named "David E Hill"

107 Publications

Electrochemical Nozaki-Hiyama-Kishi Coupling: Scope, Applications, and Mechanism.

J Am Chem Soc 2021 Jun 15;143(25):9478-9488. Epub 2021 Jun 15.

Department of Chemistry, Scripps Research, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

One of the most oft-employed methods for C-C bond formation involving the coupling of vinyl-halides with aldehydes catalyzed by Ni and Cr (Nozaki-Hiyama-Kishi, NHK) has been rendered more practical using an electroreductive manifold. Although early studies pointed to the feasibility of such a process, those precedents were never applied by others due to cumbersome setups and limited scope. Here we show that a carefully optimized electroreductive procedure can enable a more sustainable approach to NHK, even in an asymmetric fashion on highly complex medicinally relevant systems. The e-NHK can even enable non-canonical substrate classes, such as redox-active esters, to participate with low loadings of Cr when conventional chemical techniques fail. A combination of detailed kinetics, cyclic voltammetry, and in situ UV-vis spectroelectrochemistry of these processes illuminates the subtle features of this mechanistically intricate process.
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http://dx.doi.org/10.1021/jacs.1c03007DOI Listing
June 2021

Selenomethionine as an expressible handle for bioconjugations.

Proc Natl Acad Sci U S A 2021 02;118(8)

Department of Chemistry, Scripps Research, La Jolla, CA 92037;

Site-selective chemical bioconjugation reactions are enabling tools for the chemical biologist. Guided by a careful study of the selenomethionine (SeM) benzylation, we have refined the reaction to meet the requirements of practical protein bioconjugation. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, is selectively labile to glutathione, and embodies a broadly tunable cleavage profile. Specifically, a 4-bromomethylphenylacetyl (BrMePAA) linker has been applied for efficient conjugation of complex organic molecules to SeM-containing proteins. This expansion of the bioconjugation toolkit has broad potential in the development of chemically enhanced proteins.
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http://dx.doi.org/10.1073/pnas.2005164118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923357PMC
February 2021

Comprehensive characterization of protein-protein interactions perturbed by disease mutations.

Nat Genet 2021 03 8;53(3):342-353. Epub 2021 Feb 8.

Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Technological and computational advances in genomics and interactomics have made it possible to identify how disease mutations perturb protein-protein interaction (PPI) networks within human cells. Here, we show that disease-associated germline variants are significantly enriched in sequences encoding PPI interfaces compared to variants identified in healthy participants from the projects 1000 Genomes and ExAC. Somatic missense mutations are also significantly enriched in PPI interfaces compared to noninterfaces in 10,861 tumor exomes. We computationally identified 470 putative oncoPPIs in a pan-cancer analysis and demonstrate that oncoPPIs are highly correlated with patient survival and drug resistance/sensitivity. We experimentally validate the network effects of 13 oncoPPIs using a systematic binary interaction assay, and also demonstrate the functional consequences of two of these on tumor cell growth. In summary, this human interactome network framework provides a powerful tool for prioritization of alleles with PPI-perturbing mutations to inform pathobiological mechanism- and genotype-based therapeutic discovery.
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http://dx.doi.org/10.1038/s41588-020-00774-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8237108PMC
March 2021

A comprehensive library of human transcription factors for cell fate engineering.

Nat Biotechnol 2021 04 30;39(4):510-519. Epub 2020 Nov 30.

Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.
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http://dx.doi.org/10.1038/s41587-020-0742-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610615PMC
April 2021

Insights into the Role of Transient Chiral Mediators and Pyridone Ligands in Asymmetric Pd-Catalyzed C-H Functionalization.

J Org Chem 2020 11 16;85(21):13674-13679. Epub 2020 Oct 16.

Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States.

Mechanistic investigations uncover a novel role for 2-pyridone ligands and interrogate the origin of enantioselectivity in the (+)-norbornene-mediated Pd-catalyzed -C(aryl)-H functionalization of diarylmethylamines. Observations from kinetic analysis in concert with in situ F NMR monitoring allow us to propose that the pyridone ligand plays a role in enhancing the rate- and enantio-determining insertion of an arylpalladium species into a chiral norbornene derivative. The unprecedented features of 2-pyridone ligands in asymmetric 1,2 migratory insertion, and norbornene as a transient chiral mediator in relay chemistry, provide new insights into this ligand scaffold for future developments in stereoselective transition-metal-catalyzed C-H functionalization.
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http://dx.doi.org/10.1021/acs.joc.0c01798DOI Listing
November 2020

Large-scale tethered function assays identify factors that regulate mRNA stability and translation.

Nat Struct Mol Biol 2020 10 17;27(10):989-1000. Epub 2020 Aug 17.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA.

The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. Here, we develop a plasmid resource of 690 human RBPs that we subject to luciferase-based 3'-untranslated-region tethered function assays to pinpoint RBPs that regulate RNA stability or translation. Enhanced UV-cross-linking and immunoprecipitation of these RBPs identifies thousands of endogenous mRNA targets that respond to changes in RBP level, recapitulating effects observed in tethered function assays. Among these RBPs, the ubiquitin-associated protein 2-like (UBAP2L) protein interacts with RNA via its RGG domain and cross-links to mRNA and rRNA. Fusion of UBAP2L to RNA-targeting CRISPR-Cas9 demonstrates programmable translational enhancement. Polysome profiling indicates that UBAP2L promotes translation of target mRNAs, particularly global regulators of translation. Our tethering survey allows rapid assignment of the molecular activity of proteins, such as UBAP2L, to specific steps of mRNA metabolism.
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http://dx.doi.org/10.1038/s41594-020-0477-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221285PMC
October 2020

A massively parallel barcoded sequencing pipeline enables generation of the first ORFeome and interactome map for rice.

Proc Natl Acad Sci U S A 2020 05 12;117(21):11836-11842. Epub 2020 May 12.

Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, NY 14853;

Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop CR-mediated inkage of barcoded dapters o nucleic acid lements for uencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms.
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http://dx.doi.org/10.1073/pnas.1918068117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260977PMC
May 2020

ORF Capture-Seq as a versatile method for targeted identification of full-length isoforms.

Nat Commun 2020 05 11;11(1):2326. Epub 2020 May 11.

Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, 02215, USA.

Most human protein-coding genes are expressed as multiple isoforms, which greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every coding gene, the majority of alternative isoforms remains uncharacterized due to (i) vast differences of overall levels between different isoforms expressed from common genes, and (ii) the difficulty of obtaining full-length transcript sequences. Here, we present ORF Capture-Seq (OCS), a flexible method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As a proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude when compared to unenriched samples. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will accelerate mapping of the human transcriptome.
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http://dx.doi.org/10.1038/s41467-020-16174-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214433PMC
May 2020

A reference map of the human binary protein interactome.

Nature 2020 04 8;580(7803):402-408. Epub 2020 Apr 8.

Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, USA.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome, transcriptome and proteome data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.
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http://dx.doi.org/10.1038/s41586-020-2188-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7169983PMC
April 2020

Maximizing binary interactome mapping with a minimal number of assays.

Nat Commun 2019 08 29;10(1):3907. Epub 2019 Aug 29.

Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute (DFCI), 450 Brookline Avenue, Boston, MA, 02215, USA.

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.
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http://dx.doi.org/10.1038/s41467-019-11809-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715725PMC
August 2019

A genome-wide positioning systems network algorithm for in silico drug repurposing.

Nat Commun 2019 08 2;10(1):3476. Epub 2019 Aug 2.

Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA.

Recent advances in DNA/RNA sequencing have made it possible to identify new targets rapidly and to repurpose approved drugs for treating heterogeneous diseases by the 'precise' targeting of individualized disease modules. In this study, we develop a Genome-wide Positioning Systems network (GPSnet) algorithm for drug repurposing by specifically targeting disease modules derived from individual patient's DNA and RNA sequencing profiles mapped to the human protein-protein interactome network. We investigate whole-exome sequencing and transcriptome profiles from ~5,000 patients across 15 cancer types from The Cancer Genome Atlas. We show that GPSnet-predicted disease modules can predict drug responses and prioritize new indications for 140 approved drugs. Importantly, we experimentally validate that an approved cardiac arrhythmia and heart failure drug, ouabain, shows potential antitumor activities in lung adenocarcinoma by uniquely targeting a HIF1α/LEO1-mediated cell metabolism pathway. In summary, GPSnet offers a network-based, in silico drug repurposing framework for more efficacious therapeutic selections.
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http://dx.doi.org/10.1038/s41467-019-10744-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677722PMC
August 2019

Palladium(ii)-catalyzed γ-selective hydroarylation of alkenyl carbonyl compounds with arylboronic acids.

Chem Sci 2018 Nov 6;9(44):8363-8368. Epub 2018 Sep 6.

Department of Chemistry , The Scripps Research Institute , 10550 N Torrey Pines Road, La Jolla , California 92037 , USA . Email:

A catalytic γ-selective -hydroarylation of alkenyl carbonyl compounds using arylboronic acids has been developed using a substrate directivity approach with a palladium(ii) catalyst. This method tolerates a wide range of functionalized (hetero)arylboronic acids and a variety of substitution patterns on the alkene. Preliminary mechanistic studies suggest that transmetalation is rate-limiting.
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http://dx.doi.org/10.1039/c8sc03081bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247822PMC
November 2018

Protein Interactomics by Two-Hybrid Methods.

Methods Mol Biol 2018 ;1794:1-14

Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, USA.

Comprehensive identification of direct, physical interactions between biological macromolecules, such as protein-protein, protein-DNA, and protein-RNA interactions, is critical for our understanding of the function of gene products as well as the global organization and interworkings of various molecular machines within the cell. The accurate and comprehensive detection of direct interactions, however, remains a huge challenge due to the inherent structural complexity arising from various post-transcriptional and translational modifications coupled with huge heterogeneity in concentration, affinity, and subcellular location differences existing for any interacting molecules. This has created a need for developing multiple orthogonal and complementary assays for detecting various types of biological interactions. In this introduction, we discuss the methods developed for measuring different types of molecular interactions with an emphasis on direct protein-protein interactions, critical issues for generating high-quality interactome datasets, and the insights into biological networks and human diseases that current interaction mapping efforts provide. Further, we will discuss what future might lie ahead for the continued evolution of two-hybrid methods and the role of interactomics for expanding the advancement of biomedical science.
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http://dx.doi.org/10.1007/978-1-4939-7871-7_1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948107PMC
February 2019

Network Analysis of UBE3A/E6AP-Associated Proteins Provides Connections to Several Distinct Cellular Processes.

J Mol Biol 2018 03 6;430(7):1024-1050. Epub 2018 Feb 6.

Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Perturbations in activity and dosage of the UBE3A ubiquitin-ligase have been linked to Angelman syndrome and autism spectrum disorders. UBE3A was initially identified as the cellular protein hijacked by the human papillomavirus E6 protein to mediate the ubiquitylation of p53, a function critical to the oncogenic potential of these viruses. Although a number of substrates have been identified, the normal cellular functions and pathways affected by UBE3A are largely unknown. Previously, we showed that UBE3A associates with HERC2, NEURL4, and MAPK6/ERK3 in a high-molecular-weight complex of unknown function that we refer to as the HUN complex (HERC2, UBE3A, and NEURL4). In this study, the combination of two complementary proteomic approaches with a rigorous network analysis revealed cellular functions and pathways in which UBE3A and the HUN complex are involved. In addition to finding new UBE3A-associated proteins, such as MCM6, SUGT1, EIF3C, and ASPP2, network analysis revealed that UBE3A-associated proteins are connected to several fundamental cellular processes including translation, DNA replication, intracellular trafficking, and centrosome regulation. Our analysis suggests that UBE3A could be involved in the control and/or integration of these cellular processes, in some cases as a component of the HUN complex, and also provides evidence for crosstalk between the HUN complex and CAMKII interaction networks. This study contributes to a deeper understanding of the cellular functions of UBE3A and its potential role in pathways that may be affected in Angelman syndrome, UBE3A-associated autism spectrum disorders, and human papillomavirus-associated cancers.
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http://dx.doi.org/10.1016/j.jmb.2018.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866790PMC
March 2018

A framework for exhaustively mapping functional missense variants.

Mol Syst Biol 2017 12 21;13(12):957. Epub 2017 Dec 21.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740498PMC
http://dx.doi.org/10.15252/msb.20177908DOI Listing
December 2017

Dynamic Ligand Exchange as a Mechanistic Probe in Pd-Catalyzed Enantioselective C-H Functionalization Reactions Using Monoprotected Amino Acid Ligands.

J Am Chem Soc 2017 12 14;139(51):18500-18503. Epub 2017 Dec 14.

Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.

A new tool for probing enantioselective reaction mechanisms is introduced. Monitoring the temporal change in product enantiomeric excess after addition of the opposite enantiomer of the ligand during the reaction provides a means of probing dynamic ligand exchange in enantioselective C-H iodination catalyzed by Pd with monoprotected amino acid ligands (MPAAs). This work has general potential to provide insights about the dynamics of catalyst and ligand molecularity and exchange.
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http://dx.doi.org/10.1021/jacs.7b11962DOI Listing
December 2017

A Role for Pd(IV) in Catalytic Enantioselective C-H Functionalization with Monoprotected Amino Acid Ligands under Mild Conditions.

J Am Chem Soc 2017 07 27;139(27):9238-9245. Epub 2017 Jun 27.

Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.

Kinetic and mechanistic studies of the desymmetrization of benzhydrylamine using Pd/monoprotected amino acid ligands (Pd/MPAA) via C-H functionalization with molecular iodine provide mechanistic insight into the rate-determining step and the oxidation state of Pd in the C-H functionalization step. Enantiomeric excess is strikingly insensitive to temperature from ambient temperature up to over 70 °C, and reaction rate is insensitive to the electronic characteristics of the ligand's benzoyl protecting group. The reaction is highly robust with no evidence of catalyst deactivation. Intriguingly, C-H bond breaking does not occur prior to the addition of I to the reaction mixture. Electrochemical experiments demonstrate the viability of oxidative addition of I to Pd(II). Together with F NMR studies, these observations suggest that iodine oxidizes Pd prior to addition of the amine substrate. This work may lead to a better general understanding of the subtle variations in the reaction mechanisms for C-H functionalization reactions that may be extant for this ligand class depending on substrate, amino acid ligand and protecting group, and reaction conditions.
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http://dx.doi.org/10.1021/jacs.7b03716DOI Listing
July 2017

Identifying pathogenicity of human variants via paralog-based yeast complementation.

PLoS Genet 2017 May 25;13(5):e1006779. Epub 2017 May 25.

Donnelly Centre, Toronto, Ontario, Canada.

To better understand the health implications of personal genomes, we now face a largely unmet challenge to identify functional variants within disease-associated genes. Functional variants can be identified by trans-species complementation, e.g., by failure to rescue a yeast strain bearing a mutation in an orthologous human gene. Although orthologous complementation assays are powerful predictors of pathogenic variation, they are available for only a few percent of human disease genes. Here we systematically examine the question of whether complementation assays based on paralogy relationships can expand the number of human disease genes with functional variant detection assays. We tested over 1,000 paralogous human-yeast gene pairs for complementation, yielding 34 complementation relationships, of which 33 (97%) were novel. We found that paralog-based assays identified disease variants with success on par with that of orthology-based assays. Combining all homology-based assay results, we found that complementation can often identify pathogenic variants outside the homologous sequence region, presumably because of global effects on protein folding or stability. Within our search space, paralogy-based complementation more than doubled the number of human disease genes with a yeast-based complementation assay for disease variation.
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http://dx.doi.org/10.1371/journal.pgen.1006779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466341PMC
May 2017

Dynamic Role of trans Regulation of Gene Expression in Relation to Complex Traits.

Am J Hum Genet 2017 Apr 9;100(4):571-580. Epub 2017 Mar 9.

The Framingham Heart Study, 73 Mt. Wayte Avenue, Framingham, MA 01702, USA; The Population Sciences Branch, Division of Intramural Research, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

Identifying causal genetic variants and understanding their mechanisms of effect on traits remains a challenge in genome-wide association studies (GWASs). In particular, how genetic variants (i.e., trans-eQTLs) affect expression of remote genes (i.e., trans-eGenes) remains unknown. We hypothesized that some trans-eQTLs regulate expression of distant genes by altering the expression of nearby genes (cis-eGenes). Using published GWAS datasets with 39,165 single-nucleotide polymorphisms (SNPs) associated with 1,960 traits, we explored whole blood gene expression associations of trait-associated SNPs in 5,257 individuals from the Framingham Heart Study. We identified 2,350 trans-eQTLs (at p < 10); more than 80% of them were found to have cis-associated eGenes. Mediation testing suggested that for 35% of trans-eQTL-trans-eGene pairs in different chromosomes and 90% pairs in the same chromosome, the disease-associated SNP may alter expression of the trans-eGene via cis-eGene expression. In addition, we identified 13 trans-eQTL hotspots, affecting from ten to hundreds of genes, suggesting the existence of master genetic regulators. Using causal inference testing, we searched causal variants across eight cardiometabolic traits (BMI, systolic and diastolic blood pressure, LDL cholesterol, HDL cholesterol, total cholesterol, triglycerides, and fasting blood glucose) and identified several cis-eGenes (ALDH2 for systolic and diastolic blood pressure, MCM6 and DARS for total cholesterol, and TRIB1 for triglycerides) that were causal mediators for the corresponding traits, as well as examples of trans-mediators (TAGAP for LDL cholesterol). The finding of extensive evidence of genome-wide mediation effects suggests a critical role of cryptic gene regulation underlying many disease traits.
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http://dx.doi.org/10.1016/j.ajhg.2017.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384035PMC
April 2017

In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons.

Cell Syst 2017 02 25;4(2):242-250.e4. Epub 2017 Jan 25.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Synucleinopathies, including Parkinson's disease (PD), are associated with the misfolding and mistrafficking of alpha-synuclein (α-syn). Here, using an ascorbate peroxidase (APEX)-based labeling method combined with mass spectrometry, we defined a network of proteins in the immediate vicinity of α-syn in living neurons to shed light on α-syn function. This approach identified 225 proteins, including synaptic proteins, proteins involved in endocytic vesicle trafficking, the retromer complex, phosphatases and mRNA binding proteins. Many were in complexes with α-syn, and some were encoded by genes known to be risk factors for PD and other neurodegenerative diseases. Endocytic trafficking and mRNA translation proteins within this spatial α-syn map overlapped with genetic modifiers of α-syn toxicity, developed in an accompanying study (Khurana et al., this issue of Cell Systems). Our data suggest that perturbation of these particular pathways is directly related to the spatial localization of α-syn within the cell. These approaches provide new avenues to systematically examine protein function and pathology in living cells.
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http://dx.doi.org/10.1016/j.cels.2017.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578869PMC
February 2017

Proteome-scale Binary Interactomics in Human Cells.

Mol Cell Proteomics 2016 Dec 1;15(12):3624-3639. Epub 2016 Nov 1.

From the ‡Medical Biotechnology Center, VIB, Ghent, Belgium;

Because proteins are the main mediators of most cellular processes they are also prime therapeutic targets. Identifying physical links among proteins and between drugs and their protein targets is essential in order to understand the mechanisms through which both proteins themselves and the molecules they are targeted with act. Thus, there is a strong need for sensitive methods that enable mapping out these biomolecular interactions. Here we present a robust and sensitive approach to screen proteome-scale collections of proteins for binding to proteins or small molecules using the well validated MAPPIT (Mammalian Protein-Protein Interaction Trap) and MASPIT (Mammalian Small Molecule-Protein Interaction Trap) assays. Using high-density reverse transfected cell microarrays, a close to proteome-wide collection of human ORF clones can be screened for interactors at high throughput. The versatility of the platform is demonstrated through several examples. With MAPPIT, we screened a 15k ORF library for binding partners of RNF41, an E3 ubiquitin protein ligase implicated in receptor sorting, identifying known and novel interacting proteins. The potential related to the fact that MAPPIT operates in living human cells is illustrated in a screen where the protein collection is scanned for interactions with the glucocorticoid receptor (GR) in its unliganded versus dexamethasone-induced activated state. Several proteins were identified the interaction of which is modulated upon ligand binding to the GR, including a number of previously reported GR interactors. Finally, the screening technology also enables detecting small molecule target proteins, which in many drug discovery programs represents an important hurdle. We show the efficiency of MASPIT-based target profiling through screening with tamoxifen, a first-line breast cancer drug, and reversine, an investigational drug with interesting dedifferentiation and antitumor activity. In both cases, cell microarray screens yielded known and new potential drug targets highlighting the utility of the technology beyond fundamental biology.
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http://dx.doi.org/10.1074/mcp.M116.061994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141276PMC
December 2016

Mapping transcription factor interactome networks using HaloTag protein arrays.

Proc Natl Acad Sci U S A 2016 07 29;113(29):E4238-47. Epub 2016 Jun 29.

Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037;

Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.
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http://dx.doi.org/10.1073/pnas.1603229113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961138PMC
July 2016

The transcription factor ERG recruits CCR4-NOT to control mRNA decay and mitotic progression.

Nat Struct Mol Biol 2016 07 6;23(7):663-72. Epub 2016 Jun 6.

Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), University of Liège (ULg), Liège, Belgium.

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.
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http://dx.doi.org/10.1038/nsmb.3243DOI Listing
July 2016

An inter-species protein-protein interaction network across vast evolutionary distance.

Mol Syst Biol 2016 Apr 22;12(4):865. Epub 2016 Apr 22.

Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Departments of Molecular Genetics and Computer Science, University of Toronto, Toronto, ON, Canada Donnelly Centre, University of Toronto, Toronto, ON, Canada Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, ON, Canada Canadian Institute for Advanced Research, Toronto, ON, Canada

In cellular systems, biophysical interactions between macromolecules underlie a complex web of functional interactions. How biophysical and functional networks are coordinated, whether all biophysical interactions correspond to functional interactions, and how such biophysical-versus-functional network coordination is shaped by evolutionary forces are all largely unanswered questions. Here, we investigate these questions using an "inter-interactome" approach. We systematically probed the yeast and human proteomes for interactions between proteins from these two species and functionally characterized the resulting inter-interactome network. After a billion years of evolutionary divergence, the yeast and human proteomes are still capable of forming a biophysical network with properties that resemble those of intra-species networks. Although substantially reduced relative to intra-species networks, the levels of functional overlap in the yeast-human inter-interactome network uncover significant remnants of co-functionality widely preserved in the two proteomes beyond human-yeast homologs. Our data support evolutionary selection against biophysical interactions between proteins with little or no co-functionality. Such non-functional interactions, however, represent a reservoir from which nascent functional interactions may arise.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848758PMC
http://dx.doi.org/10.15252/msb.20156484DOI Listing
April 2016

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

Mol Syst Biol 2016 Apr 22;12(4):863. Epub 2016 Apr 22.

Joint IRB-BSC Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848762PMC
http://dx.doi.org/10.15252/msb.20156660DOI Listing
April 2016

Survey of variation in human transcription factors reveals prevalent DNA binding changes.

Science 2016 Mar 24;351(6280):1450-1454. Epub 2016 Mar 24.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA binding activity and used universal protein-binding microarrays to assay sequence-specific DNA binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA binding activities, which may contribute to phenotypic variation.
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http://dx.doi.org/10.1126/science.aad2257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825693PMC
March 2016

An extended set of yeast-based functional assays accurately identifies human disease mutations.

Genome Res 2016 05 14;26(5):670-80. Epub 2016 Mar 14.

Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Computer Science, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, Ontario M5G 1X5, Canada; Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA; Canadian Institute for Advanced Research, Toronto, Ontario, M5G 1Z8, Canada.

We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods.
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http://dx.doi.org/10.1101/gr.192526.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864455PMC
May 2016

Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

Cell 2016 Feb;164(4):805-17

Genomic Analysis of Network Perturbations Center of Excellence in Genomic Science (CEGS), Dana-Farber Cancer Institute, Boston, MA 02215, USA; Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms").
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http://dx.doi.org/10.1016/j.cell.2016.01.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882190PMC
February 2016

MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy.

Cancer Discov 2016 Jan 6;6(1):45-58. Epub 2015 Nov 6.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts. Department of Medicine, Harvard Medical School, Boston, Massachusetts. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. Center for Cancer Systems Biology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Unlabelled: An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers, and many cancer cell lines have amplified, overexpressed MECP2 and are dependent on MECP2 expression for growth. MECP2 copy-number gain and RAS family member alterations are mutually exclusive in several cancer types. The MECP2 splicing isoforms activate the major growth factor pathways targeted by activated RAS, the MAPK and PI3K pathways. MECP2 rescued the growth of a KRAS(G12C)-addicted cell line after KRAS downregulation, and activated KRAS rescues the growth of an MECP2-addicted cell line after MECP2 downregulation. MECP2 binding to the epigenetic modification 5-hydroxymethylcytosine is required for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action.

Significance: MECP2 is a commonly amplified oncogene in human malignancies with a unique epigenetic mechanism of action. Cancer Discov; 6(1); 45-58. ©2015 AACR.This article is highlighted in the In This Issue feature, p. 1.
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http://dx.doi.org/10.1158/2159-8290.CD-15-0341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775099PMC
January 2016
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