Menu

Close

LADL: Light-activated dynamic looping for endogenous gene expression control

Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, Margaret Dunagin, Aryeh Metzger, Wanfeng Gong, Jonathan Beagan, Arjun Raj, Jennifer E Phillips-Cremins

Preprint posted on June 18, 2018 https://www.biorxiv.org/content/early/2018/06/18/349340

Travelling over long distances: a new study uses blue light to make two (genomic) locations come together – providing an exciting new tool to study gene regulation.

Selected by Ivan Candido-Ferreira

Background

Fine-tuned control of gene expression is key for proper development and homeostasis. In multicellular organisms, the on and off switching of gene activity, as well as when and where this happen, is tightly controlled by DNA elements that can be located hundreds, thousands or millions of base-pairs away from their target genes. These DNA switches, widely known as transcriptional enhancers, break the (genomic) distances by looping within the three dimensional (3D) space of the nucleus, allowing enhancers to communicate with their target genes and control transcription.

However, our ability to manipulate these 3D interactions – especially in their endogenous context, the chromatin – was until recently hampered by the availability of methods that enable the precise editing of the genome or the epigenome. The development of CRISPR/Cas9 tools to precisely edit features of the genome has revolutionised (epi)genome engineering, but these tools are largely deployed to make constitutive changes to the genome. A new preprint from the Cremins lab elegantly tackles this limitation, by creating a new tool that enables the inducibility of 3D interactions by light in mammalian cells.

The preprint

The authors designed a light-activated dynamic looping (LADL) system, which is basically composed of four modules. They first started out by designing a synthetic architectural protein (module 1) by fusing the enzymatically-dead Cas9 (dCas9) to the CIBN protein subunit from Arabidopsis thaliana. By using sequence-specific single-guide RNAs (sgRNAs) (module 2), the dCas9-CIBN proteins bind to specific sequences, which function as anchoring points for the engineered loops. Another protein from A. thaliana, CRY2 (module 3), was used since it has the ability to heterodimerise with CIBN in response to blue light within milliseconds. Upon exposure to blue light (module 4), the LADL system forces the dynamic looping between two genomic positions, allowing scientists to engineer synthetic 3D chromatin interactions.

 

Schematic representation of the LADL system; from Figure 1A of the preprint.

 

As a proof of principle, the authors then delivered their system to mouse embryonic stem (mES) cells, with sgRNAs designed to recognise a cluster of enhancers (also called “super- enhancers”) that in native conditions controls the expression of a known stem cell gene, Klf4. They also delivered sgRNAs recognising the promoter of a gene that is not expressed in mES cells, Zfp462. Upon exposure to blue light, they observed the formation of a novel, synthetic loop together with nascent transcription of Zfp462, suggesting that their system is indeed efficient in editing the 3D genome.

Outlook

The simplicity of the authors’ approach to create novel synthetic loops in an inducible manner is very elegant. Tools for epigenome engineering are key to probe regulatory interactions and have been pivotal for the increasingly more appreciated role of gene regulatory elements in development, homeostasis and evolution. I therefore believe that the ability to test enhancers (and possibly cis-regulatory repressors) in their endogenous context, by performing induced forced-looping assays as the one reported in this study, is likely to provide new insights into the regulation of genes and their misregulation in several diseases such as developmental abnormalities. It may also represent a novel therapeutic strategy for such diseases.

Related Research
Deng, W. et al. Reactivation of developmentally silenced globin genes by forced chromatin looping. Cell 158, 849–860 (2014).

Tags: chromatin, crispr/cas9, enhancers, epigenome engineering, gene regulatory networks, transcription

Posted on: 10th August 2018

Read preprint (No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the developmental biology category:

    The visual system of the genetically tractable crustacean Parhyale hawaiensis: diversification of eyes and visual circuits associated with low-resolution vision

    Ana Patricia Ramos, Ola Gustafsson, Nicolas Labert, et al.



    Selected by Alexa Sadier

    Distinct RhoGEFs activate apical and junctional actomyosin contractility under control of G proteins during epithelial morphogenesis

    Alain Garcia De Las Bayonas, Jean-Marc Philippe, Annemarie C. Lellouch, et al.



    Selected by Ivana Viktorinová

    1

    The Hunchback temporal transcription factor determines motor neuron axon and dendrite targeting in Drosophila

    Austin Q Seroka, Chris Q Doe



    Selected by Abagael Lasseigne

    1

    Preformed Chromatin Topology Assists Transcriptional Robustness of Shh during Limb Development

    Christina Paliou, Philine Guckelberger, Robert Schöpflin, et al.



    Selected by Rafael Galupa

    1

    Molecular Logic of Spinocerebellar Tract Neuron Diversity and Connectivity

    Myungin Baek, Vilas Menon, Thomas Jessell, et al.



    Selected by Yen-Chung Chen

    In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor

    Jacob P. Keller, Jonathan S. Marvin, Haluk Lacin, et al.



    Selected by Stephan Daetwyler

    1

    Active behaviour during early development shapes glucocorticoid reactivity

    Luis A. Castillo-Ramírez, Soojin Ryu, Rodrigo J. De Marco



    Selected by Kathleen Gilmour

    A unicellular relative of animals generates an epithelium-like cell layer by actomyosin-dependent cellularization

    Omaya Dudin, Andrej Ondracka, Xavier Grau-Bové, et al.



    Selected by Paul Gerald L. Sanchez and Stefano Vianello

    1

    Members of the Arabidopsis auxin receptor gene family are essential early in embryogenesis and have broadly overlapping functions

    Michael J Prigge, Nikita Kadakia, Kathleen Greenham, et al.



    Selected by Chandra Shekhar Misra

    1

    The spindle assembly checkpoint functions during early development in non-chordate embryos

    Janet Chenevert, Marianne Roca, Lydia Besnardeau, et al.



    Selected by Maiko Kitaoka

    Over-activation of BMP signaling in neural crest cells precipitates heart outflow tract septation

    Jean Francois Darrigrand, Mariana Valente, Pauline Martinez, et al.



    Selected by Giulia Boezio

    Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

    Shih-Jui Hsu, Emily C. Stow, James R. Simmons, et al.



    Selected by Maiko Kitaoka

    1

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    Dynamic Erasure of Random X-Chromosome Inactivation during iPSC Reprogramming

    Adrian Janiszewski, Irene Talon, Juan Song, et al.



    Selected by Sergio Menchero

    MicroRNA-mediated control of developmental lymphangiogenesis

    Hyun Min Jung, Ciara Hu, Alexandra M Fister, et al.



    Selected by Rudra Nayan Das

    Endogenous CRISPR arrays for scalable whole organism lineage tracing

    James Cotterell, James Sharpe



    Selected by Irepan Salvador-Martinez

    Also in the genomics category:

    Accurate detection of m6A RNA modifications in native RNA sequences

    Huanle Liu, Oguzhan Begik, Morghan C Lucas, et al.



    Selected by Christian Bates

    1

    Crowdfunded whole-genome sequencing of the celebrity cat Lil BUB identifies causal mutations for her osteopetrosis and polydactyly

    Mike Bridavsky, Heiner Kuhl, Arthur Woodruf, et al.



    Selected by Jesus Victorino, Gabriel Aughey

    1

    Prospective, brain-wide labeling of neuronal subclasses with enhancer-driven AAVs

    Lucas T Graybuck, Adriana Sedeño-Cortés, Thuc Nghi Nguyen, et al.



    Selected by Jesus Victorino

    Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

    Arnav Moudgil, Michael N Wilkinson, Xuhua Chen, et al.



    Selected by James Briscoe

    1

    High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue-specificity

    Kaia Mattioli, Pieter-Jan Volders, Chiara Gerhardinger, et al.



    Selected by Clarice Hong

    Reconstruction of the global neural crest gene regulatory network in vivo

    Ruth M Williams, Ivan Candido-Ferreira, Emmanouela Repapi, et al.



    Selected by Hannah Brunsdon

    Charting a tissue from single-cell transcriptomes

    Mor Nitzan, Nikos Karaiskos, Nir Friedman, et al.



    Selected by Irepan Salvador-Martinez

    Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

    Mark E. Lush, Daniel C. Diaz, Nina Koenecke, et al.

    AND

    Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

    Eric D Thomas, David Raible



    Selected by Rudra Nayan Das

    2

    Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan

    Julien Rougot, Naomi D Chrispijn, Marco Aben, et al.



    Selected by Yen-Chung Chen

    1

    The embryonic transcriptome of Arabidopsis thaliana

    Falko Hofmann, Michael A Schon, Michael D Nodine



    Selected by Chandra Shekhar Misra

    1

    Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

    Mridusmita Saikia, Philip Burnham, Sara H Keshavjee, et al.

    AND

    High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

    Mandeep Singh, Ghamdan Al-Eryani, Shaun Carswell, et al.



    Selected by Samantha Seah

    The microbial basis of impaired wound healing: differential roles for pathogens, "bystanders", and strain-level diversification in clinical outcomes

    Lindsay Kalan, Jacquelyn S Meisel, Michael A Loesche, et al.



    Selected by Snehal Kadam

    Comparative analysis of droplet-based ultra-high-throughput single-cell RNA-seq systems

    Xiannian Zhang, Tianqi Li, Feng Liu, et al.



    Selected by Samantha Seah

    PUMILIO hyperactivity drives premature aging of Norad-deficient mice

    Florian Kopp, Mehmet Yalvac, Beibei Chen, et al.



    Selected by Carmen Adriaens

    LCM-seq reveals unique transcriptional adaption mechanisms of resistant neurons in spinal muscular atrophy

    Susanne Nichterwitz, Helena Storvall, Jik Nijssen, et al.

    AND

    Axon-seq decodes the motor axon transcriptome and its modulation in response to ALS

    Jik Nijssen, Julio Cesar Aguila Benitez, Rein Hoogstraaten, et al.



    Selected by Yen-Chung Chen

    LADL: Light-activated dynamic looping for endogenous gene expression control

    Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, et al.



    Selected by Ivan Candido-Ferreira

    Also in the synthetic biology category:

    The autophagic membrane tether ATG2A transfers lipids between membranes

    Shintaro Maeda, Chinatsu Otomo, Takanori Otomo



    Selected by Sandra Malmgren Hill

    Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

    Carol J. Hartley, Charlotte C. Williams, Judith A. Scoble, et al.



    Selected by Zhang-He Goh

    1

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

    Stephanie Cara Bishop, Robert Winefield, Asokan Anbanandam, et al.



    Selected by Zhang-He Goh

    Synthetic pluripotent bacterial stem cells

    Sara Molinari, David L. Shis, James Chappell, et al.



    Selected by Lorenzo Lafranchi

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Variability of bacterial behavior in the mammalian gut captured using a growth-linked single-cell synthetic gene oscillator

    David T Riglar, David L Richmond, Laurent Potvin-Trottier, et al.



    Selected by Meng Zhu

    A direct selection strategy for isolating aptamers with pH-sensitive binding activity

    Chelsea K L Gordon, Michael Eisenstein, H T Soh



    Selected by Zhang-He Goh

    SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

    Jocelyn Y. Kishi, Brian J. Beliveau, Sylvain W. Lapan, et al.



    Selected by Yen-Chung Chen

    Glutamic acid is a carrier for hydrazine during the biosyntheses of fosfazinomycin and kinamycin

    Kwo-Kwang Abraham Wang, Tai L. Ng, Peng Wang, et al.



    Selected by Ellis O'Neill

    Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

    Hannah A. Grunwald, Valentino M. Gantz, Gunnar Poplawski, et al.



    Selected by Rebekah Tillotson

    1

    Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

    Kale Kundert, James E Lucas, Kyle E Watters, et al.



    Selected by Samantha Seah

    Also in the systems biology category:

    Spreading of molecular mechanical perturbations on linear filaments

    Zsombor Balassy, Anne-Marie Lauzon, Lennart Hilbert



    Selected by Lars Hubatsch

    Lineage tracing on transcriptional landscapes links state to fate during differentiation

    Caleb Weinreb, Alejo E Rodriguez-Fraticelli, Fernando D Camargo, et al.



    Selected by Yen-Chung Chen

    1

    Short-range interactions govern cellular dynamics in microbial multi-genotype systems

    Alma Dal Co, Simon van Vliet, Daniel Johannes Kiviet, et al.

    AND

    Rapid microbial interaction network inference in microfluidic droplets

    Ryan H Hsu, Ryan L Clark, Jin Wei Tan, et al.



    Selected by Connor Rosen

    High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue-specificity

    Kaia Mattioli, Pieter-Jan Volders, Chiara Gerhardinger, et al.



    Selected by Clarice Hong

    Variability of bacterial behavior in the mammalian gut captured using a growth-linked single-cell synthetic gene oscillator

    David T Riglar, David L Richmond, Laurent Potvin-Trottier, et al.



    Selected by Meng Zhu

    Charting a tissue from single-cell transcriptomes

    Mor Nitzan, Nikos Karaiskos, Nir Friedman, et al.



    Selected by Irepan Salvador-Martinez

    Large-scale analyses of human microbiomes reveal thousands of small, novel genes and their predicted functions

    Hila Sberro, Nicholas Greenfield, Georgios Pavlopoulos, et al.



    Selected by Ganesh Kadamur

    Symmetry breaking in the embryonic skin triggers a directional and sequential front of competence during plumage patterning

    Richard Bailleul, Carole Desmarquet-Trin Dinh, Magdalena Hidalgo, et al.



    Selected by Alexa Sadier

    RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

    James M Burke, Stephanie L Moon, Evan T Lester, et al.



    Selected by Connor Rosen

    Acquired interbacterial defense systems protect against interspecies antagonism in the human gut microbiome

    Benjamin D. Ross, Adrian J. Verster, Matthew C. Radey, et al.



    Selected by Connor Rosen

    DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

    Joshua A. Weinstein, Aviv Regev, Feng Zhang



    Selected by Theo Sanderson

    2

    The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner

    Federico Germani, Daniel Hain, Denise Sternlicht, et al.



    Selected by Rohan Khadilkar

    LCM-seq reveals unique transcriptional adaption mechanisms of resistant neurons in spinal muscular atrophy

    Susanne Nichterwitz, Helena Storvall, Jik Nijssen, et al.

    AND

    Axon-seq decodes the motor axon transcriptome and its modulation in response to ALS

    Jik Nijssen, Julio Cesar Aguila Benitez, Rein Hoogstraaten, et al.



    Selected by Yen-Chung Chen

    Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

    Sydney M Shaffer, Benjamin L Emert, Ann E. Sizemore, et al.



    Selected by Leighton Daigh

    2

    Conserved phosphorylation hotspots in eukaryotic protein domain families

    Marta J Strumillo, Michaela Oplova, Cristina Vieitez, et al.



    Selected by Gautam Dey

    LADL: Light-activated dynamic looping for endogenous gene expression control

    Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, et al.



    Selected by Ivan Candido-Ferreira
    Close