Chang Chuang-Rung, Wu Ching-Shyi, Hom Yolanda, Gartenberg Marc R
Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, 08854, USA.
Genes Dev. 2005 Dec 15;19(24):3031-42. doi: 10.1101/gad.1356305. Epub 2005 Nov 30.
Eukaryotic DNA replication produces sister chromatids that are linked together until anaphase by cohesin, a ring-shaped protein complex that is thought to act by embracing both chromatids. Cohesin is enriched at centromeres, as well as discrete sites along chromosome arms where transcription positions the complex between convergent gene pairs. A relationship between cohesin and Sir-mediated transcriptional silencing has also begun to emerge. Here we used fluorescence microscopy and site-specific recombination to characterize interactions between newly replicated copies of the silent HMR mating-type locus. HMR was tagged with lac-GFP and flanked by binding sites for an inducible site-specific recombinase. Excision of the locus in cells with sister chromatids produced two chromatin circles that remained associated with one another. Pairing of the circles required silent chromatin, cohesin, and the RSC chromatin-remodeling complex. Chromatin immunoprecipitation showed that targeting of cohesin to the locus is Sir-dependent, and functional tests showed that silent chromatin acts in a continuous fashion to maintain cohesion. Remarkably, loss of silencing led to loss of cohesin from linear chromosomal templates but not from excised chromatin circles. The results are consistent with a model in which cohesin binds silent chromatin via topological linkage to individual chromatids.
真核生物的DNA复制产生姐妹染色单体，这些姐妹染色单体通过黏连蛋白连接在一起，直到后期才分开。黏连蛋白是一种环状蛋白质复合体，被认为通过环绕两条染色单体来发挥作用。黏连蛋白在着丝粒以及染色体臂上的离散位点富集，在这些位点，转录将该复合体定位在 convergent基因对之间。黏连蛋白与Sir介导的转录沉默之间的关系也已开始显现。在这里，我们使用荧光显微镜和位点特异性重组来表征沉默的HMR交配型位点新复制拷贝之间的相互作用。HMR用lac-GFP标记，并两侧带有诱导型位点特异性重组酶的结合位点。在具有姐妹染色单体的细胞中切除该位点会产生两个相互关联的染色质环。环的配对需要沉默染色质、黏连蛋白和RSC染色质重塑复合体。染色质免疫沉淀表明，黏连蛋白定位于该位点是依赖Sir的，功能测试表明沉默染色质以连续方式发挥作用来维持黏连。值得注意的是，沉默的丧失导致黏连蛋白从线性染色体模板上丢失，但不会从切除的染色质环上丢失。这些结果与一个模型一致，即黏连蛋白通过与单个染色单体的拓扑连接结合沉默染色质。
