Publications by authors named "Vidya Subramanian"

32 Publications

HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes.

Proc Natl Acad Sci U S A 2020 12 21;117(52):33404-33413. Epub 2020 Dec 21.

Broad Institute of MIT and Harvard, Cambridge, MA 02142;

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2010738117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776864PMC
December 2020

Activity-by-contact model of enhancer-promoter regulation from thousands of CRISPR perturbations.

Nat Genet 2019 12 29;51(12):1664-1669. Epub 2019 Nov 29.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases. Yet, we still do not know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-019-0538-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886585PMC
December 2019

Optimal-Transport Analysis of Single-Cell Gene Expression Identifies Developmental Trajectories in Reprogramming.

Cell 2019 02 31;176(4):928-943.e22. Epub 2019 Jan 31.

Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Systems Biology Harvard Medical School, Boston, MA 02125, USA. Electronic address:

Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them. We apply the method to reconstruct the landscape of reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18 days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402800PMC
February 2019

The NORAD lncRNA assembles a topoisomerase complex critical for genome stability.

Nature 2018 09 27;561(7721):132-136. Epub 2018 Aug 27.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex-which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)-that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression-which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0453-zDOI Listing
September 2018

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope.

Cell Host Microbe 2018 05;23(5):618-627.e6

Department of Biological Engineering, Koch Institute of Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Infectious Diseases Interdisciplinary Research Group, Singapore-MIT Alliance for Research & Technology, Singapore, Singapore. Electronic address:

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2018.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018055PMC
May 2018

An inter-residue network model to identify mutational-constrained regions on the Ebola coat glycoprotein.

Sci Rep 2017 04 11;7:45886. Epub 2017 Apr 11.

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

Recently, progress has been made in the development of vaccines and monoclonal antibody cocktails that target the Ebola coat glycoprotein (GP). Based on the mutation rates for Ebola virus given its natural sequence evolution, these treatment strategies are likely to impose additional selection pressure to drive acquisition of mutations in GP that escape neutralization. Given the high degree of sequence conservation among GP of Ebola viruses, it would be challenging to determine the propensity of acquiring mutations in response to vaccine or treatment with one or a cocktail of monoclonal antibodies. In this study, we analyzed the mutability of each residue using an approach that captures the structural constraints on mutability based on the extent of its inter-residue interaction network within the three-dimensional structure of the trimeric GP. This analysis showed two distinct clusters of highly networked residues along the GP-GP interface, part of which overlapped with epitope surfaces of known neutralizing antibodies. This network approach also permitted us to identify additional residues in the network of the known hotspot residues of different anti-Ebola antibodies that would impact antibody-epitope interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep45886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387726PMC
April 2017

Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units.

Sci Adv 2016 08 3;2(8):e1501429. Epub 2016 Aug 3.

Department of Mechanical Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA.; BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore.

Motor units are the fundamental elements responsible for muscle movement. They are formed by lower motor neurons and their muscle targets, synapsed via neuromuscular junctions (NMJs). The loss of NMJs in neurodegenerative disorders (such as amyotrophic lateral sclerosis or spinal muscle atrophy) or as a result of traumatic injuries affects millions of lives each year. Developing in vitro assays that closely recapitulate the physiology of neuromuscular tissues is crucial to understand the formation and maturation of NMJs, as well as to help unravel the mechanisms leading to their degeneration and repair. We present a microfluidic platform designed to coculture myoblast-derived muscle strips and motor neurons differentiated from mouse embryonic stem cells (ESCs) within a three-dimensional (3D) hydrogel. The device geometry mimics the spinal cord-limb physical separation by compartmentalizing the two cell types, which also facilitates the observation of 3D neurite outgrowth and remote muscle innervation. Moreover, the use of compliant pillars as anchors for muscle strips provides a quantitative functional readout of force generation. Finally, photosensitizing the ESC provides a pool of source cells that can be differentiated into optically excitable motor neurons, allowing for spatiodynamic, versatile, and noninvasive in vitro control of the motor units.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.1501429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972469PMC
August 2016

Discovery and validation of sub-threshold genome-wide association study loci using epigenomic signatures.

Elife 2016 05 10;5. Epub 2016 May 10.

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

Genetic variants identified by genome-wide association studies explain only a modest proportion of heritability, suggesting that meaningful associations lie 'hidden' below current thresholds. Here, we integrate information from association studies with epigenomic maps to demonstrate that enhancers significantly overlap known loci associated with the cardiac QT interval and QRS duration. We apply functional criteria to identify loci associated with QT interval that do not meet genome-wide significance and are missed by existing studies. We demonstrate that these 'sub-threshold' signals represent novel loci, and that epigenomic maps are effective at discriminating true biological signals from noise. We experimentally validate the molecular, gene-regulatory, cellular and organismal phenotypes of these sub-threshold loci, demonstrating that most sub-threshold loci have regulatory consequences and that genetic perturbation of nearby genes causes cardiac phenotypes in mouse. Our work provides a general approach for improving the detection of novel loci associated with complex human traits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.10557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862755PMC
May 2016

H2A.Z.1 Monoubiquitylation Antagonizes BRD2 to Maintain Poised Chromatin in ESCs.

Cell Rep 2016 Feb 21;14(5):1142-1155. Epub 2016 Jan 21.

Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA. Electronic address:

Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in embryonic stem cells (ESCs) to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 monoubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1(K3R3)). We show that H2A.Z.1(K3R3) is properly incorporated at target promoters in murine ESCs (mESCs), but loss of monoubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member BRD2, are enriched in H2A.Z.1 chromatin. We further show that BRD2 is gained at de-repressed promoters in H2A.Z.1(K3R3) mESCs, whereas BRD2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and BRD2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.12.100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764991PMC
February 2016

Chromatin Dynamics and the RNA Exosome Function in Concert to Regulate Transcriptional Homeostasis.

Cell Rep 2015 Nov 12;13(8):1610-22. Epub 2015 Nov 12.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

The histone variant H2A.Z is a hallmark of nucleosomes flanking promoters of protein-coding genes and is often found in nucleosomes that carry lysine 56-acetylated histone H3 (H3-K56Ac), a mark that promotes replication-independent nucleosome turnover. Here, we find that H3-K56Ac promotes RNA polymerase II occupancy at many protein-coding and noncoding loci, yet neither H3-K56Ac nor H2A.Z has a significant impact on steady-state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on RNA levels are revealed only in the absence of the nuclear RNA exosome. H2A.Z is also necessary for the expression of divergent, promoter-proximal noncoding RNAs (ncRNAs) in mouse embryonic stem cells. Finally, we show that H2A.Z functions with H3-K56Ac to facilitate formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher-order chromatin structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.10.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662874PMC
November 2015

A broadly neutralizing human monoclonal antibody is effective against H7N9.

Proc Natl Acad Sci U S A 2015 Sep 17;112(35):10890-5. Epub 2015 Aug 17.

Visterra, Inc., Cambridge, MA 02139;

Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (-24 h) or double-dose (-12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (-12 h) combined with oseltamivir at 50 mg/kg (-12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1502374112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568252PMC
September 2015

H2A.Z: a molecular rheostat for transcriptional control.

F1000Prime Rep 2015 5;7:01. Epub 2015 Jan 5.

Department of Biology, Massachusetts Institute of Technology 77 Massachusetts Avenue, Cambridge, MA 02139 USA.

The replacement of nucleosomal H2A with the histone variant H2A.Z is critical for regulating DNA-mediated processes across eukaryotes and for early development of multicellular organisms. How this variant performs these seemingly diverse roles has remained largely enigmatic. Here, we discuss recent mechanistic insights that have begun to reveal how H2A.Z functions as a molecular rheostat for gene control. We focus on specific examples in metazoans as a model for understanding how H2A.Z integrates information from histone post-translational modifications, other histone variants, and transcription factors (TFs) to regulate proper induction of gene expression programs in response to cellular cues. Finally, we propose a general model of how H2A.Z incorporation regulates chromatin states in diverse processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12703/P7-01DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4311278PMC
February 2015

Broadly neutralizing influenza hemagglutinin stem-specific antibody CR8020 targets residues that are prone to escape due to host selection pressure.

Cell Host Microbe 2014 May;15(5):644-51

Department of Biological Engineering, Skolkovo-MIT Center for Biomedical Engineering, Koch Institute of Integrative Cancer Research, Infectious Diseases Interdisciplinary Research Group, and Singapore-MIT Alliance for Research and Technology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

Broadly neutralizing antibodies (bNAb) that target a conserved region of a viral antigen hold significant therapeutic promise. CR8020 is a bNAb that targets the stem region of influenza A virus (IAV) hemagglutinin (HA). CR8020 is currently being evaluated for prophylactic use against group 2 IAVs in phase II studies. Structural and computational analyses reported here indicate that CR8020 targets HA residues that are prone to antigenic drift and host selection pressure. Critically, CR8020 escape mutation is seen in certain H7N9 viruses from recent outbreaks. Furthermore, the ability of the bNAb Fc region to effectively engage activating Fcγ receptors (FCγR) is essential for antibody efficacy. In this regard, our data indicate that the membrane could sterically hinder the formation of HA-CR8020-FcγRIIa/HA-IgG-FcγRIIIa ternary complexes. Altogether, our analyses suggest that epitope mutability and accessibility to immune complex assembly are important attributes to consider when evaluating bNAb candidates for clinical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2014.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258880PMC
May 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1003725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749939PMC
March 2014

Modular glycosphere assays for high-throughput functional characterization of influenza viruses.

BMC Biotechnol 2013 Apr 15;13:34. Epub 2013 Apr 15.

Singapore-MIT Alliance for Research and Technology, Singapore 138602, Singapore.

Background: The ongoing global efforts to control influenza epidemics and pandemics require high-throughput technologies to detect, quantify, and functionally characterize viral isolates. The 2009 influenza pandemic as well as the recent in-vitro selection of highly transmissible H5N1 variants have only increased existing concerns about emerging influenza strains with significantly enhanced human-to-human transmissibility. High-affinity binding of the virus hemagglutinin to human receptor glycans is a highly sensitive and stringent indicator of host adaptation and virus transmissibility. The surveillance of receptor-binding characteristics can therefore provide a strong additional indicator for the relative hazard imposed by circulating and newly emerging influenza strains.

Results: Streptavidin-coated microspheres were coated with selected biotinylated glycans to mimic either human or avian influenza host-cell receptors. Such glycospheres were used to selectively capture influenza virus of diverse subtypes from a variety of samples. Bound virus was then detected by fluorescently labelled antibodies and analyzed by quantitative flow cytometry. Recombinant hemagglutinin, inactivated virus, and influenza virions were captured and analyzed with regards to receptor specificity over a wide range of analyte concentration. High-throughput analyses of influenza virus produced dose-response curves that allow for functional assessment of relative receptor affinity and thus transmissibility.

Conclusions: Modular glycosphere assays for high-throughput functional characterization of influenza viruses introduce an important tool to augment the surveillance of clinical and veterinarian influenza isolates with regards to receptor specificity, host adaptation, and virus transmissibility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1472-6750-13-34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3751502PMC
April 2013

Expanded granulocyte/monocyte compartment in myeloid-specific triple FoxO knockout increases oxidative stress and accelerates atherosclerosis in mice.

Circ Res 2013 Mar 18;112(7):992-1003. Epub 2013 Feb 18.

Naomi Berrie Diabetes Center, 1150 St Nicholas Ave, Russ Berrie Pavilion Room 238, NY 10032, USA.

Rationale: Increased neutrophil and monocyte counts are often associated with an increased risk of atherosclerosis, but their relationship to insulin sensitivity is unknown.

Objective: To investigate the contribution of forkhead transcription factors (FoxO) in myeloid cells to neutrophil and monocyte counts, atherosclerosis, and systemic insulin sensitivity.

Methods And Results: Genetic ablation of the 3 genes encoding FoxO isoforms 1, 3a, and 4, in myeloid cells resulted in an expansion of the granulocyte/monocyte progenitor compartment and was associated with increased atherosclerotic lesion formation in low-density lipoprotein receptor knockout mice. In vivo and ex vivo studies indicate that FoxO ablation in myeloid cells increased generation of reactive oxygen species. Accordingly, treatment with the antioxidant N-acetyl-l-cysteine reversed the phenotype, normalizing atherosclerosis.

Conclusions: Our data indicate that myeloid cell proliferation and oxidative stress can be modulated via the FoxO branch of insulin receptor signaling, highlighting a heretofore-unknown link between insulin sensitivity and leukocytosis that can affect the predisposition to atherosclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.112.300749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736810PMC
March 2013

The PAD region in the mycobacterial DinB homologue MsPolIV exhibits positional heterogeneity.

Acta Crystallogr D Biol Crystallogr 2012 Aug 17;68(Pt 8):960-7. Epub 2012 Jul 17.

National Centre for Biological Sciences (NCBS-TIFR), UAS-GKVK Campus, Bellary Road, Bangalore 560 065, India.

Y-family DNA polymerases (dPols) have evolved to carry out translesion bypass to rescue stalled replication; prokaryotic members of this family also participate in the phenomenon of adaptive mutagenesis to relieve selection pressure imposed by a maladapted environment. In this study, the first structure of a member of this family from a prokaryote has been determined. The structure of MsPolIV, a Y-family dPol from Mycobacterium smegmatis, shows the presence of the characteristic finger, palm and thumb domains. Surprisingly, the electron-density map of the intact protein does not show density for the PAD region that is unique to members of this family. Analysis of the packing of the molecules in the crystals showed the existence of large solvent-filled voids in which the PAD region could be located in multiple conformations. In line with this observation, analytical gel-filtration and dynamic light-scattering studies showed that MsPolIV undergoes significant compaction upon DNA binding. The PAD region is known to insert into the major groove of the substrate DNA and to play a major role in shaping the active site. Comparison with structures of other Y-family dPols shows that in the absence of tertiary contacts between the PAD domain and the other domains this region has the freedom to adopt multiple orientations. This structural attribute of the PAD will allow these enzymes to accommodate the alterations in the width of the DNA double helix that are necessary to achieve translesion bypass and adaptive mutagenesis and will also allow regulation of their activity to prevent adventitious error-prone DNA synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S0907444912017623DOI Listing
August 2012

The many faces of the YopM effector from plague causative bacterium Yersinia pestis and its implications for host immune modulation.

Innate Immun 2011 Dec 10;17(6):548-57. Epub 2010 Aug 10.

Harvard-MIT Division of Health Sciences and Technology, Koch Institute for Integrative Cancer Research, and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

The Yersinia outer protein (Yop) M effector from the Yersinia pestis bacterium is well-known for being a critical virulence determinant; however, structural insight vis-à-vis its role in Y. pestis pathogenesis has been elusive. Here, we investigate the intact sequence of the YopM protein through our recently developed fold identification and homology modeling tools, and analyze the immune modulatory potential of its constituent domains. We identify a putative novel E3 ligase (NEL) domain towards the C-terminal tail of YopM and characterize its active site, to show that YopM could function as an autoregulated bacterial type E3 ubiquitin ligase. We further identify unreported NEL domains in several other bacteria and note remarkable similarity in sequence, structure, surface, and electrostatics for the family of NEL-containing bacterial effectors that suggests conserved function and potentially similar host targets for these proteins. Based on these observations and recent empirical evidence for degradation of the human proteins HLA-DR, thioredoxin, and NEMO/IKKγ by other members of the NEL-containing bacterial family, we discuss the potential for YopM to modulate a wide spectrum of immune signal transduction pathways. The key immune modulatory effects highlighted are suppression of MHC class II antigen presentation, dampening of nuclear factor (NF)-κB mediated inflammatory response, and intonation of mitogen-activated protein kinase (MAPK) signaling. Additionally, our analysis of the modeled YopM LRR domain reveals structural features akin to the Toll-like receptor 4 (TLR4) LRR motif. We propose that YopM LRR could be a 'molecular mimic' of TLR4 LRR, permitting reduced immunogenicity and potentially mitigating bacterial lipopolysaccharide surveillance of the innate immune system. Our identification and characterization of the YopM NEL domain, taken together with our analysis of the YopM LRR domain, provides plausible insight into subversion of host immunity by Y. pestis YopM and perhaps could set the stage for design of new therapeutic opportunities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1753425910377099DOI Listing
December 2011

Methods to characterize the effect of DNA-modifying compounds on nucleosomal DNA.

Methods Mol Biol 2010 ;613:173-92

Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA.

Eukaryotic DNA forms a complex with an equal mass of proteins to form chromatin. To fully understand the action of DNA-reactive antitumor antibiotics in the cell, their effect must be studied in a chromatin context. In particular, it is of interest to investigate how the distortion of DNA, in the context of a nucleosome, affects the action of drugs with either monoalkylation or crosslinking activity, and how modified DNA is assembled into chromatin. Here, we present experimental approaches that allow one to compare the effect of such drugs on free DNA and nucleosomes. We find significant differences that likely arise from the different geometry of nucleosomal DNA compared to free DNA and also find that drug-mediated DNA crosslinking affects nucleosome assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-60327-418-0_11DOI Listing
February 2010

CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase.

J Lipid Res 2010 Apr 2;51(4):709-19. Epub 2009 Oct 2.

Rutgers Center for Lipid Research, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA.

Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an alpha/beta-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M001917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2842141PMC
April 2010

Screening for novel regulators of embryonic stem cell identity.

Cell Stem Cell 2009 May;4(5):377-8

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

Two recent studies, including one in this issue of Cell Stem Cell, have identified novel regulators of embryonic stem cell (ESC) self-renewal using genome-wide RNAi screens in mouse ESCs (Ding et al., 2009; Hu et al., 2009) and have further expanded the repertoire of factors that regulate ESC identity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2009.04.006DOI Listing
May 2009

Obesity, inflammation, and macrophages.

Nestle Nutr Workshop Ser Pediatr Program 2009 ;63:151-9; discussion 159-62, 259-68

Department of Medicine, Columbia University, New York, NY, USA.

The World Health Organization estimates that since 1980 the prevalence of obesity has increased more than threefold throughout much of the world, and this increase is not limited to developed nations. Indeed, the incidence of obesity is increasing most rapidly among rapidly industrializing countries raising the spectre of a burgeoning epidemic in obesity-associated diseases, including diabetes, dyslipidemia, nonalcoholic fatty liver disease and atherosclerosis. Reducing the rates of obesity and its attendant complications will require both coordinated public health policy and a better understanding of the pathophysiology of obesity. Obesity is associated with low grade chronic inflammation, a common feature of many complications of obesity that appears to emanate in part from adipose tissue. In obese individuals and rodents adipose tissue macrophage accumulation is a critical component in the development of obesity-induced inflammation. The macrophages in adipose tissue are bone marrow-derived and their number is strongly correlated with bodyweight, body mass index and total body fat. The recruited macrophages in adipose tissue express high levels of inflammatory factors that contribute to systemic inflammation and insulin resistance. Interventions aimed at either reducing macrophage numbers or decreasing their inflammatory characteristics improves insulin sensitivity and decreases inflammation. Macrophage accumulation and adipose tissue inflammation are dynamic processes under the control of multiple mechanisms. Investigating the role of macrophages in adipose tissue biology and the mechanisms involved in their recruitment and activation in obesity will provide useful insights for developing therapeutic approaches to treating obesity-induced complications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000209979DOI Listing
May 2009

Perilipin A and the control of triacylglycerol metabolism.

Mol Cell Biochem 2009 Jun 31;326(1-2):15-21. Epub 2008 Dec 31.

Department of Nutritional Sciences, Rutgers, The State University of New Jersey, 96 Lipman Drive, New Brunswick, NJ 08901, USA.

Perilipin A is the most abundant protein associated with the lipid droplets of adipocytes and functions to control both basal and stimulated lipolysis. Under basal or fed conditions, perilipin A shields stored triacylglycerols from cytosolic lipases, thus promoting triacylglycerol storage. When catecholamines bind to cell surface receptors to initiate signals that activate cAMP-dependent protein kinase (PKA), phosphorylated perilipin A facilitates maximal lipolysis. Mutagenesis studies have revealed that central sequences of moderately hydrophobic amino acids are required to target nascent perilipin A to lipid droplets and provide an anchor into the hydrophobic environment of lipid droplets. Sequences of amino acids in the unique carboxyl terminus of perilipin A and those in amino terminal sequences flanking the first hydrophobic stretch are required for the barrier function of perilipin A in promoting triacylglycerol storage. Site-directed mutagenesis studies of serine residues within six PKA consensus sites of perilipin A reveal functions for phosphorylation of at least three of the sites. Phosphorylation of one or more of the serines within three amino terminal PKA sites is required to facilitate hormone-sensitive lipase access to lipid substrates. Phosphorylation of serines within two carboxyl terminal sites is also required for maximal lipolysis. Phosphorylation of serine 492 (site 5) triggers a massive remodeling of lipid droplets, whereby large peri-nuclear lipid droplets fragment into myriad lipid micro-droplets that scatter throughout the cytoplasm. We hypothesize that perilipin A binds accessory proteins to provide assistance in carrying out these functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11010-008-9998-8DOI Listing
June 2009

Alternative M2 activation of Kupffer cells by PPARdelta ameliorates obesity-induced insulin resistance.

Cell Metab 2008 Jun;7(6):496-507

Division of Endocrinology, Metabolism and Gerontology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305-5103, USA.

Macrophage infiltration and activation in metabolic tissues underlie obesity-induced insulin resistance and type 2 diabetes. While inflammatory activation of resident hepatic macrophages potentiates insulin resistance, the functions of alternatively activated Kupffer cells in metabolic disease remain unknown. Here we show that in response to the Th2 cytokine interleukin-4 (IL-4), peroxisome proliferator-activated receptor delta (PPARdelta) directs expression of the alternative phenotype in Kupffer cells and adipose tissue macrophages of lean mice. However, adoptive transfer of PPARdelta(-/-) (Ppard(-/-)) bone marrow into wild-type mice diminishes alternative activation of hepatic macrophages, causing hepatic dysfunction and systemic insulin resistance. Suppression of hepatic oxidative metabolism is recapitulated by treatment of primary hepatocytes with conditioned medium from PPARdelta(-/-) macrophages, indicating direct involvement of Kupffer cells in liver lipid metabolism. Taken together, these data suggest an unexpected beneficial role for alternatively activated Kupffer cells in metabolic syndrome and type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2008.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2587370PMC
June 2008

Effects of photochemically activated alkylating agents of the FR900482 family on chromatin.

Chem Biol 2007 May;14(5):553-63

Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.

Bioreductive alkylating agents are an important class of clinical antitumor antibiotics that crosslink and monoalkylate DNA. Here, we use a synthetic, photochemically activated derivative of FR400482 to investigate the molecular mechanism of this class of drugs in a biologically relevant context. We find that the organization of DNA into nucleosomes effectively protects it against drug-mediated crosslinking, while permitting monoalkylation. This modification has the potential to lead to the formation of covalent crosslinks between chromatin and nuclear proteins. Using in vitro approaches, we found that interstrand crosslinking of free DNA results in a significant decrease in basal and activated transcription. Finally, crosslinked plasmid DNA is inefficiently assembled into chromatin. Our studies suggest pathways for the clinical effectiveness of this class of reagents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2007.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097141PMC
May 2007

Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance.

Nature 2007 Jun 21;447(7148):1116-20. Epub 2007 May 21.

Division of Endocrinology, Metabolism and Gerontology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5103, USA.

Obesity and insulin resistance, the cardinal features of metabolic syndrome, are closely associated with a state of low-grade inflammation. In adipose tissue chronic overnutrition leads to macrophage infiltration, resulting in local inflammation that potentiates insulin resistance. For instance, transgenic expression of Mcp1 (also known as chemokine ligand 2, Ccl2) in adipose tissue increases macrophage infiltration, inflammation and insulin resistance. Conversely, disruption of Mcp1 or its receptor Ccr2 impairs migration of macrophages into adipose tissue, thereby lowering adipose tissue inflammation and improving insulin sensitivity. These findings together suggest a correlation between macrophage content in adipose tissue and insulin resistance. However, resident macrophages in tissues display tremendous heterogeneity in their activities and functions, primarily reflecting their local metabolic and immune microenvironment. While Mcp1 directs recruitment of pro-inflammatory classically activated macrophages to sites of tissue damage, resident macrophages, such as those present in the adipose tissue of lean mice, display the alternatively activated phenotype. Despite their higher capacity to repair tissue, the precise role of alternatively activated macrophages in obesity-induced insulin resistance remains unknown. Using mice with macrophage-specific deletion of the peroxisome proliferator activated receptor-gamma (PPARgamma), we show here that PPARgamma is required for maturation of alternatively activated macrophages. Disruption of PPARgamma in myeloid cells impairs alternative macrophage activation, and predisposes these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. Furthermore, gene expression profiling revealed that downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver leads to decreased insulin sensitivity in these tissues. Together, our findings suggest that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature05894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2587297PMC
June 2007

Hydrophobic sequences target and anchor perilipin A to lipid droplets.

J Lipid Res 2004 Nov 1;45(11):1983-91. Epub 2004 Sep 1.

Department of Nutritional Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA.

Perilipins regulate triacylglycerol storage and hydrolysis in adipocytes. The central 25% of the perilipin A sequence, including three hydrophobic sequences (H1, H2, and H3) and an acidic region, targets and anchors perilipins to lipid droplets. Thus, we hypothesized that H1, H2, and H3 are targeting and anchoring motifs. We now show that deletion of any single hydrophobic sequence or combinations of H1 and H3 or H2 and H3 does not prevent targeting of the mutated perilipin to lipid droplets. In contrast, mutated perilipin lacking H1 and H2 showed reduced targeting, whereas perilipin lacking H1, H2, and H3 targeted poorly to lipid droplets; thus, H3 is a weak targeting signal and either H1 or H2 is required for optimal targeting. Complete elimination of perilipin targeting was observed only when all three hydrophobic sequences were deleted in combination with either the acidic region or N-terminal sequences predicted to form amphipathic beta-strands. Unlike intact perilipin A, mutated perilipin lacking either H1 and H2 or H1, H2, and H3 was released from lipid droplets after alkaline carbonate treatment, suggesting that these forms are loosely associated with lipid droplets. The three hydrophobic sequences play a major role in targeting and anchoring perilipins to lipid droplets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.M400291-JLR200DOI Listing
November 2004

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes.

J Biol Chem 2004 Oct 2;279(40):42062-71. Epub 2004 Aug 2.

Department of Nutritional Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, USA.

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M407462200DOI Listing
October 2004