Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics
Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics
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DOI：
10.1186/1471-2121-7-19
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发表时间：
2006-04-24
期刊：
影响因子：
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通讯作者：
Silver, Pamela A.
中科院分区：
文献类型：
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作者：
Drubin, David A.;Garakani, Arman M.;Silver, Pamela A.
背景：基因转录活性与核内位置密切相关，尤其是相对于核外围，这是一个与基因沉默经典相关的区域。然而最近，在酿酒酵母中还发现活跃转录的基因位于核外围。当基因被激活时，它们会与核膜上的核孔复合体（NPC）相关联。此外，染色体不是静态结构，而是在特定基因座的实时活细胞研究中表现出受限扩散。染色体运动与转录激活和向核外围招募活性基因的关系尚未得到研究。结果：我们已经产生了一种酵母菌株，使我们能够在转录活跃和抑制条件下实时观察半乳糖诱导的 GAL 基因位点相对于核外围的运动。使用分段几何粒子追踪，我们发现受抑制的 GAL 基因座经历受限的扩散运动，并且半乳糖的转录诱导与细胞中 GAL 基因座的富集相关，这些基因座既与核外围相关，又在其运动中受到更多限制。此外，我们报告 mRNA 输出因子 Sac3 参与了半乳糖诱导的核周边 GAL 位点的富集。同时，使用一种新颖的机器视觉筛选技术，我们发现受约束的 GAL 位点的运动与半乳糖诱导的细胞中同源核的运动相关。结论：GAL 基因的转录激活与其在核外围的束缚和运动约束有关。我们描述了一个涉及基因扩散和 mRNA 输出因子 Sac3 的基因募集到核外围的模型，该模型可用作进一步实验的框架。此外，我们将机器视觉筛选方法应用于染色体运动分析，该方法使用无偏视觉数据而不是分段几何数据。这种新颖的分析方法将允许对过程进行高通量研究，这些过程可以通过染色体运动和与核外围连接的改变来监测。
Background: Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex ( NPC) at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated.Results: We have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells.Conclusion: Transcriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to the analysis of chromosome motion a machine visual screening approach that used unbiased visual data rather than segmented geometric data. This novel analytical approach will allow for high-throughput study of processes that can be monitored via alterations in chromosome motion and connectivity with the nuclear periphery.