Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus

Background: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5—a critical MYC cofactor—regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. Results: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. Conclusions: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes. We used a MYCN-amplified neuroblastoma cell line that was engineered to express a version of PDPK1 that can be degraded by addition of the dTAG47 molecule. Following 24 hr depletion of PDPK1, RNA was extracted and processed for RNA-seq.

背景：在有丝分裂过程中，细胞依赖复制后染色体的正确附着与分离，以产生两个完全相同的子代细胞。在MYC致癌基因过表达或失调的癌症中，这一过程会受损，进而引发基因组不稳定与肿瘤演进。近期研究发现，染色质调控因子WDR5——一种关键的MYC辅因子——可通过与主激酶PDPK1的直接相互作用，调控有丝分裂所需基因的表达。然而，在MYC过表达的癌症中，PDPK1与WDR5是否共同参与类似的有丝分裂基因调控，目前仍不明确。因此，为了阐明WDR5与PDPK1对高MYC水平细胞中有丝分裂基因表达的影响，我们在MYCN扩增的神经母细胞瘤细胞系中开展了对比转录组分析——MYCN扩增会导致细胞内N-MYC蛋白水平升高。
结果：通过RNA-seq分析，我们在多个工程改造的CHP-134神经母细胞瘤细胞系中鉴定出了受N-MYC与PDPK1调控的基因，并将其与此前发表的、在WDR5抑制后的CHP-134细胞中收集的基因表达数据进行对比。我们发现，正如预期，N-MYC调控大量基因，其中包括与有丝分裂相关的基因；而PDPK1则调控参与发育、信号传导与有丝分裂的特定基因集。对N-MYC与PDPK1调控基因的分析显示，存在一小簇共同受控的基因，它们与纺锤体极形成及染色体分离相关，且与同样受WDR5调控的基因存在重叠。我们还发现，N-MYC可通过WDR5-PDPK1相互作用与PDPK1发生物理结合，这提示有丝分裂基因表达的调控可能通过N-MYC-WDR5-PDPK1三重枢纽实现。
结论：综上，我们鉴定出一小簇在与有丝分裂过程相关的功能基因类别中高度富集的基因，这些基因受N-MYC、WDR5与PDPK1共同调控；并提出这三种调控因子之间的三重相互作用，可能负责调控N-MYC扩增细胞系中有丝分裂基因的表达水平。本研究为后续阐明N-MYC、WDR5与PDPK1如何协同调控细胞周期相关过程的确切机制奠定了基础。我们使用了经过工程改造的MYCN扩增神经母细胞瘤细胞系，该细胞系可表达可通过添加dTAG47分子进行降解的PDPK1变体。在PDPK1被耗竭24小时后，我们提取RNA并进行RNA-seq测序处理。