Figure 3: Chromosomal conformations tagged by TALE^Gad1Dam.

(a) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C’ chromosome conformation capture¹⁹. Position of Amplicon/primer pairs 1–8 (Supplementary Table 2) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. (b) Dam-based 3D genome mapping. TALE^Gad1Dam methylates G^(m)ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G^mATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G^mATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G^mATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G^mATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C¹⁹ and for sequences at TALE target sequence (primer pair 7) in HSV TALE^Gad1Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV^Mef2c-Dam-injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6). (c) DamID quantitative PCR for G^mATC quantification from prefrontal DNA, with primers within 100 kb from TALE^Gad1 target sequence (see a), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop¹⁹ and peak at position 7 at TALE target sequence are noteworthy. N=3 per group. (d) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE^Gad1Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a) but not at neighbouring positions 5, 6. N=3 per group. (e) Quantitative comparison of Gad1-TSS^{(−55kb) Loop} by gel densitometry from DamID–PCR products for TIME A and TIME B. N=4–5 mice per group. Data in c–e shown as mean±s.e.m.