Dynamic Analysis of Stochastic Transcription Cycles

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.

在单个哺乳动物细胞中，催乳素（prolactin）等部分基因的表达水平随时间波动幅度极大，此前有研究提出该现象以随机脉冲形式发生。为探究该行为的起源并阐明其功能相关性，我们采用全新数学工具对该表达波动性进行了定量分析，借此得以重构同一活细胞内受相同启动子调控的不同报告基因的动态转录速率。我们针对萤火虫荧光素酶（firefly luciferase）与不稳定增强型绿色荧光蛋白（destabilized EGFP）这两种报告基因开展定量显微分析，以此解析活单个克隆垂体细胞与原代垂体细胞中催乳素启动子调控的基因表达动态，观测时长最长可达25小时。我们对转录脉冲的时间依赖性与周期性进行了定量表征，并估算了转录激活相与非激活相的时长及波动幅度。我们测得平均周期时长约为11小时，并证实：尽管转录激活相的实测时间分布与学界公认的转录模型相符，但非激活相的分布模式存在差异，且表现出显著的记忆效应，转录失活的不应期接近3小时。在同一单个克隆或原代细胞内，受催乳素启动子调控的两个不同报告基因均存在转录周期，但二者的周期时序彼此独立且相位相反。本研究首次在单个活细胞中对两个等效基因座的转录动态进行了实时分析。在未受刺激的条件下，细胞在每个基因座上均表现出独立的转录动态。本次分析的一项核心结果，是证实了转录非激活相存在最短不应期。细胞对急性信号的响应以及组蛋白乙酰化调控实验的结果，均与"该不应期对应染色质重塑阶段、且该阶段显著提升了转录周期性"这一假说相符。因此，组织中看似随机的部分细胞所发生的随机时序转录爆发，或许可形成整体协调但异质性的表型，该表型能够对刺激产生急性响应。