Department of Experimental Mouse Genetics, Helmholtz Centre for Infection Research, Braunschweig, Germany.
PLoS One. 2010 Oct 29;5(10):e13769. doi: 10.1371/journal.pone.0013769.
Methylation of residues in histone tails is part of a network that regulates gene expression. JmjC domain containing proteins catalyze the oxidative removal of methyl groups on histone lysine residues. Here, we report studies to test the involvement of Jumonji domain-containing protein 6 (Jmjd6) in histone lysine demethylation. Jmjd6 has recently been shown to hydroxylate RNA splicing factors and is known to be essential for the differentiation of multiple tissues and cells during embryogenesis. However, there have been conflicting reports as to whether Jmjd6 is a histone-modifying enzyme.
METHODOLOGY/PRINCIPAL FINDINGS: Immunolocalization studies reveal that Jmjd6 is distributed throughout the nucleoplasm outside of regions containing heterochromatic DNA, with occasional localization in nucleoli. During mitosis, Jmjd6 is excluded from the nucleus and reappears in the telophase of the cell cycle. Western blot analyses confirmed that Jmjd6 forms homo-multimers of different molecular weights in the nucleus and cytoplasm. A comparison of mono-, di-, and tri-methylation states of H3K4, H3K9, H3K27, H3K36, and H4K20 histone residues in wildtype and Jmjd6-knockout cells indicate that Jmjd6 is not involved in the demethylation of these histone lysine residues. This is further supported by overexpression of enzymatically active and inactive forms of Jmjd6 and subsequent analysis of histone methylation patterns by immunocytochemistry and western blot analysis. Finally, treatment of cells with RNase A and DNase I indicate that Jmjd6 may preferentially associate with RNA/RNA complexes and less likely with chromatin.
CONCLUSIONS/SIGNIFICANCE: Taken together, our results provide further evidence that Jmjd6 is unlikely to be involved in histone lysine demethylation. We confirmed that Jmjd6 forms multimers and showed that nuclear localization of the protein involves association with a nucleic acid matrix.
组蛋白尾部残基的甲基化是调节基因表达的网络的一部分。JmjC 结构域蛋白催化组蛋白赖氨酸残基上甲基基团的氧化去除。在这里，我们报告了测试 JmjC 结构域蛋白 6（Jmjd6）在组蛋白赖氨酸去甲基化中的作用的研究。最近已经表明，Jmjd6 羟化 RNA 剪接因子，并且在胚胎发生过程中对于多种组织和细胞的分化是必不可少的。然而，关于 Jmjd6 是否是组蛋白修饰酶，存在相互矛盾的报道。
方法/主要发现：免疫定位研究表明，Jmjd6 分布在核质中，位于异染色质 DNA 区域之外，偶尔在核仁中定位。在有丝分裂期间，Jmjd6 从核中排除，并在细胞周期的末期重新出现。Western blot 分析证实，Jmjd6 在核和细胞质中形成不同分子量的同源多聚体。野生型和 Jmjd6 敲除细胞中 H3K4、H3K9、H3K27、H3K36 和 H4K20 组蛋白残基的单、二和三甲基化状态的比较表明，Jmjd6 不参与这些组蛋白赖氨酸残基的去甲基化。这进一步得到了用酶活性和非活性形式过表达 Jmjd6 以及随后通过免疫细胞化学和 Western blot 分析分析组蛋白甲基化模式的支持。最后，用 RNase A 和 DNase I 处理细胞表明，Jmjd6 可能优先与 RNA/RNA 复合物而不是染色质结合。
结论/意义：综上所述，我们的结果进一步证明 Jmjd6 不太可能参与组蛋白赖氨酸去甲基化。我们证实 Jmjd6 形成多聚体，并表明该蛋白的核定位涉及与核酸基质的结合。
