Figure 4. Cap-G binds to DNA in NSCs and neurons.

(A) Cap-G binding at example loci in NSCs (wor-GAL4), all neurons including immature neurons (elav-GAL4) and mature (nSyb-GAL4) neurons. Light grey boxes on the gene annotation tracks represent other genes. Horizontal grey bars on the data tracks indicate statistically significant peaks. Y-axes display normalised ratio of log₂ Cap-G-Dam/Dam. N.b. since TaDa data is normalised to Dam-only signal it is normal to observe ‘negative peaks’ at sites of depleted binding that have higher background Dam-only methylation. (B) Venn diagram showing unique genes bound by Cap-G and the total overlap between all cell types. (C) Genomic annotation of Cap-G peaks shows enrichment in gene bodies (introns and exons) whilst binding at non-coding regions are depleted. (D) Cap-G binding is depleted at known Drosophila Regulatory Elements (REDfly, http://redfly.ccr.buffalo.edu/index.php). Profiles plotted against centre of enhancer region and 10 Kb up/downstream. (E) Average Cap-G binding is depleted at accessible chromatin for all cell types. Plot shows 2 Kb up/downstream centre of an open chromatin region. Note that a subset of sites appear enriched for Cap-G binding (blue lines on heatmap) (F) Average Cap-G binding in different chromatin states in NSCs. Cap-G binding is enriched in repressive states (Black, HP1, TrxG-repressive, and PcG repressive), and depleted in permissive chromatin states (non-TrxG, TrxG, and PcG). Heatmap is shown for the most highly enriched ‘black’ state (non-HP1, non-PcG repressive state).

Figure 4—figure supplement 1. Analysis of Cap-G binding.

(A) Scatterplots indicating correlation between replicates for each cell-type. (B) Principal component analysis indicating close relationship between replicates. (C) Average binding of Cap-G across scaled gene-regions (all genes). Binding is relatively increased within the gene body. (D) Cap-G binding at example loci. Light grey boxes on the gene annotation tracks represent other genes. Horizontal grey bars on the data tracks indicate statistically significant peaks. Note increased binding of Cap-G to stg upstream regulatory region and ase up and downstream regions in mature neurons. Unc-89 (not expressed in CNS) shows strong Cap-G binding in all cell stages.

Figure 4—figure supplement 2. Comparisons between chromatin accessibility and Cap-G binding.

(A) Correlation heatmap between Cap-G binding and Accessible Chromatin (AC) per cell type. Cap-G binding negatively correlates with AC in each cell type. Numbers represent Spearman’s rank correlation coefficient. Cap-G binding and AC samples cluster separately by k-means. (B) PCA of Cap-G binding and AC per cell type. Cap-G binding (red) and AC (grey) cluster separately. Elav and NSC cluster in proximity of each other reflecting their greater similarity, whilst nSyb is more distant, observed in both AC and Cap-G binding samples.