Publications by authors named "Mitchell Guttman"

56 Publications

Integrated spatial genomics reveals global architecture of single nuclei.

Nature 2021 02 27;590(7845):344-350. Epub 2021 Jan 27.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies. Because individual cells appear to be highly variable at all these levels, it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.
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http://dx.doi.org/10.1038/s41586-020-03126-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878433PMC
February 2021

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses.

Cell 2020 11 8;183(5):1325-1339.e21. Epub 2020 Oct 8.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA. Electronic address:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.
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http://dx.doi.org/10.1016/j.cell.2020.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543886PMC
November 2020

Publisher Correction: A protein assembly mediates Xist localization and gene silencing.

Nature 2020 Oct;586(7830):E30

Department of Biological Chemistry at the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-020-2790-yDOI Listing
October 2020

High-Resolution Mapping of Multiway Enhancer-Promoter Interactions Regulating Pathogen Detection.

Mol Cell 2020 10 28;80(2):359-373.e8. Epub 2020 Sep 28.

Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, USA; Department of Dermatology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. Electronic address:

Eukaryotic gene expression regulation involves thousands of distal regulatory elements. Understanding the quantitative contribution of individual enhancers to gene expression is critical for assessing the role of disease-associated genetic risk variants. Yet, we lack the ability to accurately link genes with their distal regulatory elements. To address this, we used 3D enhancer-promoter (E-P) associations identified using split-pool recognition of interactions by tag extension (SPRITE) to build a predictive model of gene expression. Our model dramatically outperforms models using genomic proximity and can be used to determine the quantitative impact of enhancer loss on gene expression in different genetic backgrounds. We show that genes that form stable E-P hubs have less cell-to-cell variability in gene expression. Finally, we identified transcription factors that regulate stimulation-dependent E-P interactions. Together, our results provide a framework for understanding quantitative contributions of E-P interactions and associated genetic variants to gene expression.
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http://dx.doi.org/10.1016/j.molcel.2020.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572724PMC
October 2020

A protein assembly mediates Xist localization and gene silencing.

Nature 2020 11 9;587(7832):145-151. Epub 2020 Sep 9.

Department of Biological Chemistry at the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus and binding diverse proteins to achieve X-chromosome inactivation (XCI). The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off. Here we show that the Xist RNA-binding proteins PTBP1, MATR3, TDP-43 and CELF1 assemble on the multivalent E-repeat element of Xist and, via self-aggregation and heterotypic protein-protein interactions, form a condensate in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.
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http://dx.doi.org/10.1038/s41586-020-2703-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644664PMC
November 2020

Xist drives spatial compartmentalization of DNA and protein to orchestrate initiation and maintenance of X inactivation.

Curr Opin Cell Biol 2020 06 11;64:139-147. Epub 2020 Jun 11.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA. Electronic address:

X chromosome inactivation (XCI) is the process whereby one of the X chromosomes in female mammalian cells is silenced to equalize X-linked gene expression with males. XCI depends on the long noncoding RNA Xist, which coats the inactive X chromosome in cis and triggers a cascade of events that ultimately lead to chromosome-wide transcriptional silencing that is stable for the lifetime of an organism. In recent years, the discovery of proteins that interact with Xist have led to new insights into how the initiation of XCI occurs. Nevertheless, there are still various unknowns about the mechanisms by which Xist orchestrates and maintains stable X-linked silencing. Here, we review recent work elucidating the role of Xist and its protein partners in mediating chromosome-wide transcriptional repression, as well as discuss a model by which Xist may compartmentalize proteins across the inactive X chromosome to enable both the initiation and maintenance of XCI.
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http://dx.doi.org/10.1016/j.ceb.2020.04.009DOI Listing
June 2020

Approaches for Understanding the Mechanisms of Long Noncoding RNA Regulation of Gene Expression.

Cold Spring Harb Perspect Biol 2019 12 2;11(12). Epub 2019 Dec 2.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125.

Mammalian genomes encode tens of thousands of long noncoding RNAs (lncRNAs) that have been implicated in a diverse array of biological processes and human diseases. In recent years, the development of new tools for studying lncRNAs has enabled important progress in defining the mechanisms by which Xist and other lncRNAs function. This collective work provides a framework for how to define the mechanisms by which lncRNAs act. This includes defining lncRNA function, identifying and characterizing lncRNA-protein interactions, and lncRNA localization in the cell. In this review, we discuss various experimental approaches for deciphering lncRNA mechanisms and discuss issues and limitations in interpreting these results. We explore what these data can reveal about lncRNA function and mechanism as well as emerging insights into lncRNA biology that have been derived from these studies.
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http://dx.doi.org/10.1101/cshperspect.a032151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886450PMC
December 2019

The bipartite TAD organization of the X-inactivation center ensures opposing developmental regulation of Tsix and Xist.

Nat Genet 2019 06 27;51(6):1024-1034. Epub 2019 May 27.

Institut Curie, CNRS UMR3215, INSERM U934, Paris, France.

The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.
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http://dx.doi.org/10.1038/s41588-019-0412-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551226PMC
June 2019

Phase separation drives X-chromosome inactivation: a hypothesis.

Nat Struct Mol Biol 2019 05;26(5):331-334

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.

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http://dx.doi.org/10.1038/s41594-019-0223-0DOI Listing
May 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.
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http://dx.doi.org/10.1038/s41586-018-0453-zDOI Listing
September 2018

Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus.

Cell 2018 07 7;174(3):744-757.e24. Epub 2018 Jun 7.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA. Electronic address:

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.
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http://dx.doi.org/10.1016/j.cell.2018.05.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548320PMC
July 2018

RAP-MS: A Method to Identify Proteins that Interact Directly with a Specific RNA Molecule in Cells.

Methods Mol Biol 2018 ;1649:473-488

Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Blvd, MC 156-29, Pasadena, CA, 91125, USA.

RNA molecules interact with proteins to perform a variety of functions in living cells. The binding partners of many RNAs, in particular the newly discovered class of long noncoding RNAs (lncRNAs), remain largely unknown. RNA antisense purification coupled with mass spectrometry (RAP-MS) is a method that enables the identification of direct and specific protein interaction partners of a specific RNA molecule. Because RAP-MS uses direct RNA-protein cross-linking methods coupled along with highly denaturing purification conditions, RAP-MS provides a short list of high confidence protein interactors.
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http://dx.doi.org/10.1007/978-1-4939-7213-5_31DOI Listing
June 2018

The 4D nucleome project.

Nature 2017 09;549(7671):219-226

Department of Bioengineering, University of California San Diego, La Jolla, California 92093, USA.

The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.
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http://dx.doi.org/10.1038/nature23884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617335PMC
September 2017

Response to Comment on "Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing".

Science 2017 06;356(6343)

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

Wang question whether Lamin B receptor is required for Xist-mediated silencing because they claim that our cells contain an inversion rather than a deletion. We present evidence that these cells contain a proper deletion and that the confusion is caused by DNA probes used in the experiment. Accordingly, the points raised have no effect on the conclusions in our paper.
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http://dx.doi.org/10.1126/science.aam5439DOI Listing
June 2017

Re-evaluating the foundations of lncRNA-Polycomb function.

EMBO J 2017 04 15;36(8):964-966. Epub 2017 Mar 15.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

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http://dx.doi.org/10.15252/embj.201796796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391134PMC
April 2017

Linking Protein and RNA Function within the Same Gene.

Cell 2017 02;168(5):753-755

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA. Electronic address:

Exposure to ultraviolet light leads to a cell-wide DNA damage response that includes a global reduction in transcription. Williamson et al., identify a protein involved in this process as well as a noncoding RNA produced by alternative processing of RNA transcribed from the same gene that promotes recovery from the repressed state.
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http://dx.doi.org/10.1016/j.cell.2017.02.014DOI Listing
February 2017

Quantitative predictions of protein interactions with long noncoding RNAs.

Nat Methods 2016 12;14(1):5-6

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.

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http://dx.doi.org/10.1038/nmeth.4100DOI Listing
December 2016

Local regulation of gene expression by lncRNA promoters, transcription and splicing.

Nature 2016 11 26;539(7629):452-455. Epub 2016 Oct 26.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.

Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs). A few of these lncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes, and it has been suggested that many other lncRNAs can also act as local regulators. Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes. However, these correlations have been challenging to dissect and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves. For example, some gene promoters have been proposed to have dual functions as enhancers, and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes. Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis. Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour. These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes.
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http://dx.doi.org/10.1038/nature20149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853796PMC
November 2016

Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression.

Nat Rev Mol Cell Biol 2016 12 26;17(12):756-770. Epub 2016 Oct 26.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.

Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples - including Xist, which orchestrates X chromosome inactivation - has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus.
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http://dx.doi.org/10.1038/nrm.2016.126DOI Listing
December 2016

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing.

Science 2016 10 4;354(6311):468-472. Epub 2016 Aug 4.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

The Xist long noncoding RNA orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the three-dimensional (3D) structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here, we show that Xist directly interacts with the Lamin B receptor, an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing by recruiting the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the 3D structure of DNA, enabling Xist and its silencing proteins to spread across the X to silence transcription.
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http://dx.doi.org/10.1126/science.aae0047DOI Listing
October 2016

m(6)A RNA methylation promotes XIST-mediated transcriptional repression.

Nature 2016 09 7;537(7620):369-373. Epub 2016 Sep 7.

Department of Pharmacology, Weill-Cornell Medical College, Cornell University, New York, New York 10065, USA.

The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N-methyladenosine (mA) residues-a reversible base modification of unknown function in long non-coding RNAs. We show that mA formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the mA-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent mA consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an mA methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of mA-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes mA residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of mA. These data reveal a pathway of mA formation and recognition required for XIST-mediated transcriptional repression.
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http://dx.doi.org/10.1038/nature19342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509218PMC
September 2016

A Guide to Packing Your DNA.

Cell 2016 Apr;165(2):259-61

Genetic material is not randomly organized within the nucleus of a cell. How this organization occurs and why it matters are questions that Cell editor Marta Koch posed to Mitchell Guttman, Job Dekker, and Stavros Lomvardas. Excerpts from this Conversation are presented below, and an audio file of the full discussion is available with the article online.
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http://dx.doi.org/10.1016/j.cell.2016.03.039DOI Listing
April 2016

Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).

Nat Methods 2016 06 28;13(6):508-14. Epub 2016 Mar 28.

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

As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity.
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http://dx.doi.org/10.1038/nmeth.3810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887338PMC
June 2016

Evolutionary analysis across mammals reveals distinct classes of long non-coding RNAs.

Genome Biol 2016 Feb 2;17:19. Epub 2016 Feb 2.

Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, 01655, USA.

Background: Recent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation.

Results: We present slncky, a lncRNA discovery tool that produces a high-quality set of lncRNAs from RNA-sequencing data and further uses evolutionary constraint to prioritize lncRNAs that are likely to be functionally important. Our automated filtering pipeline is comparable to manual curation efforts and more sensitive than previously published computational approaches. Furthermore, we developed a sensitive alignment pipeline for aligning lncRNA loci and propose new evolutionary metrics relevant for analyzing sequence and transcript evolution. Our analysis reveals that evolutionary selection acts in several distinct patterns, and uncovers two notable classes of intergenic lncRNAs: one showing strong purifying selection on RNA sequence and another where constraint is restricted to the regulation but not the sequence of the transcript.

Conclusion: Our results highlight that lncRNAs are not a homogenous class of molecules but rather a mixture of multiple functional classes with distinct biological mechanism and/or roles. Our novel comparative methods for lncRNAs reveals 233 constrained lncRNAs out of tens of thousands of currently annotated transcripts, which we make available through the slncky Evolution Browser.
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http://dx.doi.org/10.1186/s13059-016-0880-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4739325PMC
February 2016

The NIH BD2K center for big data in translational genomics.

J Am Med Inform Assoc 2015 Nov 13;22(6):1143-7. Epub 2015 Jul 13.

UC Santa Cruz Genomics Institute, University of California, Santa Cruz, CA, USA Howard Hughes Medical Institute, Bethesda, MD, USA

The world's genomics data will never be stored in a single repository - rather, it will be distributed among many sites in many countries. No one site will have enough data to explain genotype to phenotype relationships in rare diseases; therefore, sites must share data. To accomplish this, the genetics community must forge common standards and protocols to make sharing and computing data among many sites a seamless activity. Through the Global Alliance for Genomics and Health, we are pioneering the development of shared application programming interfaces (APIs) to connect the world's genome repositories. In parallel, we are developing an open source software stack (ADAM) that uses these APIs. This combination will create a cohesive genome informatics ecosystem. Using containers, we are facilitating the deployment of this software in a diverse array of environments. Through benchmarking efforts and big data driver projects, we are ensuring ADAM's performance and utility.
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http://dx.doi.org/10.1093/jamia/ocv047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009913PMC
November 2015

The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.

Nature 2015 May 27;521(7551):232-6. Epub 2015 Apr 27.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA.

Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins--SHARP, SAF-A and LBR--are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome.
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http://dx.doi.org/10.1038/nature14443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516396PMC
May 2015

Simultaneous generation of many RNA-seq libraries in a single reaction.

Nat Methods 2015 Apr 2;12(4):323-5. Epub 2015 Mar 2.

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

Although RNA-seq is a powerful tool, the considerable time and cost associated with library construction has limited its utilization for various applications. RNAtag-Seq, an approach to generate multiple RNA-seq libraries in a single reaction, lowers time and cost per sample, and it produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches.
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http://dx.doi.org/10.1038/nmeth.3313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712044PMC
April 2015

RNA antisense purification (RAP) for mapping RNA interactions with chromatin.

Methods Mol Biol 2015 ;1262:183-97

Broad Institute of Harvard and MIT, 415 Main St., Cambridge, MA, 02142, USA,

RNA-centric biochemical purification is a general approach for studying the functions and mechanisms of noncoding RNAs. Here, we describe the experimental procedures for RNA antisense purification (RAP), a method for selective purification of endogenous RNA complexes from cell extracts that enables mapping of RNA interactions with chromatin. In RAP, the user cross-links cells to fix endogenous RNA complexes and purifies these complexes through hybrid capture with biotinylated antisense oligos. DNA loci that interact with the target RNA are identified using high-throughput DNA sequencing.
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http://dx.doi.org/10.1007/978-1-4939-2253-6_11DOI Listing
September 2015
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