Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase
Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase
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DOI：
10.1093/nar/gki635
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Pradhan, S
中科院分区：
文献类型：
--
作者：
Estève, PO;Patnaik, D;Pradhan, S
染色质中组蛋白H3（H3）N-末端尾部的赖氨酸9（K9）的甲基化与转录沉默基因相关，并且由组蛋白甲基转移酶介导。小鼠G9 a是具有特征性SET结构域和ANK重复序列的1263个氨基酸的H3-K9甲基转移酶。在本文中，我们已经使用了一系列的绿色荧光蛋白标记的缺失构建体，以确定两个核定位信号（NLS），第一个NLS之间的氨基酸24和109嵌入和第二个氨基酸394和401之间的小鼠G9 a。我们的数据表明，长和短G9 a亚型都能够进入细胞核甲基化染色质。全长或N-末端缺失的G9 a同种型也是甲基化重组H3或代表H3的N-末端尾的合成肽的催化活性酶。使用N-末端尾肽的体外甲基化反应导致K9的三甲基化，其保持进行性，即使在通过缺失而缺乏N-末端区域的G9 a酶中也是如此。G19 a和H3的共表达导致H3-K9的二甲基化和三甲基化，而HeLa细胞中siRNA介导的G9 a敲低导致H3-K9的二甲基化和三甲基化的整体减少。与麦芽糖结合蛋白融合的重组缺失突变体酶（MBPG 9a Delta 634）用于各种底物的稳态动力学分析，并与全长G9 a（G9 aFL）进行比较。MBP-G9 a Delta 634对各种底物的转换数与G9 aFL相比接近3倍，而它们对重组H3的米氏常数Δ Met（Km）相似。酸克雷对于MBP-G9 a，Delta 634在各种底物下为约2.3 - 2.65 μ M。MBP-G9 a Delta 634和G9 aFL的催化效率（k（cat）/K-m）相似，表明N-末端对于催化不是必需的。此外，保守氨基酸R1097 A、W1103 A、Y1120 A、Y1138 A和R1162 A或催化区中的金属结合C1168 A的突变导致催化受损的酶，从而证实G9 a的C-末端参与催化。因此，不同的结构域调节G9 a的核靶向和催化功能。
Methylation of lysine 9 (K9) in the N-terminus tail of histone H3 (H3) in chromatin is associated with transcriptionally silenced genes and is mediated by histone methyltransferases. Murine G9a is a 1263 amino acid H3-K9 methyltransferase that possesses characteristic SET domain and ANK repeats. In this paper, we have used a series of green fluorescent protein-tagged deletion constructs to identify two nuclear localization signals (NLS), the first NLS embedded between amino acids 24 and 109 and the second between amino acids 394 and 401 of murine G9a. Our data show that both long and short G9a isoforms were capable of entering the nucleus to methylate chromatin. Full-length or N-terminus-deleted G9a isoforms were also catalytically active enzymes that methylated recombinant H3 or synthetic peptides representing the N-terminus tail of H3. In vitro methylation reactions using N-terminus tail peptides resulted in tri-methylation of K9 that remained processive, even in G9a enzymes that lacked an N-terminus region by deletion. Co-expression of G19a and H3 resulted in di- and tri-methylation of H3-K9, while siRNA-mediated knockdown of G9a in HeLa cells resulted in reduction of global H3-K9 di- and tri-methylation. A recombinant deletion mutant enzyme fused with maltose-binding protein (MBPG9a Delta 634) was used for steady-state kinetic analysis with various substrates and was compared with full-length G9a (G9aFL). Turnover numbers of MBP-G9a Delta 634 for various substrates was similar to 3-fold less compared with G9aFL, while their Michaelis constants AdoMet (Km) for recombinant H3 were similar. The K. for MBP-G9a Delta 634 was -2.3-2.65 mu M with various substrates. Catalytic efficiencies (k(cat)/K-m) for both MBP-G9a Delta 634 and G9aFL were similar, suggesting that the N-terminus is not essential for catalysis. Furthermore, mutation of conserved amino acids R1097A, W1103A, Y1120A, Y1138A and R1162A, or the metal binding C1168A in the catalytic region, resulted in catalytically impaired enzymes, thereby confirming the involvement of the C-terminus of G9a in catalysis. Thus, distinct domains modulate nuclear targeting and catalytic functions of G9a.