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

Posted on: 10 August 2018

Preprint posted on 18 June 2018

Article now published in Nature Methods at http://dx.doi.org/10.1038/s41592-019-0436-5

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

Categories: developmental biology, genomics, synthetic biology, systems biology

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

doi: https://doi.org/10.1242/prelights.4308

Read preprint