Levin May, Stark Michal, Assaraf Yehuda G
The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
Oncotarget. 2018 Mar 30;9(24):16861-16882. doi: 10.18632/oncotarget.24717.
Histone methylation is regulated to shape the epigenome by modulating DNA compaction, thus playing central roles in fundamental chromatin-based processes including transcriptional regulation, DNA repair and cell proliferation. Histone methylation is erased by demethylases including the well-established KDM4 subfamily members, however, little is known about their dimerization capacity and its impact on their demethylase activity. Using the powerful bimolecular fluorescence complementation technique, we herein show the formation of human KDM4A and KDM4C homodimers and heterodimers in nuclei of live transfectant cells and evaluate their H3K9me3 demethylation activity. Using size exclusion HPLC as well as Western blot analysis, we show that endogenous KDM4C undergoes dimerization under physiological conditions. Importantly, we identify the JmjN domain as the KDM4C dimerization interface and pin-point specific charged residues therein to be essential for this dimerization. We further demonstrate that KDM4A/C dimerization is absolutely required for their demethylase activity which was abolished by the expression of free JmjN peptides. In contrast, KDM4B does not dimerize and functions as a monomer, and hence was not affected by free JmjN expression. KDM4 proteins are overexpressed in numerous malignancies and their pharmacological inhibition or depletion in cancer cells was shown to impair tumor cell proliferation, invasion and metastasis. Thus, the KDM4 dimer-interactome emerging from the present study bears potential implications for cancer therapeutics via selective inhibition of KDM4A/C demethylase activity using JmjN-based peptidomimetics.
组蛋白甲基化通过调节DNA压缩来调控，从而塑造表观基因组，因此在基于染色质的基本过程中发挥核心作用，包括转录调控、DNA修复和细胞增殖。组蛋白甲基化可被包括成熟的KDM4亚家族成员在内的去甲基化酶消除，然而，关于它们的二聚化能力及其对去甲基化酶活性的影响知之甚少。利用强大的双分子荧光互补技术，我们在此展示了人KDM4A和KDM4C同二聚体和异二聚体在活转染细胞细胞核中的形成，并评估了它们的H3K9me3去甲基化活性。利用尺寸排阻高效液相色谱以及蛋白质免疫印迹分析，我们表明内源性KDM4C在生理条件下会发生二聚化。重要的是，我们确定JmjN结构域为KDM4C二聚化界面，并指出其中特定的带电荷残基对这种二聚化至关重要。我们进一步证明，KDM4A/C二聚化对于它们的去甲基化酶活性是绝对必需的，而游离JmjN肽的表达会消除这种活性。相比之下，KDM4B不会二聚化，而是作为单体发挥作用，因此不受游离JmjN表达的影响。KDM4蛋白在多种恶性肿瘤中过表达，并且在癌细胞中对它们进行药理学抑制或清除已显示会损害肿瘤细胞的增殖、侵袭和转移。因此，本研究中出现的KDM4二聚体相互作用组通过使用基于JmjN的拟肽选择性抑制KDM4A/C去甲基化酶活性，对癌症治疗具有潜在意义。
