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 *

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

    Register here

    Also in the developmental biology category:

    Quantitative, real-time, single cell analysis in tissue reveals expression dynamics of neurogenesis

    Cerys S Manning, Veronica Biga, James Boyd, et al.



    Selected by Teresa Rayon

    The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila



    Selected by Clarice Hong

    1

    Optogenetic dissection of mitotic spindle positioning in vivo

    Lars-Eric Fielmich, Ruben Schmidt, Daniel J Dickinson, et al.



    Selected by Angika Basant

    1

    Excitable RhoA dynamics drive pulsed contractions in the early C. elegans embryo.

    Jonathan B Michaux, Francois B Robin, William M McFadden, et al.



    Selected by Sundar Naganathan

    A transition from SoxB1 to SoxE transcription factors is essential for progression from pluripotent blastula cells to neural crest cells

    Elsy Buitrago-Delgado, Elizabeth Schock, Kara Nordin, et al.



    Selected by Amanda Haage

    Moving beyond P values: Everyday data analysis with estimation plots

    Joses Ho, Tayfun Tumkaya, Sameer Aryal, et al.



    Selected by Gautam Dey

    1

    Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages is necessary for functional spinal cord regeneration in zebrafish

    Themistoklis M. Tsarouchas, Daniel Wehner, Leonardo Cavone, et al.



    Selected by Shikha Nayar

    1

    JNK-mediated spindle reorientation in stem cells promotes dysplasia in the aging intestine

    Daniel Hu, Heinrich Jasper



    Selected by Maiko Kitaoka

    ER-to-Golgi trafficking of procollagen in the absence of large carriers.

    Janine McCaughey, Nicola Stevenson, Stephen Cross, et al.



    Selected by Gautam Dey

    1

    Mechanosensitive binding of p120-Catenin at cell junctions regulates E-Cadherin turnover and epithelial viscoelasticity

    K. Venkatesan Iyer, Romina Piscitello-Gómez, Frank Jülicher, et al.



    Selected by Ivana Viktorinová

    A novel mechanism of gland formation in zebrafish involving transdifferentiation of renal epithelial cells and live cell extrusion

    Richard W Naylor, Alan J Davidson



    Selected by Giuliana Clemente

    1

    An intrinsic cell cycle timer terminates limb bud outgrowth

    Joseph Pickering, Kavitha Chinnaiya, Constance A Rich, et al.



    Selected by Ashrifia Adomako-Ankomah

    1

    Fbxw7 is a critical regulator of Schwann cell myelinating potential

    Breanne L Harty, Fernanda Coelho, Sarah D Ackerman, et al.



    Selected by Yen-Chung Chen

    Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene

    Augusto Berrocal, Nicholas C Lammers, Hernan G Garcia, et al.



    Selected by Erik Clark

    TORC1 modulation in adipose tissue is required for organismal adaptation to hypoxia in Drosophila.

    Byoungchun Lee, Elizabeth C Barretto, Savraj S Grewal



    Selected by Sarah Bowling

    The Ly6/uPAR protein Bouncer is necessary and sufficient for species-specific fertilization

    Sarah Herberg, Krista R Gert, Alexander Schleiffer, et al.



    Selected by James Gagnon

    Also in the genomics category:

    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

    Single-cell Map of Diverse Immune Phenotypes Driven by the Tumor Microenvironment

    Elham Azizi, Ambrose J. Carr, George Plitas, et al.



    Selected by Tim Fessenden

    Cell type-specific interchromosomal interactions as a mechanism for transcriptional diversity

    Adan Horta, Kevin Monahan, Lisa Bashkirova, et al.



    Selected by Boyan Bonev

    PDX Finder: A Portal for Patient-Derived tumor Xenograft Model Discovery

    Nathalie Conte, Jeremy Mason, Csaba Halmagyi, et al.



    Selected by Carmen Adriaens

    An atlas of silencer elements for the human and mouse genomes

    Naresh Doni Jayavelu, Ajay Jajodia, Arpit Mishra, et al.



    Selected by Rafael Galupa

    1

    Capturing the onset of PRC2-mediated repressive domain formation

    Ozgur Oksuz, Varun Narendra, Chul-Hwan Lee, et al.



    Selected by Boyan Bonev

    Heterochromatin drives organization of conventional and inverted nuclei

    Martin Falk, Yana Feodorova, Natasha Naumova, et al.



    Selected by Boyan Bonev

    The ancestral animal genetic toolkit revealed by diverse choanoflagellate transcriptomes

    Daniel Richter, Parinaz Fozouni, Michael Eisen, et al.



    Selected by Rafael Galupa

    Genome-wide selection scans integrated with association mapping reveal mechanisms of physiological adaptation across a salinity gradient in killifish

    Reid S. Brennan, Timothy M. Healy, Heather J. Bryant, et al.



    Selected by Andy Turko

    Precise temporal regulation of alternative splicing during neural development

    Sebastien M Weyn-Vanhentenryck, Huijuan Feng, Dmytro Ustianenko, et al.



    Selected by James Gagnon
    Close