Loop engineering techniques that modulate long-range chromosomal interactions play a pivotal part in deciphering the relationship between 3D genome architecture and function. Now, Kim, Rege et al. present the light-activated dynamic looping (LADL) system, which is based on a synthetic architectural protein that rapidly induces long-range chromatin interactions in response to blue light.
The LADL system comprises two plasmids, one encoding a LADL ‘anchor’ and one encoding sequence-specific CRISPR guide RNAs (gRNAs) designed to target two genomic regions to be joined through loop formation. The LADL anchor is formed of an enzymatically inactive Cas9 (dCas9) that is fused to a truncated version of the CIB1 protein (CIBN; also known as transcription factor bHLH63) from Arabidopsis thaliana. gRNAs guide the LADL anchor to the two desired genomic anchoring sites. The gRNA-encoding plasmid also includes CRY2, whose protein product acts as an inducible bridging factor that joins the two anchoring sites upon activation with blue light (470 nm wavelength) through heterodimerization with CIBN.
Using mouse embryonic stem (ES) cells, the authors compared transfected cells exposed to 24 h of blue light or dark and found no differences in dCas9 and CRY2 transcript levels nor in the expression of pluripotency markers between the two conditions. This finding suggests that transfection and light induction had no impact on transgene expression or the ES cell state. To test the LADL system, the authors chose a ~800-kb-sized region around the Klf4 and Zfp462 genes, reasoning that loop engineering would redirect the interaction between Klf4 and its long-range enhancer to Zfp462. Recruitment of the LADL system to its target locations was measured by chromatin immunoprecipitation followed by quantitative PCR (ChIP–qPCR), which confirmed the presence of dCas9–CIBN at both the Klf4 enhancer element and the Zfp462 promoter under dark conditions.
Blue light activation led to a doubling in intensity of the dCas9–CIBN ChIP signal at the Zfp462 promoter and a slight signal decrease at the Klf4 enhancer. A high-resolution map of long-range chromatin interactions generated by chromosome conformation capture carbon copy (5C) showed the formation of a new contact between Zfp462 and the Klf4 enhancer upon blue light illumination. The loop between Klf4 and its enhancer remained largely unperturbed. Notably, the newly formed loop between Zfp462 and the enhancer was detectable 4 h after the start of the light stimulus, which is substantially faster than current chemical loop engineering approaches. Finally, single-molecule RNA fluorescence in situ hybridization showed a modest increase in Zfp462 expression.
“synthetic architectural protein that rapidly induces long-range chromatin interactions in response to blue light”
While the approach requires improvements to increase the strength of LADL-induced contacts, the method should prove useful for 3D optoepigenetic engineering of chromatin at short time scales.
Kim, J. H. & Rege, M. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019)
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Koch, L. Optoepigenetics for 3D genome engineering. Nat Rev Genet 20, 501 (2019). https://doi.org/10.1038/s41576-019-0154-y