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:

    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

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    A direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

    Christopher M Uyehara, Daniel J McKay



    Selected by Natalie Dye

    A metabolic switch from OXPHOS to glycolysis is essential for cardiomyocyte proliferation in the regenerating heart

    Hessel Honkoop, Dennis de Bakker, Alla Aharonov, et al.



    Selected by Andreas van Impel

    1

    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

    Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissue

    Pauline E Jullien, Stefan Grob, Antonin Marchais, et al.



    Selected by Chandra Shekhar Misra

    1

    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

    1

    Force inference predicts local and tissue-scale stress patterns in epithelia

    Weiyuan Kong, Olivier Loison, Pruthvi Chavadimane Shivakumar, et al.



    Selected by Sundar Naganathan

    Embryo geometry drives formation of robust signaling gradients through receptor localization

    Zhechun Zhang, Steven Zwick, Ethan Loew, et al.



    Selected by Diana Pinheiro

    Unlimited genetic switches for cell-type specific manipulation

    Jorge Garcia-Marques, Ching-Po Yang, Isabel Espinosa-Medina, et al.



    Selected by Rafael Almeida

    1

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    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 coordination of terminal differentiation and cell cycle exit is mediated through the regulation of chromatin accessibility

    Yiqin Ma, Daniel J McKay, Laura Buttitta



    Selected by Gabriel Aughey

    1

    Embryo geometry drives formation of robust signaling gradients through receptor localization

    Zhechun Zhang, Steven Zwick, Ethan Loew, et al.



    Selected by Paul Gerald L. Sanchez and Stefano Vianello

    1

    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

    A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis

    Saiko Yoshida, Alja van der Schuren, Maritza van Dop, et al.



    Selected by Martin Balcerowicz

    1

    Also in the genomics category:

    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

    1

    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

    Precise tuning of gene expression output levels in mammalian cells

    Yale S. Michaels, Mike B Barnkob, Hector Barbosa, et al.



    Selected by Tim Fessenden

    1

    Template switching causes artificial junction formation and false identification of circular RNAs

    Chong Tang, Tian Yu, Yeming Xie, et al.



    Selected by Fabio Liberante

    The genomic basis of colour pattern polymorphism in the harlequin ladybird

    Mathieu Gautier, Junichi Yamaguchi, Julien Foucaud, et al.



    Selected by Fillip Port

    Widespread inter-individual gene expression variability in Arabidopsis thaliana

    Sandra Cortijo, Zeynep Aydin, Sebastian Ahnert, et al.



    Selected by Martin Balcerowicz

    Also in the systems biology category:

    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

    A minimal "push-pull" bistability model explains oscillations between quiescent and proliferative cell states.

    Sandeep Krishna, Sunil Laxman



    Selected by Lauren Neves

    1

    Close