Publications by authors named "Stuart S Levine"

45 Publications

Quantitative mapping of the cellular small RNA landscape with AQRNA-seq.

Nat Biotechnol 2021 Apr 15. Epub 2021 Apr 15.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.

Current next-generation RNA-sequencing (RNA-seq) methods do not provide accurate quantification of small RNAs within a sample, due to sequence-dependent biases in capture, ligation and amplification during library preparation. We present a method, absolute quantification RNA-sequencing (AQRNA-seq), that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for all small RNAs in a sample. Library preparation and data processing were optimized and validated using a 963-member microRNA reference library, oligonucleotide standards of varying length, and RNA blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied during cancer progression, while application to bacterial transfer RNA pools, with the challenges of secondary structure and abundant modifications, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced, tRNA-driven, codon-biased translation. AQRNA-seq thus provides a versatile means to quantitatively map the small RNA landscape in cells.
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http://dx.doi.org/10.1038/s41587-021-00874-yDOI Listing
April 2021

High-throughput Minitaturized RNA-Seq Library Preparation.

J Biomol Tech 2020 12;31(4):151-156

Department of Biology, MIT BioMicro Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Advances in next-generation sequencing technologies have allowed RNA sequencing to become an increasingly time efficient, cost-effective, and accessible tool for genomic research. We present here an automated and miniaturized workflow for RNA library preparation that minimizes reagent usage and processing time required per sample to generate Illumina compatible libraries for sequencing. The reduced-volume libraries show similar behavior to full-scale libraries with comparable numbers of genes detected and reproducible clustering of samples.
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http://dx.doi.org/10.7171/jbt.20-3104-004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566612PMC
December 2020

Cross-Site Evaluation of Commercial Sanger Sequencing Chemistries.

J Biomol Tech 2020 09;31(3):88-93

Genomics Core Facility, Van Andel Institute, Grand Rapids, Michigan, USA.

Sanger sequencing remains an essential tool utilized by researchers. Despite competition from commercial sequencing providers, many academic sequencing core facilities continue to offer these services based on a model of competitive pricing, knowledgeable technical support, and rapid turnaround time. In-house Sanger sequencing remains a viable core service and, until recently, Applied Biosystems BigDye Terminator chemistry was the only commercially available solution for Sanger DNA sequencing on Applied Biosystems (ABI) instruments; however, several new products employing novel dye chemistries and reaction configurations have entered the market. As a result, there is a need to benchmark the performance of these new chemistries on various DNA templates, including difficult-to-sequence templates, and their amenability to commonly employed cost-saving measures, such as dye dilution and reaction miniaturization. To evaluate these new reagents, a study was designed to compare the quality of Sanger sequencing data produced by ABI BigDye and commercially available kits from 2 other vendors using both control and difficult-to-sequence DNA templates under various reaction conditions. This study will serve as a valuable resource to core facilities conducting Sanger sequencing that wish to evaluate the use of an alternative chemistry in their sequencing core.
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http://dx.doi.org/10.7171/jbt.20-3103-002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351326PMC
September 2020

Bioinformatics Core Survey Highlights the Challenges Facing Data Analysis Facilities.

J Biomol Tech 2020 07;31(2):66-73

Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Over the last decade, the cost of -omics data creation has decreased 10-fold, whereas the need for analytical support for those data has increased exponentially. Consequently, bioinformaticians face a second wave of challenges: novel applications of existing approaches (, single-cell RNA sequencing), integration of -omics data sets of differing size and scale (, spatial transcriptomics), as well as novel computational and statistical methods, all of which require more sophisticated pipelines and data management. Nonetheless, bioinformatics cores are often asked to operate under primarily a cost-recovery model, with limited institutional support. Seeing the need to assess bioinformatics core operations, the Association of Biomolecular Resource Facilities Genomics Bioinformatics Research Group conducted a survey to answer questions about staffing, services, financial models, and challenges to better understand the challenges bioinformatics core facilities are currently faced with and will need to address going forward. Of the respondent groups, we chose to focus on the survey data from smaller cores, which made up the majority. Although all cores indicated similar challenges in terms of changing technologies and analysis needs, small cores tended to have the added challenge of funding their operations largely through cost-recovery models with heavy administrative burdens.
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http://dx.doi.org/10.7171/jbt.20-3102-005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192196PMC
July 2020

Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.

J Biomol Tech 2020 07;31(2):47-56

Interdisciplinary Center for Biotechnology Research Gene Expression and Genotyping, University of Florida, Gainsville, Florida, USA.

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs . Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.
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http://dx.doi.org/10.7171/jbt.20-3102-001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953595PMC
July 2020

SPRI Beads-based Size Selection in the Range of 2-10kb.

J Biomol Tech 2020 04;31(1):7-10

Department of Biology, BioMicro Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Application of solid-phase reversible immobilization (SPRI) beads for size selection in molecular biology should be expanded, in light of the property of the beads to accommodate to high MW intervals of DNA fragment size selection, depending on composition of bead-suspension buffer. Here we show how the conventional size selection interval of 150-800 bp be shifted to 1.5-7 Kbp with by adjusting the concentration of NaCl in the stock suspension buffer. The MW capacity of SPRI beads also change when NaCl replaced with other cations and when the concentration of polyethylene glycol (PEG) 8000 is decreased. Testing the limits of SPRI beads revealed cuts as high as 10 Kbp are possible for some salt/PEG combinations of modified SPRI beads-suspension buffers.
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http://dx.doi.org/10.7171/jbt.20-3101-002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944320PMC
April 2020

Single-cell transcriptomic profiling of the aging mouse brain.

Nat Neurosci 2019 10 24;22(10):1696-1708. Epub 2019 Sep 24.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.
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http://dx.doi.org/10.1038/s41593-019-0491-3DOI Listing
October 2019

Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.

Cell 2019 08;178(5):1115-1131.e15

Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital Boston and Harvard Medical School, Boston, MA 02114, USA; Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (βOHB), distinguishes self-renewing Lgr5 stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes βOHB levels in Lgr5 ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous βOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, βOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through βOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of βOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.
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http://dx.doi.org/10.1016/j.cell.2019.07.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732196PMC
August 2019

H3K27me3-mediated silencing of structural genes is required for zebrafish heart regeneration.

Development 2019 10 9;146(19). Epub 2019 Oct 9.

Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA

Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3, that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3-expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity.
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http://dx.doi.org/10.1242/dev.178632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803378PMC
October 2019

HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis.

Elife 2019 04 29;8. Epub 2019 Apr 29.

Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, United States.

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis.
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http://dx.doi.org/10.7554/eLife.46976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533081PMC
April 2019

Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription.

Cell Chem Biol 2019 05 14;26(5):711-723.e14. Epub 2019 Mar 14.

Division of Oncology, Departments of Medicine and Pathology Stanford School of Medicine, Stanford, CA 94305, USA.

The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in vivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.
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http://dx.doi.org/10.1016/j.chembiol.2019.02.009DOI Listing
May 2019

Global transcriptional regulation of innate immunity by ATF-7 in C. elegans.

PLoS Genet 2019 02 21;15(2):e1007830. Epub 2019 Feb 21.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

The nematode Caenorhabditis elegans has emerged as a genetically tractable animal host in which to study evolutionarily conserved mechanisms of innate immune signaling. We previously showed that the PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates innate immunity of C. elegans through phosphorylation of the CREB/ATF bZIP transcription factor, ATF-7. Here, we have undertaken a genomic analysis of the transcriptional response of C. elegans to infection by Pseudomonas aeruginosa, combining genome-wide expression analysis by RNA-seq with ATF-7 chromatin immunoprecipitation followed by sequencing (ChIP-Seq). We observe that PMK-1-ATF-7 activity regulates a majority of all genes induced by pathogen infection, and observe ATF-7 occupancy in regulatory regions of pathogen-induced genes in a PMK-1-dependent manner. Moreover, functional analysis of a subset of these ATF-7-regulated pathogen-induced target genes supports a direct role for this transcriptional response in host defense. The genome-wide regulation through PMK-1- ATF-7 signaling reveals a striking level of control over the innate immune response to infection through a single transcriptional regulator.
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http://dx.doi.org/10.1371/journal.pgen.1007830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400416PMC
February 2019

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

BMC Genomics 2018 Mar 15;19(1):199. Epub 2018 Mar 15.

MIT BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA, USA.

Background: Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples.

Results: We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency.

Conclusions: These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.
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http://dx.doi.org/10.1186/s12864-018-4585-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389247PMC
March 2018

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

Cell Host Microbe 2018 Mar 22;23(3):395-406.e4. Epub 2018 Feb 22.

Harvard-MIT Department of Health Sciences and Technology, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Boston, MA 02142, USA; Broad Institute, Boston, MA 02142, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; Koch Institute for Integrative Cancer Research, Boston, MA 02142, USA; Department of Medicine, Brigham and Women's Hospital Boston, Boston, MA 02115, USA. Electronic address:

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.
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http://dx.doi.org/10.1016/j.chom.2018.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048090PMC
March 2018

Host proteostasis modulates influenza evolution.

Elife 2017 09 26;6. Epub 2017 Sep 26.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, United States.

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host-pathogen interactions that shape viral evolution, and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance.
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http://dx.doi.org/10.7554/eLife.28652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614556PMC
September 2017

The Hox proteins Ubx and AbdA collaborate with the transcription pausing factor M1BP to regulate gene transcription.

EMBO J 2017 10 4;36(19):2887-2906. Epub 2017 Sep 4.

Aix Marseille Université, CNRS, IBDM, UMR 7288, Marseille, France

In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Hox transcription factors control transcription, we uncovered a molecular mechanism that increases productive transcription. We found that the Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. We found that AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription.
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http://dx.doi.org/10.15252/embj.201695751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623858PMC
October 2017

Mutational spectra of aflatoxin B in vivo establish biomarkers of exposure for human hepatocellular carcinoma.

Proc Natl Acad Sci U S A 2017 04 28;114(15):E3101-E3109. Epub 2017 Mar 28.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139;

Aflatoxin B (AFB) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB, and, as such, is an early detection metric for AFB-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.
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http://dx.doi.org/10.1073/pnas.1700759114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393230PMC
April 2017

Monitoring Error Rates In Illumina Sequencing.

J Biomol Tech 2016 12 16;27(4):125-128. Epub 2016 Sep 16.

BioMicro Center, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Guaranteeing high-quality next-generation sequencing data in a rapidly changing environment is an ongoing challenge. The introduction of the Illumina NextSeq 500 and the depreciation of specific metrics from Illumina's Sequencing Analysis Viewer (SAV; Illumina, San Diego, CA, USA) have made it more difficult to determine directly the baseline error rate of sequencing runs. To improve our ability to measure base quality, we have created an open-source tool to construct the Percent Perfect Reads (PPR) plot, previously provided by the Illumina sequencers. The PPR program is compatible with HiSeq 2000/2500, MiSeq, and NextSeq 500 instruments and provides an alternative to Illumina's quality value (Q) scores for determining run quality. Whereas Q scores are representative of run quality, they are often overestimated and are sourced from different look-up tables for each platform. The PPR's unique capabilities as a cross-instrument comparison device, as a troubleshooting tool, and as a tool for monitoring instrument performance can provide an increase in clarity over SAV metrics that is often crucial for maintaining instrument health. These capabilities are highlighted.
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http://dx.doi.org/10.7171/jbt.16-2704-002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026502PMC
December 2016

RNA polymerase II promoter-proximal pausing in mammalian long non-coding genes.

Genomics 2016 08 16;108(2):64-77. Epub 2016 Jul 16.

Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC 27705, USA.

Mammalian genomes encode a large number of non-coding RNAs (ncRNAs) that greatly exceed mRNA genes. While the physiological and pathological roles of ncRNAs have been increasingly understood, the mechanisms of regulation of ncRNA expression are less clear. Here, our genomic study has shown that a significant number of long non-coding RNAs (lncRNAs, >1000 nucleotides) harbor RNA polymerase II (Pol II) engaged with the transcriptional start site. A pausing and transcriptional elongation factor for protein-coding genes, tripartite motif-containing 28 (TRIM28) regulates the transcription of a subset of lncRNAs in mammalian cells. In addition, the majority of lncRNAs in human and murine cells regulated by Pol II promoter-proximal pausing appear to function in stimulus-inducible biological pathways. Our findings suggest an important role of Pol II pausing for the transcription of mammalian lncRNA genes.
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http://dx.doi.org/10.1016/j.ygeno.2016.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524647PMC
August 2016

Transportable, Chemical Genetic Methodology for the Small Molecule-Mediated Inhibition of Heat Shock Factor 1.

ACS Chem Biol 2016 Jan 19;11(1):200-10. Epub 2015 Nov 19.

Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.

Proteostasis in the cytosol is governed by the heat shock response. The master regulator of the heat shock response, heat shock factor 1 (HSF1), and key chaperones whose levels are HSF1-regulated have emerged as high-profile targets for therapeutic applications ranging from protein misfolding-related disorders to cancer. Nonetheless, a generally applicable methodology to selectively and potently inhibit endogenous HSF1 in a small molecule-dependent manner in disease model systems remains elusive. Also problematic, the administration of even highly selective chaperone inhibitors often has the side effect of activating HSF1 and thereby inducing a compensatory heat shock response. Herein, we report a ligand-regulatable, dominant negative version of HSF1 that addresses these issues. Our approach, which required engineering a new dominant negative HSF1 variant, permits dosable inhibition of endogenous HSF1 with a selective small molecule in cell-based model systems of interest. The methodology allows us to uncouple the pleiotropic effects of chaperone inhibitors and environmental toxins from the concomitantly induced compensatory heat shock response. Integration of our method with techniques to activate HSF1 enables the creation of cell lines in which the cytosolic proteostasis network can be up- or down-regulated by orthogonal small molecules. Selective, small molecule-mediated inhibition of HSF1 has distinctive implications for the proteostasis of both chaperone-dependent globular proteins and aggregation-prone intrinsically disordered proteins. Altogether, this work provides critical methods for continued exploration of the biological roles of HSF1 and the therapeutic potential of heat shock response modulation.
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http://dx.doi.org/10.1021/acschembio.5b00740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891198PMC
January 2016

Next-generation sequencing reveals the biological significance of the N(2),3-ethenoguanine lesion in vivo.

Nucleic Acids Res 2015 Jun 2;43(11):5489-500. Epub 2015 Apr 2.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States

Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N(2),3-ethenoguanine (N(2),3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2'-fluoro-2'-deoxyribose analog of N(2),3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N(2),3-εG and its isomer 1,N(2)-ethenoguanine (1,N(2)-εG) were evaluated in various repair and replication backgrounds. We found that N(2),3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N(2)-εG induces various substitutions and frameshifts. We also found that N(2),3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N(2),3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.
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http://dx.doi.org/10.1093/nar/gkv243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477646PMC
June 2015

Diverse cell stresses induce unique patterns of tRNA up- and down-regulation: tRNA-seq for quantifying changes in tRNA copy number.

Nucleic Acids Res 2014 Dec 27;42(22):e170. Epub 2014 Oct 27.

Department of Biological Engineering and Infectious Diseases Interdisciplinary Research Group, Singapore-MIT Alliance for Research & Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Emerging evidence points to roles for tRNA modifications and tRNA abundance in cellular stress responses. While isolated instances of stress-induced tRNA degradation have been reported, we sought to assess the effects of stress on tRNA levels at a systems level. To this end, we developed a next-generation sequencing method that exploits the paucity of ribonucleoside modifications at the 3'-end of tRNAs to quantify changes in all cellular tRNA molecules. Application of this tRNA-seq method to Saccharomyces cerevisiae identified all 76 expressed unique tRNA species out of 295 coded in the yeast genome, including all isoacceptor variants, with highly precise relative (fold-change) quantification of tRNAs. In studies of stress-induced changes in tRNA levels, we found that oxidation (H2O2) and alkylation (methylmethane sulfonate, MMS) stresses induced nearly identical patterns of up- and down-regulation for 58 tRNAs. However, 18 tRNAs showed opposing changes for the stresses, which parallels our observation of signature reprogramming of tRNA modifications caused by H2O2 and MMS. Further, stress-induced degradation was limited to only a small proportion of a few tRNA species. With tRNA-seq applicable to any organism, these results suggest that translational control of stress response involves a contribution from tRNA abundance.
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http://dx.doi.org/10.1093/nar/gku945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267671PMC
December 2014

Polycomb Repressive Complex 2 regulates lineage fidelity during embryonic stem cell differentiation.

PLoS One 2014 21;9(10):e110498. Epub 2014 Oct 21.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, making it difficult to determine the precise roles of PRC2 during lineage commitment. Moreover, while studies suggest that PRC2 prevents DNA methylation, how these two epigenetic regulators coordinate to regulate lineage programs is poorly understood. Using several PRC2 mutant ESC lines that maintain varying levels of H3K27me3, we found that partial maintenance of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). In contrast, genes that function to specify other lineages failed to be repressed in these cells, suggesting that PRC2 is also necessary for lineage fidelity. We also found that loss of H3K27me3 leads to a modest gain in DNA methylation at PRC2 target regions in both ESCs and in SMNs. Our study demonstrates a critical role for PRC2 in safeguarding lineage decisions and in protecting genes against inappropriate DNA methylation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110498PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204901PMC
June 2015

Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences.

Nat Commun 2014 Jun 5;5:3951. Epub 2014 Jun 5.

Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.
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http://dx.doi.org/10.1038/ncomms4951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4322921PMC
June 2014

H2A.Z acidic patch couples chromatin dynamics to regulation of gene expression programs during ESC differentiation.

PLoS Genet 2013 22;9(8):e1003725. Epub 2013 Aug 22.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z(AP3)) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z(AP3) interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z(AP3) was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z(AP3) ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z(AP3) ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z(AP3) displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z(AP3) mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.
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http://dx.doi.org/10.1371/journal.pgen.1003725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749939PMC
March 2014

Lane-by-lane sequencing using Illumina's Genome Analyzer II.

Biotechniques 2013 May;54(5):265-9

Department of Biology, Koch Institute for Integrative Cancer Research and MIT Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.

Next-generation sequencing has become an essential tool in molecular biology that has been successfully applied to a broad variety of experimental approaches. While several platforms for next-generation sequencing exist, the most commonly used approach is sequencing-by-synthesis, implemented on Illumina's Genome Analyzer II (GAII) and HiSeq2000 systems. A key constraint of these sequencers is the need to run multiple lanes of samples with identical parameters as part of a single flowcell. Here, we present a series of modifications to the Illumina Genome Analyzer II, along with a script generating tool, that allow users to run the GAII in a lane-by-lane manner. Any number of lanes can be run at one time. Repeated use of the same flowcell on multiple sequencing runs does not appreciably reduce the intensity, cluster density, or accuracy of the run. These modifications will enable smaller-scale experiments with unusual design parameters to be run routinely on the GAII.
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http://dx.doi.org/10.2144/000114032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345167PMC
May 2013

Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage.

Cell 2012 Sep 12;151(1):206-20. Epub 2012 Sep 12.

Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, USA.

Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel preactivation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease.
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http://dx.doi.org/10.1016/j.cell.2012.07.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462286PMC
September 2012

Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity.

Proc Natl Acad Sci U S A 2012 Aug 30;109(33):13458-63. Epub 2012 Jul 30.

Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Toxoplasma gondii is a highly successful protozoan parasite that infects all warm-blooded animals and causes severe disease in immunocompromised and immune-naïve humans. It has an unusual global population structure: In North America and Europe, isolated strains fall predominantly into four largely clonal lineages, but in South America there is great genetic diversity and the North American clonal lineages are rarely found. Genetic variation between Toxoplasma strains determines differences in virulence, modulation of host-signaling pathways, growth, dissemination, and disease severity in mice and likely in humans. Most studies on Toxoplasma genetic variation have focused on either a few loci in many strains or low-resolution genome analysis of three clonal lineages. We use whole-genome sequencing to identify a large number of SNPs between 10 Toxoplasma strains from Europe and North and South America. These were used to identify haplotype blocks (genomic regions) shared between strains and construct a Toxoplasma haplotype map. Additional SNP analysis of RNA-sequencing data of 26 Toxoplasma strains, representing global diversity, allowed us to construct a comprehensive genealogy for Toxoplasma gondii that incorporates sexual recombination. These data show that most current isolates are recent recombinants and cannot be easily grouped into a limited number of haplogroups. A complex picture emerges in which some genomic regions have not been recently exchanged between any strains, and others recently spread from one strain to many others.
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http://dx.doi.org/10.1073/pnas.1117047109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421188PMC
August 2012

miR-132, an experience-dependent microRNA, is essential for visual cortex plasticity.

Nat Neurosci 2011 Sep 4;14(10):1240-2. Epub 2011 Sep 4.

Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Using quantitative analyses, we identified microRNAs (miRNAs) that were abundantly expressed in visual cortex and that responded to dark rearing and/or monocular deprivation. The most substantially altered miRNA, miR-132, was rapidly upregulated after eye opening and was delayed by dark rearing. In vivo inhibition of miR-132 in mice prevented ocular dominance plasticity in identified neurons following monocular deprivation and affected the maturation of dendritic spines, demonstrating its critical role in the plasticity of visual cortex circuits.
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http://dx.doi.org/10.1038/nn.2909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183341PMC
September 2011

Mediator and cohesin connect gene expression and chromatin architecture.

Nature 2010 Sep 18;467(7314):430-5. Epub 2010 Aug 18.

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.

Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator-cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.
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http://dx.doi.org/10.1038/nature09380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953795PMC
September 2010
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