Figure 9
Chromatin immunoprecipitation analysis of Hdac1 binding to cis -regulatory regions of Hdac1-regulated genes. (A, B) Hdac1 is stably and specifically associated with DNA sequences close to the transcription start site of the ascl1b gene in (A) 12 hpf and (B) 27 hpf zebrafish embryos. Chromatin was immunoprecipitated with anti-Hdac1 antibody (blue) and control IgG (red) and DNA content analysed by Q-PCR, which revealed Hdac1 is stably associated with DNA sequences between -785bp and -85bp upstream of ascl1b in wild-type embryos. In contrast, Hdac1 was not detectably associated with similarly positioned DNA sequences close to neurod4, dlb or her6. (C) Immunoprecipitation of ascl1b promoter sequences from chromatin with an Hdac1-specific antibody is abolished in 27 hpf hdac1 mutant embryos. Comparison of Hdac1 protein binding to chromatin encompassing the ascl1b transcription start site in 27 hpf wild-type sibling (SIB) and hdac1 mutant (MUT) embryos. Chromatin was immunoprecipitated with anti-Hdac1 antibody (blue, green bars) or control IgG (red, purple bars) and DNA content analysed by Q-PCR. Physical association of Hdac1 with DNA sequences between -785bp and -85bp upstream of ascl1b was detected in wild-type sibling embryos, but not in hdac1 mutant embryos. Bar graphs show mean values with standard deviations for technical triplicates. Asterisks indicate statistical significance (P < 0.05).