Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York, USA.
PLoS One. 2012;7(8):e44656. doi: 10.1371/journal.pone.0044656. Epub 2012 Aug 31.
In the yeast Saccharomyces cerevisiae, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of HMT1 and the synthetic genetic array (SGA) methodology. This screen revealed interactions between HMT1 and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both RPD3 and HMT1 deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual rpd3/hmt1-null mutant behaves like the rpd3-null single mutant with respect to silencing behavior, indicating that RPD3 is epistatic to HMT1. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.
在酵母酿酒酵母中,端粒处沉默染色质的建立和维持需要组蛋白修饰酶的拮抗活性之间的微妙平衡。先前,我们证明了蛋白质精氨酸甲基转移酶 Hmt1 在酵母沉默染色质的形成中发挥作用。为了更好地理解有助于这种现象的 Hmt1 相互作用的性质,我们使用 HMT1 的 null 等位基因和合成遗传阵列 (SGA) 方法进行了系统的反向遗传筛选。该筛选揭示了 HMT1 与编码组蛋白去乙酰化酶复合物 Rpd3L(大)组成部分的基因之间的相互作用。与仅携带 Hmt1 缺失的单突变体相比,携带 RPD3 和 HMT1 缺失的双突变体在端粒边界区域显示出增加的端粒沉默和 Sir2 占据。然而,与 rpd3 缺失的单突变体相比,双 rpd3/hmt1 缺失突变体的沉默行为类似于 rpd3 缺失的单突变体,表明 RPD3 对 HMT1 具有上位性。缺乏 Hmt1 或其催化活性的突变体显示组蛋白去乙酰化酶 Rpd3 向端粒边界区域的募集增加。此外,在这些功能丧失突变体中,Rpd3 的乙酰化 H4K5 的水平(Rpd3 的底物)在端粒边界区域发生改变。相比之下,乙酰化 H4K16(组蛋白去乙酰化酶 Sir2 的靶标)的水平在这些区域增加。有趣的是,缺失 Rpd3 或 Sir2 的突变体在这些端粒边界区域中显示出 H4R3 的二甲基化水平降低的不同程度。总的来说,这些数据提供了对 Hmt1 促进端粒处沉默染色质的正确建立和维持的机制的深入了解。