Extended Data Figure 1: 4C-seq in Drosophila embryos.
From: Enhancer loops appear stable during development and are associated with paused polymerase
a, Example of classification into distal and promoter-proximal enhancer viewpoints. Distal enhancers are located >1 kb away from the closest TSS and devoid of H3K4me3 chromatin signature (green), a mark characteristic of gene promoters, while promoter-proximal enhancers are located within 1 kb of the closest TSS. WE, whole embryo. b, Outline of BiTS-4C-seq: genetically modified embryos expressing a tagged nuclear protein exclusively in the mesoderm were collected, aged and crosslinked. Fixed nuclei were extracted and digested with DpnII (whole embryo analysis) or sorted by FACS before DpnII digestion (tissue-specific analysis). After 4C template preparation, ligation junctions were amplified and sequenced. Count values for each DpnII fragment were transformed and fitted using a monotone fit (red line) facilitating an analysis of significant interactions (blue arrowheads). In addition to the 103 viewpoints used at both time-points, four additional viewpoints were used for reciprocal 4C experiments in whole embryos at 6–8 h. c, e, g–l, 4C interaction maps (viewpoint, red arrowhead) at the E2f (c), pdm2 (ref. 51) (e), Con (g), eya (h), stumps (i), Mef2 (j), sli (k) and slp1 (l) loci. The expected interaction with the promoter (blue arrowhead) of the genes is observed. Known enhancers are indicated. MESO, mesoderm (generated by FACS sorting). d, f, Expression (double in situ hybridization) of the E2f (d) or pdm2 (f) genes (red) and the expression driven by their interacting enhancer (green) at stage 11.
