Glowczewski Lynn, Waterborg Jakob H, Berman Judith G
Department of Genetics, Cell Biology and Development, University of Minnesota, 6-170 MCB Building, 420 Washington Ave. SE, Minneapolis, MN 55455, USA.
Mol Cell Biol. 2004 Dec;24(23):10180-92. doi: 10.1128/MCB.24.23.10180-10192.2004.
In yeast, the establishment and maintenance of a transcriptionally silent chromatin state are dependent upon the acetylation state of the N terminus of histone proteins. Histone H4 proteins that contain mutations in N-terminal lysines disrupt heterochromatin and result in yeast that cannot mate. Introduction of a wild-type copy of histone H4 restores mating, despite the presence of the mutant protein, suggesting that mutant H4 protein is either excluded from, or tolerated in, chromatin. To understand how the cell differentiates wild-type histone and mutant histone in which the four N-terminal lysines were replaced with alanine (H4-4A), we analyzed silencing, growth phenotypes, and the histone composition of chromatin in yeast strains coexpressing equal amounts of wild-type and mutant H4 proteins (histone H4 heterozygote). We found that histone H4 heterozygotes have defects in heterochromatin silencing and growth, implying that mutations in H4 are not completely recessive. Nuclear preparations from histone H4 heterozygotes contained less mutant H4 than wild-type H4, consistent with the idea that cells exclude some of the mutant histone. Surprisingly, the N-terminal nuclear localization signal of H4-4A fused to green fluorescent protein was defective in nuclear localization, while a mutant in which the four lysines were replaced with arginine (H4-4R) appeared to have normal nuclear import, implying a role for the charged state of the acetylatable lysines in the nuclear import of histones. The biased partial exclusion of H4-4A was dependent upon Cac1p, the largest subunit of yeast chromatin assembly factor 1 (CAF-1), as well as upon the karyopherin Kap123p, but was independent of Cac2p, another CAF-1 component, and other chromatin assembly proteins (Hir3p, Nap1p, and Asf1p). We conclude that N-terminal lysines of histone H4 are important for efficient histone nuclear import. In addition, our data support a model whereby Cac1p and Kap123 cooperate to ensure that only appropriately acetylated histone H4 proteins are imported into the nucleus.
在酵母中，转录沉默染色质状态的建立和维持取决于组蛋白N端的乙酰化状态。N端赖氨酸发生突变的组蛋白H4会破坏异染色质，导致酵母无法进行交配。尽管存在突变蛋白，但引入野生型组蛋白H4的拷贝可恢复交配能力，这表明突变的H4蛋白要么被排除在染色质之外，要么在染色质中被容忍。为了了解细胞如何区分野生型组蛋白和四个N端赖氨酸被丙氨酸取代的突变组蛋白（H4-4A），我们分析了共表达等量野生型和突变型H4蛋白的酵母菌株（组蛋白H4杂合子）中的沉默、生长表型以及染色质的组蛋白组成。我们发现组蛋白H4杂合子在异染色质沉默和生长方面存在缺陷，这意味着H4中的突变并非完全隐性。组蛋白H4杂合子的细胞核提取物中突变型H4的含量低于野生型H4，这与细胞排除部分突变组蛋白的观点一致。令人惊讶的是，与绿色荧光蛋白融合的H4-4A的N端核定位信号在核定位方面存在缺陷，而四个赖氨酸被精氨酸取代的突变体（H4-4R）似乎具有正常的核输入，这意味着可乙酰化赖氨酸的带电状态在组蛋白的核输入中起作用。H4-4A的偏向性部分排除依赖于酵母染色质组装因子1（CAF-1）的最大亚基Cac1p以及核转运蛋白Kap123p，但不依赖于CAF-1的另一个组分Cac2p和其他染色质组装蛋白（Hir3p、Nap1p和Asf1p）。我们得出结论，组蛋白H4的N端赖氨酸对于有效的组蛋白核输入很重要。此外，我们的数据支持一种模型，即Cac1p和Kap123协同作用以确保只有适当乙酰化的组蛋白H4蛋白被导入细胞核。
