Dynamic analysis of stochastic transcription cycles.
Dynamic analysis of stochastic transcription cycles.
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DOI：
10.1371/journal.pbio.1000607
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发表时间：
2011-04
期刊：
影响因子：
9.8
通讯作者：
White MR
中科院分区：
文献类型：
--
作者：
Harper CV;Finkenstädt B;Woodcock DJ;Friedrichsen S;Semprini S;Ashall L;Spiller DG;Mullins JJ;Rand DA;Davis JR;White MR
单个细胞中基因表达的循环是由动态染色质重塑控制的。在单个哺乳动物细胞中，催乳素等一些基因的表达随时间变化很大，并以随机脉冲的形式出现。为了研究这种行为的起源并了解其功能相关性，我们使用新的数学工具定量分析了这种变异性，使我们能够重建同一活细胞中由相同启动子控制的不同报告基因的动态转录率。通过荧光素酶和不稳定EGFP两个报告基因的定量显微镜分析，我们分析了催乳素启动子导向基因在活的个体克隆和初级垂体细胞中长达25小时的表达动态。我们量化了转录脉冲的时间依赖性和周期性，并估计了活性和非活性转录期的长度和变化。我们发现平均周期约为11小时，并证明虽然活性阶段的测量时间分布与普遍接受的转录模型一致，但非活性阶段的分布不同，并且具有很强的记忆性，转录失活的不应期接近3小时。转录周期发生在同一个体克隆或原代细胞中两个不同的催乳素启动子控制的报告基因上。然而，周期的时间是独立的和不同步的。我们首次在单细胞中实时分析了两个等效位点的转录动力学。在非刺激条件下，细胞在每个位点表现出独立的转录动力学。这些分析的一个关键结果是转录不活跃期存在最小不应期的证据。对急性信号的反应和组蛋白乙酰化操作的结果与假设一致，即不应期对应于染色质重塑的一个阶段，该阶段显着增加了循环性。因此，在组织中明显随机的细胞亚群中，随机时间的转录爆发可能产生总体协调但异质性的表型，能够对刺激产生急性反应。基因转录等生物过程的定时是确保细胞和组织对环境做出适当反应的关键。直到最近，人们才假设组织中的大多数细胞都以类似的方式作出反应，并且细胞活动的变化相对稳定。然而，单细胞中信使RNA和蛋白质水平的研究表明存在相当大的异质性。这表明，随着时间的推移，单细胞中的转录可能是高度动态的。采用实验和理论相结合的方法，在25小时的周期内对报告基因表达进行延时成像，我们测量了催乳素基因在单个垂体细胞中的转录率，并检测到转录活性的清晰周期。数学分析，使用二元模型，假设转录是开或关，表明这些周期的特征是一个最小的不应期，涉及染色质重塑。在同一细胞中，由相同启动子驱动的两种不同报告基因结构的转录时间是相异的，这表明基因表达的脉冲是由于特定基因表达的内在过程，而不是环境影响。我们进一步表明，转录脉冲在每个基因位点是独立的染色质周期。因此，基因表达的异质性模式可能促进完整组织内细胞中灵活的转录反应，同时在静息状态下保持良好的平均基因表达水平。
Cycling of gene expression in individual cells is controlled by dynamic chromatin remodeling. In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h. We quantified the time-dependence and cyclicity of the transcription pulses and estimated the length and variation of active and inactive transcription phases. We showed an average cycle period of approximately 11 h and demonstrated that while the measured time distribution of active phases agreed with commonly accepted models of transcription, the inactive phases were differently distributed and showed strong memory, with a refractory period of transcriptional inactivation close to 3 h. Cycles in transcription occurred at two distinct prolactin-promoter controlled reporter genes in the same individual clonal or primary cells. However, the timing of the cycles was independent and out-of-phase. For the first time, we have analyzed transcription dynamics from two equivalent loci in real-time in single cells. In unstimulated conditions, cells showed independent transcription dynamics at each locus. A key result from these analyses was the evidence for a minimum refractory period in the inactive-phase of transcription. The response to acute signals and the result of manipulation of histone acetylation was consistent with the hypothesis that this refractory period corresponded to a phase of chromatin remodeling which significantly increased the cyclicity. Stochastically timed bursts of transcription in an apparently random subset of cells in a tissue may thus produce an overall coordinated but heterogeneous phenotype capable of acute responses to stimuli. Timing of biological processes such as gene transcription is crucial to ensure that cells and tissues respond appropriately to their environment. Until recently it was assumed that most cells in a tissue responded in a similar way, and that changes in cellular activity were relatively stable. However, studies of messenger RNA and protein levels in single cells have shown the presence of considerable heterogeneity. This suggested that transcription in single cells may be highly dynamic over time. Using a combined experimental and theoretical approach, with time-lapse imaging of reporter gene expression over 25 h periods, we measured the rate of prolactin gene transcription in single pituitary cells and detected clear cycles of transcriptional activity. Mathematical analysis, using a binary model that assumed transcription was on or off, showed that these cycles were characterized by a minimum refractory period that involved chromatin remodeling. The timing of transcription from two different reporter constructs driven by identical promoters in the same cell was out-of-phase, suggesting that the pulses of gene expression are due to processes intrinsic to expression of a particular gene and not to environmental effects. We further show that the pulses of transcription are independent chromatin cycles at each gene locus. Therefore, heterogeneous patterns of gene expression may facilitate flexible transcriptional responses in cells within intact tissue, while maintaining a well-regulated average level of gene expression in the resting state.
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