Toteva Tea, Mason Bethany, Kanoh Yutaka, Brøgger Peter, Green Daniel, Verhein-Hansen Janne, Masai Hisao, Thon Geneviève
Department of Biology, BioCenter, University of Copenhagen, 2200 Copenhagen N, Denmark.
Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamkitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):1093-1098. doi: 10.1073/pnas.1614837114. Epub 2017 Jan 17.
The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identify cis- and trans-acting factors capable of creating heterochromatin-euchromatin boundaries in fission yeast. The preponderance of cis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin-euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains.
迄今为止，在所有已研究的真核生物中，端粒保护蛋白复合体成分Rif1已成为复制时间程序的全局调节因子，可能是通过调节基因组的三维结构来实现的。在裂殖酵母中，另一个端粒保护蛋白复合体成分Taz1可能具有类似的功能。在这里，我们通过进行高通量遗传筛选，鉴定了Rif1和Taz1出人意料的特性，该筛选旨在鉴定能够在裂殖酵母中产生异染色质-常染色质边界的顺式和反式作用因子。筛选中鉴定出的顺式作用元件大多源自与Taz1结合并与复制起点相关的基因组位点，其起始受到Taz1和Rif1的抑制。这些元件形成边界和基因沉默需要Taz1和Rif1，并与该区域复制时间的改变同时发生。因此，对Taz1和Rif1敏感的小染色体元件（STAR）可以在一个大的结构域上同时调节基因表达和DNA复制，在其边缘建立异染色质-常染色质边界。当与DNA相连时，Taz1、Rif1以及与Rif1相关的蛋白磷酸酶Sds21和Dis2各自都足以建立一个边界。此外，有效的边界形成需要Mcm4复制解旋酶的氨基末端结构域，据信促进复制的依赖Dbf4的激酶和Rif1招募的磷酸酶的拮抗活性在此结构域上汇聚以控制复制起点的起始。这些观察结果共同为DNA复制的协调控制以及基因组组织成表达结构域提供了深入了解。
