Like any live-action remake, vibrant colors are essential for a new epigenetic editing tool that simultaneously introduces H3K9me3 at hundreds of targeted loci while also allowing for HD visualization of the heterochromatic results.
Now, let’s meet the stars of this show that were cast by a team led by Hanhui Ma from ShanghaiTech University. H3K9me3 is a mark of heterochromatin formation with a role in silencing repeat structures. This histone post-translational modification can be precisely deposited at target loci by catalytically dead Cas9 (dCas9) fused to a KRAB protein domain. The magic of color comes from fluorescent guide RNAs (CRISPRainbow) that allow for DNA visualization. To visualize association with heterochromatin, a HaloTag for the heterochromatin protein HP1α was knocked-in using CRISPR-Cas9.Together, these celebs form EpiGo-KRAB – Epigenetic perturbation induced genome organization-KRAB. This system involves:
First, the bright team identified a target repeat class with 836 copies in the q-arm of chromosome 19. Using ChIP-seq, they confirmed that their target region acquired H3K9me3, successfully inducing epigenetic alterations via EpiGo-KRAB. Next, they examined the spatiotemporal dynamics of the region using live-cell tracking, finding:
- The target region interacts with the heterochromatin protein in EpiGo-KRAB cell lines
- These regions condense and coalesce together over two hours
While these results showed that EpiGo-KRAB was sufficient to induce genome reorganization, the radiant researchers wanted to show that this was a result of methyltransferase action. By knocking down SETDB1, they found a marked reduction in this genome clustering in the EpiGo-KRAB cell lines. Next, the luminous lab mates checked downstream effects of these newly acquired H3K9me3 marks, finding:
- New genome clustering doesn’t result in widespread gene silencing
- Some genes with promoters close to the dCas9 target are repressed, with decreased expression, increased H3K9me3, as well as the loss of H3K4me3 and H3K27ac
- Activity influences H3K9me3 spread – it spreads greatly throughout transcriptionally inactive regions, but very little in active regions
- New H3K9me3 marks induced extensive rearrangements of chromatin compartmentalization (Hi-C)
This EpiGo-KRAB tool is powerful in its ability to track spatiotemporal changes following the addition of new H3K9me3. That we can now observe heterochromatin formation in living cells while imaging the genomic architecture is exciting.
To see the paper in living color, check it out at Genome Biology, December 2020.