DHX36 regulates the level of G4-DNA at the Gphn and Chl1 locus. Integrated genome browser (IGV) plot of G4-DNA IP with infusions of either DHX36 shRNA or SC shRNA into the mPFC, as well as DHX36 IP for (A) Chl1 loci and (B) Gphn loci. C, Control experiments with FLAG-only controls were performed alongside G4-IP on SC and DHX36 shRNA-treated EXT animals. Analysis at the Chl1 site revealed that there was a significantly higher enrichment in the DHX36-treated G4-IPs but not DHX36-treated FLAG controls (F(3,17) = 10.68, ***p < 0.001; Dunnett's post hoc test; FLAG SC shRNA vs FLAG DHX36 shRNA, p = 0.9934; FLAG-G4 SC shRNA vs G4-IP SC shRNA, **p = 0.093; FLAG-G4 SC shRNA vs G4-IP DHX36 shRNA, ***p = 0.0007). Similarly, there was a significant enrichment at the Gphn site only for the G4-IP, not FLAG control (F(3,17) = 4.900, *p < 0.01; Dunnett's post hoc test; FLAG SC shRNA vs FLAG DHX36 shRNA, p = 0.9480; FLAG-G4 SC shRNA vs G4-IP SC shRNA, p = 0.4913; FLAG-G4 SC shRNA vs G4-IP DHX36 shRNA, **p = 0.0072). SC versus DHX36 shRNA-treated animals that underwent extinction training followed by G4-IP with and without reverse crosslinking were also collected. Without reverse crosslinking, FAIRE allows for the assessment of histone- and protein-free regions. Assessment of the Chl1 gene revealed that much of the G4 peak had protein bound as the signal decreased and was significantly increased following DHX36 shRNA (F(3,25) = 10.32, ***p < 0.001; Dunnett's post hoc test; G4-IP SC shRNA vs FAIRE-G4 SC shRNA, p = 0.4978; G4-IP SC shRNA vs FAIRE-G4 DHX36 shRNA, p = 0.4531; G4-IP SC shRNA vs G4-IP DHX shRNA, **p = 0.0098). D, Although the Gphn G4 peak appears to be mainly protein free as there was no decrease in signal in the FAIRE groups, there was also a significant increase in G4 following DHX36 knockdown (F(3,25) = 1.759, p > 0.05; Dunnett's post hoc test; G4-IP SC shRNA vs FAIRE-G4 SC shRNA, p = 0.9403; G4-IP SC shRNA vs FAIRE-G4 DHX36 shRNA, p = 0.9403; G4-IP SC shRNA vs G4-IP DHX shRNA, *p = 0.0234). E, In Arc+/NeuN+ cells extracted from the mPFC after fear and extinction training, there was a significant increase in G4 signal at the Gphn locus during extinction (F(4,11) = 4.290, *p < 0.05; Dunnett's post hoc test; RC 10 vs EXT 30, *p = 0.0133). F, There was also a significant increase at this locus for RNA from this Gphn G4 locus in unsorted cells derived from the mPFC following extinction training (F(4,31) = 1.232, *p < 0.05; Dunnett's post hoc test; RC 10 vs EXT 10, *p = 0.0437). G, In Arc+/NeuN+ cells extracted from the mPFC after fear and extinction training, there was also a significant increase in G4 signal at the Chl1 locus during extinction (F(4,11) = 9.907, **p < 0.01; Dunnett's post hoc test; RC 10 vs EXT 30, ***p = 0.0004). H, There was no significant change in RNA expression from the Chl1 G4 locus in unsorted cells derived from the mPFC following extinction training (F(4,31) = 1.989, p = 0.1208).
