Co-Localization of the Oncogenic Transcription Factor MYCN and the DNA Methyl Binding Protein MeCP2 at Genomic Sites in Neuroblastoma

BackgroundMYCN is a transcription factor that is expressed during the development of the neural crest and its dysregulation plays a major role in the pathogenesis of pediatric cancers such as neuroblastoma, medulloblastoma and rhabdomyosarcoma. MeCP2 is a CpG methyl binding protein which has been associated with a number of cancers and developmental disorders, particularly Rett syndrome. Methods and FindingsUsing an integrative global genomics approach involving chromatin immunoprecipitation applied to microarrays, we have determined that MYCN and MeCP2 co-localize to gene promoter regions, as well as inter/intragenic sites, within the neuroblastoma genome (MYCN amplified Kelly cells) at high frequency (70.2% of MYCN sites were also positive for MeCP2). Intriguingly, the frequency of co-localization was significantly less at promoter regions exhibiting substantial hypermethylation (8.7%), as determined by methylated DNA immunoprecipitation (MeDIP) applied to the same microarrays. Co-immunoprecipitation of MYCN using an anti-MeCP2 antibody indicated that a MYCN/MeCP2 interaction occurs at protein level. mRNA expression profiling revealed that the median expression of genes with promoters bound by MYCN was significantly higher than for genes bound by MeCP2, and that genes bound by both proteins had intermediate expression. Pathway analysis was carried out for genes bound by MYCN, MeCP2 or MYCN/MeCP2, revealing higher order functions. ConclusionsOur results indicate that MYCN and MeCP2 protein interact and co-localize to similar genomic sites at very high frequency, and that the patterns of binding of these proteins can be associated with significant differences in transcriptional activity. Although it is not yet known if this interaction contributes to neuroblastoma disease pathogenesis, it is intriguing that the interaction occurs at the promoter regions of several genes important for the development of neuroblastoma, including ALK, AURKA and BDNF.背景：MYCN是一种在神经嵴发育过程中表达的转录因子，其失调在神经母细胞瘤、髓母细胞瘤和横纹肌肉瘤等儿童癌症的发病机制中发挥关键作用。甲基CpG结合蛋白2（MeCP2）是一种CpG甲基结合蛋白，与多种癌症及发育障碍相关，尤其是雷特综合征（Rett syndrome）。 方法与结果：本研究采用整合式全基因组学策略，将染色质免疫共沉淀技术结合微阵列分析，证实于MYCN扩增的Kelly细胞来源的神经母细胞瘤基因组中，MYCN与MeCP2可高频共定位至基因启动子区域及基因间/基因内位点（70.2%的MYCN结合位点同时存在MeCP2结合）。有趣的是，经甲基化DNA免疫共沉淀（MeDIP）联合相同微阵列分析证实，在存在显著高甲基化的启动子区域，二者的共定位频率显著降低至8.7%。使用抗MeCP2抗体进行MYCN的免疫共沉淀实验证实，MYCN与MeCP2在蛋白层面存在相互作用。mRNA表达谱分析显示，仅被MYCN结合的启动子所调控的基因的中位表达水平显著高于仅被MeCP2结合的基因，而同时被两种蛋白结合的基因则呈现中等表达水平。对MYCN、MeCP2单独结合或二者共同结合的基因进行通路富集分析，揭示了其高阶生物学功能。 结论：本研究结果表明，MYCN与MeCP2蛋白存在相互作用，并以极高频率共定位至相似的基因组位点，且二者的结合模式与转录活性的显著差异密切相关。尽管目前尚不清楚该相互作用是否参与神经母细胞瘤的疾病发病机制，但值得关注的是，该相互作用发生在多个与神经母细胞瘤发育密切相关的基因的启动子区域，包括ALK、AURKA及BDNF。