Background: CHD5 is a tumor suppressor gene located on 1p36 able to form a NuRD-type chromatin-remodeling complex. It is preferentially expressed in the nervous system and testis, and expression is very low or absent in high-risk NBs, especially those with 1p deletion and/or MYCN amplification. EZH2, a Polycomb group protein and subunit of the Polycomb repressive complex 2 (PRC2), binds to gene promoters and causes histone-3 lysine-27 trimethylation (H3K27me3), leading to transcriptional suppression. Recent evidence suggests that MYCN contributes to the regulation of PRC2. We analyzed the H3K27me3 status, as well as the binding of EZH2 and MYCN at the proximal CHD5 promoter.
Methods: ChIP assays were performed using nuclear proteins prepared from NB cell lines NLF (1p deletion), NGP (1p translocation, no deletion), NBLS and SY5Y (no 1p deletion) by crosslinking and sonication. Immunoprecipitation was performed with antibodies EZH2, H3K27me3, MYCN and control IgG. Bound DNA samples were analyzed by PCR and qPCR with primers designed around the CHD5 transcription start site with appropriate negative control primers.
Results: H3K27 trimethylation was found in CHD5 promoter -250 bp and inside intron 1 in NLF and NGP cell lines. Both cell lines showed very low CHD5 expression. EZH2 binding was also found, consistent with the H3K27me3 in both NLF and NGP. We also observed MYCN binding to the E-boxes around -250bp and -800 bp of the CHD5 promoter in NLF and NGP. These 3 factors (H3K27me3, EZH2, and MYCN) were not found around the CHD5 promoter of the NBLS cell line, which shows high CHD5 expression.
Conclusion: Our data strongly suggest that H3K27 trimethylation by EZH2 contributes to the epigenetic suppression of CHD5 expression, and that MYCN binding may also contribute to the regulation of CHD5 expression.