Person: Lareau, Caleb
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Lareau
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Caleb
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Lareau, Caleb
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Publication The SARS-CoV-2 RNA–protein Interactome in Infected Human Cells(Springer Science and Business Media LLC, 2020-12-21) Schmidt, Nora; Lareau, Caleb; Keshishian, Hasmik; Ganskih, Sabina; Schneider, Cornelius; Hennig, Thomas; Melanson, Randy; Werner, Simone; Wei, Yuanjie; Zimmer, Matthias; Ade, Jens; Kirschner, Luisa; Zielinski, Sebastian; Dölken, Lars; Lander, Eric; Caliskan, Neva; Fischer, Utz; Vogel, Jörg; Carr, Steven A.; Bodem, Jochen; Munschauer, MathiasCharacterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.Publication The ATPase Module of Mammalian SWI/SNF Family Complexes Mediates Subcomplex Identity and Catalytic Activity–independent Genomic Targeting(Springer Science and Business Media LLC, 2019-03-11) Mashtalir, Nazar; Wang, Lu; Kadoch, Cigall; Pan, Joshua; McKenzie, Zachary; D’Avino, Andrew; Lareau, Caleb; St. Pierre, Roodolph; Shilatifard, AliPerturbations to mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complexes have been widely implicated as driving events in cancer1. One such perturbation is the dual loss of the SMARCA4 and SMARCA2 ATPase subunits in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT)2-5, SMARCA4-deficient thoracic sarcomas6 and dedifferentiated endometrial carcinomas7. However, the consequences of dual ATPase subunit loss on mSWI/SNF complex subunit composition, chromatin targeting, DNA accessibility and gene expression remain unknown. Here we identify an ATPase module of subunits that is required for functional specification of BAF and PBAF subcomplexes. Using SMARCA4/2 ATPase mutant variants, we define the catalytic activity -dependent and -independent contributions of the ATPase module to the targeting of BAF and PBAF complexes genome-wide. Finally, by linking distinct mSWI/SNF complex target sites to tumor-suppressive gene expression programs, we clarify the transcriptional consequences of SMARCA4/2 dual loss in SCCOHT.Publication Transcriptome-Wide Off-Target RNA Editing Induced by CRISPR-Guided DNA Base Editors(Springer Science and Business Media LLC, 2019-04-17) Grünewald, Julian; Zhou, Ronghao; Garcia, Sara; Iyer, Sowmya; Lareau, Caleb; Aryee, Martin; Joung, KeithCRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.Publication Fine mapping of chromosome 15q25 implicates ZNF592 in neurosarcoidosis patients(John Wiley & Sons, Ltd, 2015) Lareau, Caleb; Adrianto, Indra; Levin, Albert M; Iannuzzi, Michael C; Rybicki, Benjamin A; Montgomery, Courtney GNeurosarcoidosis is a clinical subtype of sarcoidosis characterized by the presence of granulomas in the nervous system. Here, we report a highly significant association with a variant (rs75652600, P = 3.12 × 10−8, odds ratios = 4.34) within a zinc finger gene, ZNF592, from an imputation-based fine-mapping study of the chromosomal region 15q25 in African-Americans with neurosarcoidosis. We validate the association with ZNF592, a gene previously shown to cause cerebellar ataxia, in a cohort of European-Americans with neurosarcoidosis by uncovering low-frequency variants with a similar risk effect size (chr15:85309284, P = 0.0021, odds ratios = 5.36).Publication Polygenic Risk Assessment Reveals Pleiotropy between Sarcoidosis and Inflammatory Disorders in the Context of Genetic Ancestry(2016) Lareau, Caleb; DeWeese, Colby F.; Adrianto, Indra; Lessard, Christopher J.; Gaffney, Patrick M.; Iannuzzi, Michael C.; Rybicki, Benjamin A.; Levin, Albert M.; Montgomery, Courtney G.Sarcoidosis is a complex disease of unknown etiology characterized by the presence of granulomatous inflammation. Though various immune system pathways have been implicated in disease, the relationship between the genetic determinants of sarcoidosis and other inflammatory disorders has not been characterized. Herein, we examined the degree of genetic pleiotropy common to sarcoidosis and other inflammatory disorders to identify shared pathways and disease systems pertinent to sarcoidosis onset. To achieve this, we quantify the association of common variant polygenic risk scores from nine complex inflammatory disorders with sarcoidosis risk. Enrichment analyses of genes implicated in pleiotropic associations were further used to elucidate candidate pathways. In European-Americans, we identify significant pleiotropy between risk of sarcoidosis and risk of asthma (R2=2.03%; p=8.89×10−9), celiac disease (R2=2.03%; p=8.21×10−9), primary biliary cirrhosis (R2=2.43%; p=2.01×10−10), and rheumatoid arthritis (R2=4.32%; p=2.50×10−17). These associations validate in African Americans only after accounting for the proportion of genome-wide European ancestry, where we demonstrate similar effects of polygenic risk for African-Americans with the highest levels of European ancestry. Variants and genes implicated in European-American pleiotropic associations were enriched for pathways involving interleukin-12, interleukin-27, and cell adhesion molecules, corroborating the hypothesized immunopathogenesis of disease.Publication Common genes associated with antidepressant response in mouse and man identify key role of glucocorticoid receptor sensitivity(Public Library of Science, 2017) Carrillo-Roa, Tania; Labermaier, Christiana; Weber, Peter; Herzog, David P.; Lareau, Caleb; Santarelli, Sara; Wagner, Klaus V.; Rex-Haffner, Monika; Harbich, Daniela; Scharf, Sebastian H.; Nemeroff, Charles B.; Dunlop, Boadie W.; Craighead, W. Edward; Mayberg, Helen S.; Schmidt, Mathias V.; Uhr, Manfred; Holsboer, Florian; Sillaber, Inge; Binder, Elisabeth B.; Müller, Marianne B.Response to antidepressant treatment in major depressive disorder (MDD) cannot be predicted currently, leading to uncertainty in medication selection, increasing costs, and prolonged suffering for many patients. Despite tremendous efforts in identifying response-associated genes in large genome-wide association studies, the results have been fairly modest, underlining the need to establish conceptually novel strategies. For the identification of transcriptome signatures that can distinguish between treatment responders and nonresponders, we herein submit a novel animal experimental approach focusing on extreme phenotypes. We utilized the large variance in response to antidepressant treatment occurring in DBA/2J mice, enabling sample stratification into subpopulations of good and poor treatment responders to delineate response-associated signature transcript profiles in peripheral blood samples. As a proof of concept, we translated our murine data to the transcriptome data of a clinically relevant human cohort. A cluster of 259 differentially regulated genes was identified when peripheral transcriptome profiles of good and poor treatment responders were compared in the murine model. Differences in expression profiles from baseline to week 12 of the human orthologues selected on the basis of the murine transcript signature allowed prediction of response status with an accuracy of 76% in the patient population. Finally, we show that glucocorticoid receptor (GR)-regulated genes are significantly enriched in this cluster of antidepressant-response genes. Our findings point to the involvement of GR sensitivity as a potential key mechanism shaping response to antidepressant treatment and support the hypothesis that antidepressants could stimulate resilience-promoting molecular mechanisms. Our data highlight the suitability of an appropriate animal experimental approach for the discovery of treatment response-associated pathways across species.Publication diffloop: a computational framework for identifying and analyzing differential DNA loops from sequencing data(Oxford University Press, 2017) Lareau, Caleb; Aryee, MartinAbstract Summary The 3D architecture of DNA within the nucleus is a key determinant of interactions between genes, regulatory elements, and transcriptional machinery. As a result, differences in DNA looping structure are associated with variation in gene expression and cell state. To systematically assess changes in DNA looping architecture between samples, we introduce diffloop, an R/Bioconductor package that provides a suite of functions for the quality control, statistical testing, annotation, and visualization of DNA loops. We demonstrate this functionality by detecting differences between ENCODE ChIA-PET samples and relate looping to variability in epigenetic state. Availability and implementation Diffloop is implemented as an R/Bioconductor package available at https://bioconductor.org/packages/release/bioc/html/diffloop.html Contact aryee.martin@mgh.harvard.edu Supplementary information Supplementary data are available at Bioinformatics online.Publication CRISPR DNA Base Editors With Reduced RNA Off-Target and Self-Editing Activities(Springer Science and Business Media LLC, 2019-09) Grünewald, Julian; Zhou, Ronghao; Iyer, Sowmya; Lareau, Caleb; Garcia, Sara; Aryee, Martin; Joung, KeithCytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1,2,3,4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.Publication Activity-by-Contact model of enhancer specificity from thousands of CRISPR perturbations(Cold Spring Harbor Laboratory, 2019-01-26) Fulco, Charles P.; Nasser, Joseph; Jones, Thouis; Munson, Glen; Bergman, Drew T.; Subramanian, Vidya; Grossman, Sharon; Anyoha, Rockwell; Doughty, Benjamin; Patwardhan, Tejal A.; Nguyen, Tung H.; Kane, Michael; Perez, Elizabeth; Durand, Neva C.; Lareau, Caleb; Stamenova, Elena K.; Aiden, Erez Lieberman; Lander, Eric; Engreitz, JesseMammalian genomes harbor millions of noncoding elements called enhancers that quantitatively regulate gene expression, but it remains unclear which enhancers regulate which genes. Here we describe an experimental approach, based on CRISPR interference, RNA FISH, and flow cytometry (CRISPRi-FlowFISH), to perturb enhancers in the genome, and apply it to test >3,000 potential regulatory enhancer-gene connections across multiple genomic loci. A simple equation based on a mechanistic model for enhancer function performed remarkably well at predicting the complex patterns of regulatory connections we observe in our CRISPR dataset. This Activity-by-Contact (ABC) model involves multiplying measures of enhancer activity and enhancer-promoter 3D contacts, and can predict enhancer-gene connections in a given cell type based on chromatin state maps. Together, CRISPRi-FlowFISH and the ABC model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.Publication Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing(The Rockefeller University Press, 2017) Tang, Qin; Iyer, Sowmya; Lobbardi, Riadh; Moore, John C.; Chen, Huidong; Lareau, Caleb; Hebert, Christine; Shaw, McKenzie L.; Neftel, Cyril; Suva, Mario; Ceol, Craig J.; Bernards, Andre; Aryee, Martin; Pinello, Luca; Drummond, Iain; Langenau, DavidRecent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish.