Fig. 3.

Integrating β-catenin ChIP-seq and RNA-seq analysis to identify direct Wnt8a/β-catenin target genes. (A) Venn diagram illustrating overlap between genes near β-peaks (red) and the wnt8a positively regulated genes (as in Fig. 2B). Note that from among the longlist of 274 potential wnt8a-regulated genes, 179 are associated with identified β-peaks (amber border around lens-shaped area), representing the longlist of probable direct Wnt8a/β-catenin target genes. Also note that all but one (xmcl2) of the validated shortlist of wnt8a positively regulated genes are among these and therefore represent the shortlist of 13 direct Wnt8a/β-catenin target genes (yellow). Also note that the majority of gene loci near β-peaks are not correlated with wnt8a-regulated genes and, conversely, that more than one-third of wnt8a-regulated genes in the longlist are not associated with identified β-peaks (most likely representing indirect wnt8a targets). (B) β-catenin ChIP-qPCR of identified β-peaks of our shortlist in chromatin extracted from control (uninjected) and wnt8a MO-injected embryos. Note that β-catenin association is reduced in the wnt8a loss-of-function experiment for most of the 15 β-peaks analysed. IgG antibodies were used as control. Error bars represent s.e.m. of three to five biological replicates. (C) Luciferase assays of reporter constructs containing sequences near identified β-peaks of wnt8a-regulated genes. Error bars represent s.d. of three biological replicates. (D) GO analysis suggests that β-peak-associated genes tend to encode transcription factors and also cell-to-cell signalling components, and to function in developmental processes, with different emphasis between wnt8a-regulated (purple and amber) and non-regulated genes (red). (E) DNA occupancy level of β-catenin around the peak summit shows higher enrichment in direct Wnt8a/β-catenin target gene loci [shortlist (purple) and longlist (amber)] compared with non-wnt8a-regulated genes (red). Read density was analysed using HOMER (bin size 100 bp). (F) TCF/LEF consensus motif is enriched under all 58 β-peaks associated with all 13 shortlisted Wnt8a/β-catenin target genes.