Figure 6

Wnt3a changes occupancy of Setdb1 at certain target gene promoters. (a) Setdb1 genome-wide recruitment is reduced after Wnt3a stimulation. ChIP-seq was performed in proliferating C2C12 myoblasts non-treated (Control) or treated with Wnt3a (Wnt3a). Graphic presents average Setdb1 binding density of genomic regions surrounding (±5 kb) Setdb1-binding sites (as in Figure 2a, FDR<1%, fourfold enrichment over the input and P-value<10⁻⁵). Setdb1 occupancy and input density are plotted as average of reads density (every 50 bp) and normalised to total number of reads. (b) Setdb1 and canonical Wnt signalling have common target genes in myoblasts. Deregulated genes upon Setdb1 acute knockdown in proliferating C2C12 myoblasts (red), analysed by RNA-sequencing, were crossed with deregulated genes upon Wnt3a stimulation in proliferating primary myoblasts (grey), analysed by microarray. Results are presented as Venn diagram. The correlation is determined as highly significant by hypergeometric test. P-value<10⁻¹⁶. (c) GO analysis of 245 (out of 270) commonly (in the same way) deregulated genes after Setdb1 acute knockdown in proliferating C2C12 myoblasts and Wnt3a stimulation in primary myoblasts as described in (b). Fisher's exact test was performed to proof significance. P-value between 10⁻³⁰ and 10⁻¹⁰⁰. (d) Genome Browser presentation of Setdb1-binding profile at the Ankrd1 enhancer in proliferating C2C12 myoblasts non-treated (control) or treated with Wnt3a for 24 h (Wnt3a). (e) Wnt3a increases Ankrd1 expression in proliferating primary and C2C12 myoblasts. Cells were untreated or stimulated with Wnt3a protein for 24 h (primary myoblasts) and 50–72 h (C2C12 myoblasts). Relative mRNA expression analysis of Ankrd1 was performed. Data are represented as fold change relative to untreated cells and normalised to CycloA and TBP. Data are presented as mean±s.e.m. of three independent experiments. For significance Student paired t-test was applied. *P-values <0.05 and are considered significant. (f) Model of Wnt3a-dependent nuclear export of Setdb1 and subsequent gene activation. In myoblasts (left) Setdb1 is located in the nucleus, where it occupies its target genes, such as Ankrd1, and represses their transcription by methylating H3K9. Hereby, interaction with other proteins are likely and require further investigation. When canonical Wnt signalling is repressed, β-Catenin is phosphorylated and degraded. In myotubes (right) Setdb1 is released from certain target genes and exported from the nucleus in a Wnt3a-dependent manner. Owing to the lack of H3K9me3 a subset of target genes are transcribed. It is possible that Setdb1 is replaced at certain promoters/enhancers by β-Catenin due to its translocation to the nucleus. The exported Setdb1 could be marked for nuclear export by certain post-translational modifications, such as phosphorylation. Proteasomal degradation of Setdb1 in the cytoplasm is possible. P, phosphrylation; X, any transcription factor; me in red circle, methylation. See also Supplementary Figure S6.