Enomoto S, McCune-Zierath P D, Gerami-Nejad M, Sanders M A, Berman J
Department of Plant Biology, University of Minnesota, St. Paul 55108, USA.
Genes Dev. 1997 Feb 1;11(3):358-70. doi: 10.1101/gad.11.3.358.
In the yeast Saccharomyces cerevisiae, telomere repeat DNA is assembled into a specialized heterochromatin-like complex that silences the transcription of adjacent genes. The general DNA-binding protein Rap1p binds telomere DNA repeats, contributes to telomere length control and to telomeric silencing, and is a major component of telomeric chromatin. We identified Rap1p localization factor 2 (RLF2) in a screen for genes that alleviate antagonism between telomere and centromere sequences on plasmids. In rlf2 mutants, telomeric chromatin is perturbed: Telomeric silencing is reduced and Rap1p localization is altered. In wild-type cells, Rap1p and telomeres localize to bright perinuclear foci. In rlf2 strains, the number of Rap1p foci is increased, Rap1p staining is more diffuse throughout the nucleus, Rap1p foci are distributed in a much broader perinuclear domain, and nuclear volume is 50% larger. Despite the altered distribution of Rap1p in rlf2 mutant cells, fluorescence in situ hybridization to subtelomeric repeats shows that the distribution of telomeric DNA is similar in wild-type and mutant cells. Thus in rlf2 mutant cells, the distribution of Rap1p does not reflect the distribution of telomeric DNA. RLF2 encodes a highly charged coiled-coil protein that has significant similarity to the p150 subunit of human chromatin assembly factor-I(hCAF-I), a complex that is required for the DNA replication-dependent assembly of nucleosomes from newly synthesized histones in vitro. Furthermore, RLF2 is identical to CAC1, a subunit of yeast chromatin assembly factor-I (yCAF-I) which assembles nucleosomes in vitro. In wild-type cells, epitope-tagged Rlf2p expressed from the GAL10 promoter localizes to the nucleus with a pattern distinct from that of Rap1p, suggesting that Rlf2p is not a component of telomeric chromatin. This study provides evidence that yCAF-I is required for the function and organization of telomeric chromatin in vivo. We propose that Rlf2p facilitates the efficient and timely assembly of histones into telomeric chromatin.
在酿酒酵母中，端粒重复DNA被组装成一种特殊的类异染色质复合体，该复合体可使相邻基因的转录沉默。通用DNA结合蛋白Rap1p结合端粒DNA重复序列，有助于端粒长度控制和端粒沉默，并且是端粒染色质的主要成分。我们在筛选可缓解质粒上的端粒与着丝粒序列之间拮抗作用的基因时，鉴定出了Rap1p定位因子2（RLF2）。在rlf2突变体中，端粒染色质受到干扰：端粒沉默减弱，Rap1p定位改变。在野生型细胞中，Rap1p和端粒定位于明亮的核周焦点。在rlf2菌株中，Rap1p焦点数量增加，Rap1p染色在整个细胞核中更加弥散，Rap1p焦点分布在更广泛的核周区域，并且核体积大50%。尽管rlf2突变体细胞中Rap1p的分布发生了改变，但对亚端粒重复序列进行的荧光原位杂交显示，野生型和突变体细胞中端粒DNA的分布相似。因此，在rlf2突变体细胞中，Rap1p的分布并不反映端粒DNA的分布。RLF2编码一种高度带电的卷曲螺旋蛋白，该蛋白与人染色质组装因子-I（hCAF-I）的p150亚基具有显著相似性，hCAF-I是一种在体外从新合成的组蛋白进行依赖DNA复制的核小体组装所必需的复合体。此外，RLF2与CAC1相同，CAC1是酵母染色质组装因子-I（yCAF-I）的一个亚基，yCAF-I在体外组装核小体。在野生型细胞中，由GAL10启动子表达的带有表位标签的Rlf2p定位于细胞核，其模式与Rap1p不同，这表明Rlf2p不是端粒染色质的组成成分。这项研究提供了证据，证明yCAF-I在体内端粒染色质的功能和组织中是必需的。我们提出，Rlf2p促进组蛋白高效且及时地组装到端粒染色质中。
