The binding of Chp2’s chromodomain to methylated H3K9 is essential for Chp2’s role in heterochromatin assembly in fission yeast

The binding of heterochromatin protein 1 (HP1) to lysine 9–methylated histone H3 (H3K9me) is an essential step in heterochromatin assembly. Chp2, an HP1-family protein in the fission yeast Schizosaccharomyces pombe, is required for heterochromatic silencing. Chp2 recruits SHREC, a multifunctional protein complex containing the nucleosome remodeler Mit1 and the histone deacetylase Clr3. Although the targeting of SHREC to chromatin is thought to occur via two distinct modules regulated by the SHREC components Chp2 and Clr2, it is not clear how Chp2’s chromatin binding regulates SHREC function. Here, we show that H3K9me binding by Chp2’s chromodomain (CD) is essential for Chp2’s silencing function and for SHREC’s targeting to chromatin. Cells expressing a Chp2 mutant with defective H3K9me binding (Chp2-W199A) have a silencing defect, with a phenotype similar to that of chp2-null cells. Genetic analysis using a synthetic silencing system revealed that a Chp2 mutant and SHREC-component mutants had similar phenotypes, suggesting that Chp2’s function also affects SHREC’s chromatin binding. Size-exclusion chromatography of native protein complexes showed that Chp2-CD’s binding of H3K9me3 ensures Clr3’s chromatin binding, and suggested that SHREC’s chromatin binding is mediated by separable functional modules. Interestingly, we found that the stability of the Chp2 protein depended on the Clr3 protein’s histone deacetylase activity. Our findings demonstrate that Chp2’s H3K9me binding is critical for SHREC function and that the two modules within the SHREC complex are interdependent.

异染色质蛋白1（heterochromatin protein 1, HP1）与赖氨酸9甲基化组蛋白H3（H3K9me）的结合是异染色质组装过程中的核心步骤。粟酒裂殖酵母（Schizosaccharomyces pombe）中的HP1家族蛋白Chp2是异染色质沉默所必需的因子。Chp2可招募SHREC——一种兼具多种功能的蛋白复合物，该复合物包含核小体重塑蛋白Mit1与组蛋白去乙酰化酶Clr3。尽管学界普遍认为，SHREC被靶向招募至染色质的过程依赖于两个受SHREC组分Chp2与Clr2调控的独立模块，但目前尚不明确Chp2的染色质结合活性如何调控SHREC的功能。本研究证实，Chp2的染色质结构域（chromodomain, CD）结合H3K9me的能力，对Chp2的沉默功能以及SHREC靶向染色质均具有决定性作用。表达H3K9me结合缺陷型Chp2突变体（Chp2-W199A）的细胞会出现沉默缺陷，其表型与chp2基因敲除（chp2-null）细胞高度相似。通过人工合成沉默系统开展的遗传分析显示，Chp2突变体与SHREC组分突变体的表型一致，这表明Chp2的功能同样会影响SHREC的染色质结合能力。对天然蛋白复合物进行的尺寸排阻色谱分析结果表明，Chp2-CD结合H3K9me3能够保障Clr3的染色质结合活性，同时提示SHREC的染色质结合过程由可分离的功能模块介导。值得注意的是，本研究还发现Chp2蛋白的稳定性依赖于Clr3蛋白的组蛋白去乙酰化酶活性。本研究结果证实，Chp2的H3K9me结合能力对SHREC的功能至关重要，且SHREC复合物内的两个功能模块存在相互依存关系。