Control of stochastic gene expression by host factors at the HIV promoter.
Control of stochastic gene expression by host factors at the HIV promoter.
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DOI：
10.1371/journal.ppat.1000260
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发表时间：
2009-01
期刊：
影响因子：
6.7
通讯作者：
Schaffer DV
中科院分区：
文献类型：
--
作者：
Burnett JC;Miller-Jensen K;Shah PS;Arkin AP;Schaffer DV
病毒长末端重复序列（LTR）内的HIV启动子协调了病毒生命周期的许多方面，从病毒基因表达和复制的动态到潜伏状态的建立。特别是，在病毒整合到宿主基因组后，Tat正反馈回路放大的病毒基因表达的随机波动可能有助于形成生产性、反激活状态或非活性状态。在相当一部分携带HIV-1模型原病毒（LTR-GFP-IRES-Tat）整合拷贝的细胞中，这种双峰基因表达谱是动态的，因为细胞会自发地在活跃（Bright）和不活跃（Off）表达模式之间持续切换。此外，这些转换动力学可能有助于前病毒潜伏期的建立和维持，因为在病毒整合后，基因表达的长时间延迟可能在病毒转激活之前发生。HIV-1启动子包含顺式作用Sp1和NF-κB元件，通过激活和抑制复合物的募集来调节基因表达。我们假设，这些积极因素和消极因素的相互作用可以调节Bright和Off模式的稳定性，从而改变病毒基因表达对Tat反馈回路随机波动的敏感性。利用Sp1和NF-κB元件引入突变的慢病毒模型变体，我们采用流式细胞术、mRNA定量、药理学扰动和染色质免疫沉淀来揭示每个位点对病毒基因调控的显著功能差异。具体来说，Sp1位点明显稳定了Bright和Off状态，因此它们的突变促进了嘈杂的基因表达，减少了组蛋白乙酰化和去乙酰化的调节。此外，NF-κB位点表现出不同的特性，其中κB位点I比κB位点II具有更强的激活作用。此外，Sp1位点III在募集p300和RelA到启动子中起着特别重要的作用。最后，对感染病毒变体的362个克隆细胞群体的分析显示，Sp1位点的任何突变产生的克隆分叉频率比野生型启动子高6倍。因此，每个Sp1和NF-κB位点对病毒基因表达的调控有不同的贡献，Sp1位点在功能上“抑制”转录噪声，从而调节这种病毒潜伏期模型的频率和维持。这些结果可能对治疗HIV潜伏期具有生物医学意义。HIV基因组整合到宿主染色体后，病毒启动子协调一系列复杂的输入来控制病毒潜伏期的建立、病毒基因表达的开始以及随后的基因表达水平。这些输入包括整合位点的染色质结构、宿主转录因子和病毒编码的转录调节因子Tat。重要的是，来自宿主和病毒转录调节因子的转录噪声可能在复制与潜伏期之间的决定中发挥关键作用，因为基因表达的随机波动被tat介导的正转录反馈回路放大。为了评估关键转录因子结合元件在基因表达动力学中的个体贡献，我们采用了在多个启动子元件中引入突变的模型HIV病毒。对基因表达动态和转录因子募集到病毒启动子的广泛分析表明，每个位点对病毒基因表达和低表达状态的建立有不同的贡献，而低表达状态可能导致病毒潜伏期。这种系统级的方法阐明了宿主和病毒因素对病毒基因表达的动态、大小和随机效应的协同作用，并提供了对导致病毒前潜伏期的机制的见解。
The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-κB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-κB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-κB sites exhibit distinct properties, with κB site I serving a stronger activating role than κB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-κB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency. After HIV genome integration into the host chromosome, the viral promoter coordinates a complex set of inputs to control the establishment of viral latency, the onset of viral gene expression, and the ensuing gene expression levels. Among these inputs are chromatin structure at the site of integration, host transcription factors, and the virally encoded transcriptional regulator Tat. Importantly, transcriptional noise from host and viral transcriptional regulators may play a critical role in the decision between replication versus latency, because stochastic fluctuations in gene expression are amplified by a Tat-mediated positive transcriptional feedback loop. To evaluate the individual contributions of key transcription factor binding elements in gene expression dynamics, we employ model HIV viruses with mutations introduced into numerous promoter elements. Extensive analysis of gene expression dynamics and transcription factor recruitment to the viral promoter reveals that each site differentially contributes to viral gene expression and to the establishment of a low expression state that may contribute to viral latency. This systems-level approach elucidates the synergistic contributions of host and viral factors to the dynamics, magnitudes, and stochastic effects in viral gene expression, as well as provides insights into mechanisms that contribute to proviral latency.
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