Adelman Karen, Kennedy Megan A, Nechaev Sergei, Gilchrist Daniel A, Muse Ginger W, Chinenov Yurii, Rogatsky Inez
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18207-12. doi: 10.1073/pnas.0910177106. Epub 2009 Oct 9.
The kinetics and magnitude of cytokine gene expression are tightly regulated to elicit a balanced response to pathogens and result from integrated changes in transcription and mRNA stability. Yet, how a single microbial stimulus induces peak transcription of some genes (TNFalpha) within minutes whereas others (IP-10) require hours remains unclear. Here, we dissect activation of several lipopolysaccharide (LPS)-inducible genes in macrophages, an essential cell type mediating inflammatory response in mammals. We show that a key difference between the genes is the step of the transcription cycle at which they are regulated. Specifically, at TNFalpha, RNA Polymerase II initiates transcription in resting macrophages, but stalls near the promoter until LPS triggers rapid and transient release of the negative elongation factor (NELF) complex and productive elongation. In contrast, no NELF or polymerase is detectible near the IP-10 promoter before induction, and LPS-dependent polymerase recruitment is rate limiting for transcription. We further demonstrate that this strategy is shared by other immune mediators and is independent of the inducer and signaling pathway responsible for gene activation. Finally, as a striking example of evolutionary conservation, the Drosophila homolog of the TNFalpha gene, eiger, displayed all of the hallmarks of NELF-dependent polymerase stalling. We propose that polymerase stalling ensures the coordinated, timely activation the inflammatory gene expression program from Drosophila to mammals.
细胞因子基因表达的动力学和幅度受到严格调控，以引发对病原体的平衡反应，这是转录和mRNA稳定性综合变化的结果。然而，单一微生物刺激如何在几分钟内诱导某些基因（TNFα）达到转录峰值，而其他基因（IP-10）则需要数小时，目前尚不清楚。在这里，我们剖析了巨噬细胞中几种脂多糖（LPS）诱导基因的激活过程，巨噬细胞是哺乳动物介导炎症反应的一种重要细胞类型。我们表明，这些基因之间的一个关键差异在于它们在转录周期中受到调控的步骤。具体而言，对于TNFα，RNA聚合酶II在静息巨噬细胞中启动转录，但在启动子附近停滞，直到LPS触发负性延伸因子（NELF）复合物的快速和短暂释放以及有效的延伸。相比之下，在诱导之前，IP-10启动子附近未检测到NELF或聚合酶，并且LPS依赖性聚合酶的募集是转录的限速步骤。我们进一步证明，这种策略也适用于其他免疫介质，并且与负责基因激活的诱导剂和信号通路无关。最后，作为进化保守性的一个显著例子，TNFα基因的果蝇同源物eiger表现出所有NELF依赖性聚合酶停滞的特征。我们提出，聚合酶停滞确保从果蝇到哺乳动物的炎症基因表达程序得到协调、及时的激活。
