Extended Data Fig. 7: Perturbation of gene expression and CRM loops by enhancer deletion and Zld depletion.
a. Scheme of the wild type doc locus (+/+) and the doc locus after CRISPR/Cas9 genome editing (ΔCRMc/ΔCRMc). doc enhancer/CRMc, FRT sequence and primers used for genotyping are in teal, yellow and blue, respectively. Genotyping PCR products on agarose gel electrophoresis are shown in the lower panel. Orange and green stars correspond to the bands of the expected sizes after amplification using primers flanking the doc enhancer/CRMc sequence. See Methods for further details. b, RNA-FISH imaging of doc1 and doc2 in the CRMc-deletion mutant. Scale bars: 50 𝜇m/ 5 𝜇m (inset). c, RNA-FISH imaging of doc1, doc2 and sna in control (RNAi white) and RNAi Zld embryos. Black arrows show the doc1 and doc2 expression patterns in the anterior part of the embryo. Grey arrows indicate the absence (doc1, doc2) or perturbation (sna) of gene expression patterns in RNAi-Zld embryos. Scale bar: 50 𝜇m. d, Tracks for pioneer factor binding (Zelda) and RNA Pol2 binding in the doc-TAD. See Supplementary Table 1 for assignment of CRMb-d. e, Transcription levels (RNAseq) of doc1, doc2 and doc3 in wild-type versus zld- embryos69. f, Hi-C matrix for a genomic region containing doc-TAD in wild-type and Zld-depleted embryos. Data from Hug et al. (2017)40. g, Distribution of radius of gyration for the doc-TAD in Zld-depleted embryos (see Extended Fig. 1i for wild-type). h-j, Log2(observed/expected) average contact frequencies between Zelda bound regions at long-range distances (> 250 kb) ranked by increasing Zelda enrichment in nc14 (panel g), nc14 zld-RNAi (panel h) and at short-range (⩽ 250 kb) in nc14 triptolide-treated embryos (panel i). k, Violin plot of intragroup Log2(observed/expected) distribution between 62 selected pre-MBT enhancers and neighbouring sequences (± 5 kb) in nc14 (upper panel) and nc14 zld-RNAi (lower panel). The central white marker indicates the median and the vertical black lines indicate the extreme values of the distribution. The coordinates of enhancers and closest pre-MBT genes are listed in Supplementary Table 8.
