A cis-regulatory map of the Drosophila genome

Abstract

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.

Figure 1. Chromatin dynamics across Drosophila development

(A) Enrichment of CBP and H3K4me1 (rows) within regions marked by other chromatin modifications, factors, or annotated enhancers (columns). Note that (i) CBP is enriched within all active marks (H3K4me3, H3K27Ac, H3K9Ac, H3K4me1 and PolII) at all stages of development and (ii) early embryo (0–16h) CBP and H3K4me1 marked regions are enriched within H3K27me3 domains and annotated enhancers (right panel). (B) Heatmap depicting fold enrichment of CBP bound regions (columns) at different developmental stages for each of the 22 clusters of TSS-distal regions (rows) grouped by their protein binding profiles. A subset of the clusters shows significant enrichment for CBP at different developmental stages. (C) Enrichment of enhancer categories (columns) for each of the 22 clusters of TSS-distal regions (rows). Many clusters enriched for CBP binding in early development are also strongly enriched for enhancers (rows with *). (D) Embryo-specific CBP binding predicts unannotated enhancers. RNA in situs with a Gal4 probe were used to stain transgenic embryos representing five different enhancer predictions (rows), at four to five different stages (columns). EO044 overlaps the known expression pattern for the neighboring gene, CG8745 (FlyExpress Database). (E) Enrichment of enhancer annotations (rows) within the binding sites of each transcription factor (columns). For panels C and G gray boxes indicate no overlap. For panels D and E all values greater or less than zero are significant, FDR < 0.01.

Figure 2. Transcription factor binding site complexity

(A) Number of TFBS (left y-axis, black circles) and distribution of genomic annotation classes (right y-axis, colors) as a function of TFBS complexity (x-axis). (B) TFBS enrichment (color scale, depleted in blue, enriched in red) of TFBS sorted by TF binding site complexity (x-axis) within annotated enhancers (CM: cardiac mesoderm, Ht: heart muscle, SM: somatic muscle; VM: visceral muscle.), HDAC binding sites, early embryo chromatin marks. At top is a heatmap depicting nucleosome density as a function of TFBS complexity.

Figure 3. Transcription factor binding site overlap

Pairwise TFBS enrichment/depletion (colour coded by Z-score). TFBS datasets labeled at left. Selected interactions described in the text are highlighted.