So, your therapist says you have boundary issues? Well you’re not alone; CTCF is known to play a critical role in setting the boundaries of topologically associating domains (TADs), but there’s been no therapist to uncover their definitive impact. Thankfully, new research from the lab of Bradley Bernstein at the Harvard Medical School (Boston, USA) provides us with a new tool for digging deep into the methyl sensitive personalities of individual CTCF sites — no psychoanalysis required.
To study the contribution of a specific CTCF site to gene regulation, the talented team set out to disrupt it. It was previously shown that catalytically dead Cas9 (dCas9) fused to the KRAB protein domain can be targeted to a specific locus using a guide RNA, where it catalyzes histone H3 lysine 9 methylation and represses gene expression. The team repurposed this epigenome editing system to target CTCF DNA motifs and displace the CTCF protein.
Here are the details on this useful tool:
- dCas9-KRAB effectively induces H3K9 methylation and displaces CTCF at 8 of the 10 tested sites, with minimal off-target effects
- This effect requires the continuous presence of dCas9-KRAB
- Fusing dCas9 to a DNA methyltransferase (DNMT3A3L) also induces H3K9 methylation and displaces CTCF, but to a lesser degree than dCas9-KRAB
- The effects of dCas9-DNMT3A3L are semi-heritable, such that some level of methylation and CTCF displacement persist throughout serial passaging of cells, even once dCas9-DNMT3A3L is no longer detected
- A combination approach using dCas9-KRAB and dCas9-DNMT3A3L results in the best of both fusions: there is robust H3K9 methylation and CTCF displacement, in a semi-heritable manner
- Some level of CTCF displacement is retained as far as 27 days post-transfection
This tireless team immediately put their newly-made tool to work modeling IDH-mutant gliomas, which are characterized by reduced CTCF binding and insulator dysfunction at the PDGFRA domain boundary. They mimicked the disease-associated CTCF loss in a gliomasphere cell model (GSC6) by targeting dCas9-KRAB to the PDGFRA insulator, resulting in a 95% reduction in CTCF binding.
The effect on TAD interactions was examined using 4C-seq, where they found that there is:
- A significant decrease in the interaction between the insulator and the PDGFRA promoter
- An increased interaction between the PDGFRA promoter and enhancers in neighboring TADs, likely driving a measured increase in PDGFRA expression
These results demonstrate the exciting potential of this approach, not only for expanding our knowledge of insulators in gene expression, but also for understanding their role in disease.