Abstract
BackgroundMethylation 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 FindingsImmunolocalization 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/SignificanceTaken 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.
Highlights
Covalent histone modifications play an important role in regulating chromatin structure, gene expression and epigenetic inheritance [1,2]
Histone methylation dependent patterns did not differ in wildtype and Jumonji domain-containing protein 6 (Jmjd6) knockout mouse embryonic fibroblasts (MEFs) when these histone residues were analyzed by immunocytochemistry (Figure 8 and Figures S4, S5 and S6). These results demonstrate that Jmjd6 is very likely not directly involved either in mono, di, or tri-demethylation of histone lysine residues H3K4, H3K9, H3K27, H3K36, or H4K20
We have investigated the expression of Jmjd6 in nuclear compartments and analyzed regions of the protein required for its nuclear expression
Summary
Covalent histone modifications play an important role in regulating chromatin structure, gene expression and epigenetic inheritance [1,2]. The methylation of histone tails is a modification that regulates important processes such as heterochromatin formation, X-chromosome inactivation, imprinting and DNA repair [6]. This posttranslational modification can occur on either lysine or arginine residues in the tails of histones 2, 3, and 4, with lysines being either mono-, di-, or tri-methylated and arginines being mono-methylated or symmetrically or asymmetrically di-methylated [7]. Methylation of residues in histone tails is part of a network that regulates gene expression. We report studies to test the involvement of Jumonji domain-containing protein 6 (Jmjd6) in histone lysine demethylation. There have been conflicting reports as to whether Jmjd is a histone-modifying enzyme
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