Enomoto S, Berman J
Department of Plant Biology and Plant Molecular Genetics Institute, University of Minnesota, St. Paul, Minnesota 55108 USA.
Genes Dev. 1998 Jan 15;12(2):219-32. doi: 10.1101/gad.12.2.219.
CAC1/RLF2 encodes the largest subunit of chromatin assembly factor I (CAF-I), a complex that assembles newly synthesized histones onto recently replicated DNA in vitro. In vivo, cac1/rlf2 mutants are defective in telomeric silencing and mislocalize Rap1p, a telomere-binding protein. Here, we report that in cells lacking CAF-I the silent mating loci are derepressed partially. MATa cac1 cells exhibit an unusual response to alpha-factor: They arrest and form mating projections (shmoos) initially, but are unable to sustain the arrest state, giving rise to clusters of shmooing cells. cac1 MATa HMLa HMRa strains do not form these shmoo clusters, indicating that derepression of HMLalpha causes the shmoo cluster phenotype in cac1 cells. When SIR3 is reintroduced into sir1 sir3 cells, HML remains derepressed indicating that SIR1 is required for the re-establishment of silencing at HML. In contrast, when SIR3 is reintroduced into cac1 sir3 cells, silencing is restored to HML, indicating that CAF-I is not required for the re-establishment of silencing. Loss of the other CAF-I subunits (Cac2p and Cac3p/Msi1p) also results in the shmoo cluster phenotype, implying that loss of CAF-I activity gives rise to this unstable repression of HML. Strains carrying certain mutations in the amino terminus of histone H4 and strains with limiting amounts of Sir2p or Sir3p also form shmoo clusters, implying that the shmoo cluster phenotype is indicative of defects in maintenance of the structural integrity of silent chromatin. MATa cac- sir1 double mutants have a synergistic mating defect, suggesting that the two silencing mechanisms, establishment and maintenance, function cooperatively. We propose a model to explain the distinctions between the establishment and the maintenance of silent chromatin.
CAC1/RLF2编码染色质组装因子I（CAF-I）的最大亚基，该复合物可在体外将新合成的组蛋白组装到最近复制的DNA上。在体内，cac1/rlf2突变体在端粒沉默方面存在缺陷，并且端粒结合蛋白Rap1p的定位错误。在此，我们报道在缺乏CAF-I的细胞中，沉默的交配位点会部分去抑制。MATa cac1细胞对α因子表现出异常反应：它们最初会停滞并形成交配突起（shmoos），但无法维持停滞状态，从而产生成群的形成shmoos的细胞。cac1 MATa HMLa HMRa菌株不会形成这些shmoo簇，这表明HMLα的去抑制导致了cac1细胞中的shmoo簇表型。当将SIR3重新引入sir1 sir3细胞时，HML仍然处于去抑制状态，这表明SIR1是在HML处重新建立沉默所必需的。相反，当将SIR3重新引入cac1 sir3细胞时，HML的沉默得以恢复，这表明重新建立沉默不需要CAF-I。其他CAF-I亚基（Cac2p和Cac3p/Msi1p）的缺失也会导致shmoo簇表型，这意味着CAF-I活性的丧失会导致HML这种不稳定的抑制。在组蛋白H4氨基末端携带某些突变的菌株以及Sir2p或Sir3p含量有限的菌株也会形成shmoo簇，这意味着shmoo簇表型表明沉默染色质的结构完整性维持存在缺陷。MATa cac- sir1双突变体具有协同交配缺陷，这表明建立和维持这两种沉默机制协同发挥作用。我们提出了一个模型来解释沉默染色质建立和维持之间的区别。
