Intersection of ChIP and FLIP, genomic methods to study the dynamics of the cohesin proteins
Intersection of ChIP and FLIP, genomic methods to study the dynamics of the cohesin proteins
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DOI：
10.1007/s10577-008-9007-9
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发表时间：
2009-02-01
影响因子：
2.6
通讯作者：
Gerton, Jennifer L.
中科院分区：
文献类型：
--
作者：
McNairn, Adrian J.;Gerton, Jennifer L.
进化上保守的粘着蛋白Smc 1、Smc 3、Rad 21（Mcd 1）和Scc 3在粘着蛋白复合体中发挥作用，该粘着蛋白复合体为染色体的粘着提供基础，并参与基因调控。了解这些蛋白质如何将基因组连接在一起需要使用全基因组方法来研究这些必需蛋白质的分子机制。虽然染色质免疫沉淀，随后的DNA微阵列（ChIP芯片）的研究提供了一个快照的时间，这些蛋白质与各种基因组相关联，粘蛋白是动态的，在染色质上的定位和相互作用。研究这些蛋白质的动态性质需要活细胞成像等方法。我们提出的证据，从荧光损失在光漂白（FLIP）实验中的芽殖酵母，每个凝聚素亚基的衰减常数是类似的，类似于60-90秒的间期。从G（1）期到S期再到中期，染色质上的衰变常数增加，这与一旦染色体凝聚后与染色质的相互作用变得更加稳定一致。一个小人口的Smc 3在一个位置与着丝粒位置一致具有较长的衰减常数比散装Smc 3。粘着蛋白与染色质的相互作用，在其位置和动力学方面的表征，可能是理解这种蛋白质复合物如何有助于染色体分离和基因调控的关键。
The evolutionarily conserved cohesin proteins Smc1, Smc3, Rad21 (Mcd1), and Scc3 function in the cohesin complex that provides the basis for chromosome cohesion and is involved in gene regulation. Understanding how these proteins link together the genome requires the use of whole-genome approaches to study the molecular mechanisms of these essential proteins. While chromatin immunoprecipitation followed by DNA microarray (ChIP-chip) studies have provided a snapshot in time of where these proteins associate with various genomes, the cohesin proteins are dynamic in their localization and interactions on chromatin. Study of the dynamic nature of these proteins requires approaches such as live cell imaging. We present evidence from fluorescence loss in photobleaching (FLIP) experiments in budding yeast that the decay constant of each cohesin subunit is similar to similar to 60-90 s in interphase. The decay constant on chromatin increases from G(1) to S phase to metaphase, consistent with the interaction with chromatin becoming more stable once chromosomes are cohered. A small population of Smc3 at a position consistent with centromeric location has a longer decay constant than bulk Smc3. The characterization of the interaction of cohesin with chromatin, in terms of both its position and its dynamics, may be key to understanding how this protein complex contributes to chromosome segregation and gene regulation.