Figure 1.

Overview of experimental strategies. (A) Schematic of transgenic lines generated in each species of Drosophila. Three plasmids, containing either a Dichaete-Dam fusion protein, a SoxN-Dam fusion protein, or Dam only, were injected. Dichaete-Dam and Dam-only lines were created for each species, while SoxN-Dam lines were only created in D. melanogaster and D. simulans. The dendrogram represents the evolutionary relationships between the species used. Photographs of flies are from N. Gompel (http://www.ibdml.univ-mrs.fr/equipes/BP_NG/Illustrations/melanogaster%20subgroup.html). Abbreviations: mel, Drosophila melanogaster; sim, Drosophila simulans; yak, Drosophila yakuba; pse, Drosophila pseudoobscura. (B) Outline of the DamID-seq experimental protocol. A TF-Dam fusion protein or a Dam-only control is expressed in embryos, leading to methylation of GATC sites in the vicinity of binding events. Genomic DNA is extracted and methylated fragments are isolated via digestion with DpnI, which recognises only methylated GATC. These fragments are purified through ligation of adapters, further digestion of non-methylated DNA with DpnII and PCR amplification [96]. DNA from both TF-Dam fusion samples and control Dam-only samples is sequenced and mapped to the genome, and the relative enrichment of reads is compared between conditions to generate binding profiles. Orange lines represent GATC sites, grey ovals represent methylation, blue diamonds represent Dam protein and pink ovals represent TFs. Figure adapted from Carl and Russell (in press).