For your daily serving of epigenetics brain food, look no further than the entwined spaghetti-like chromatin. Although it may look like a bowl full of chaos, there are distinct domains and long-range genomic interactions that function in a variety of regulatory roles. A team at the University of Pennsylvania (USA) led by Jennifer Phillips-Cremins and Arjun Raj have been stewing on some new techniques to study these interactions, and now they’re dishing out a new solution: light-activated dynamic looping (LADL).
LADL was designed as an inducible optoepigenetic editing system to generate de novo long-range contacts within the genome. The LADL system is contained on two transfectable vectors and can be endlessly modified to join two unique sections of the genome. The system is comprised of four discrete parts:
- The “anchor”, which consists of an enzymatically dead Cas9 (dCas9) linked to the CIBN protein from A. thaliana
- The guide RNAs (gRNAs), designed to target the anchor to the specific genomic regions of interest
- The CRY2 protein from A. thaliana, which is known to interact with CIBN in response to blue light
- Blue light exposure, which induces the CIBN/CRY2 interaction and loop formation
After ensuring that there were no off-target effects, the team went on to test LADL’s capabilities in mouse embryonic stem cells by inducing a novel interaction between the stretch enhancer of Klf4 and the Zpf462 promoter (~800 kb span). The gRNA plasmids were designed to Klf4 and Zpf462, and transfected alongside the anchor plasmid. To determine LADLs efficacy, they used chromosome conformation capture carbon copy (5C) to verify higher-order chromatin structure, and single-molecule RNA-Fluorescence in situ hybridization (FISH) to assess changes in Zpf462 expression.
Here’s what they found:
- With as little as four hours of blue light exposure, LADL successfully generates a de novo contact and loop between the Klf4 enhancer and the Zpf462 promoter, with minimal effects on the endogenous chromatin interactions
- The LADL-induced interaction between the Klf4 enhancer and the Zpf462 promoter results in increased numbers of Zpf462 mRNA transcripts
- Overall, ~2.5 fold increase in Klf4-Zfp462 interaction frequency, and ~1.2 fold increase in Zfp462 gene expression
This system has several benefits over other loop formation methods: a short induction time, blue light rather than small molecule induction, and reversibility. These advantages make the LADL system an exciting new tool for manipulating chromatin architecture and studying the effects on gene expression.