Figure 6. Dynamic changes in H3K9me3, H3K36me3 and JmjD2A occupancy on neural crest specifier genes during chick development in vivo.

(A) Chromatin immunoprecipiation (ChIP) assay was used to assess the H3K9me3 and H3K36me3 occupancy around the transcriptional start site (TSS) of Sox10, Snail2 and β-actin. Three independent experiments were performed for each stage. The vertical axis represents % input (ChIP enriched/input) and horizontal axis the distance from the TSS in kilobases (Kb). Schematic diagrams represent primer location over the three analyzed genes and numbers represent approximate distance in Kb from the TSS. Cartoons schematize the two stages (stages 4–5 and 10–11) of analysis, with red indicating the region of tissue dissected and collected for ChIP. One representative sample is depicted per stage. The results show high occupancy of H3K9me3 at −0.5kb from the TSS of Sox10 and Snail2 genes at stage 3–4. This repressive mark appears to be eliminated at stage 10–11. In contrast, both Snail2 and β-actin genes shown high abundance of the active mark H3K36me3, mostly downstream of the TSS. (B) ChIP assays assessed binding of JmjD2A at -0.5Kb from the Sox10 and Snail2 TSS. The graphs show a mean ± SD of three independent experiments for each stage. At stage 3–4, JmjD2A significantly binds to both Sox10 (0.006% input) and Snail2 (0.007% input), when compared with two distant, control regions in their respective chromosome 1 (Chr1nc, 0.002% input) and 2 (Chr2nc, 0.003% input), as well as with β-actin locus (0.003% input). At stage 10–11, this binding was greatly reduced to 0.002 and 0.003% input on the Sox10 and Snail2 genes, respectively. Asterisk indicates P<0.05 versus respective negative control by paired t-student test. See also Figure S6.