Vaňková Hausnerová Viola, Lanctôt Christian
Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University, Prague, Czech Republic.
BIOCEV and Department of Cell Biology, Faculty of Science, Charles University, Vestec u Prahy, 252 50, Czech Republic.
Biol Cell. 2017 Jan;109(1):65-79. doi: 10.1111/boc.201600032. Epub 2016 Oct 18.
The levels of chromatin condensation usually correlate inversely with the levels of transcription. The mechanistic links between chromatin condensation and RNA polymerase II activity remain to be elucidated. In the present work, we sought to experimentally determine whether manipulation of chromatin condensation levels can have a direct effect on transcriptional activity.
We generated a U-2-OS cell line in which the nascent transcription of a reporter gene could be imaged alongside chromatin compaction levels in living cells. The transcripts were tagged at their 5' end with PP7 stem loops, which can be detected upon expression of a PP7 capsid protein fused to green fluorescent protein. Cycles of global chromatin hypercondensation and decondensation were performed by perfusing culture media of different osmolarities during imaging. We used the fluorescence recovery after photobleaching technique to analyse the transcriptional dynamics in both conditions. Surprisingly, we found that, despite a drop in signal intensity, nascent transcription appeared to continue at the same rate in hypercondensed chromatin. Furthermore, quantification of transcriptional profiles revealed that chromatin decondensation was accompanied by a brief and transient spike in transcriptional output.
We propose a model whereby the initiation of transcription is not impaired in condensed chromatin, but inefficient elongation in these conditions leads to the accumulation of RNA polymerase II at the transcription site. Upon chromatin decondensation, release of the RNA polymerase II halt triggers a wave of transcription, which we detect as a transient spike in activity.
The results presented here shed light on the activity of RNA polymerase II during chromatin condensation and decondensation. As such, they point to a new level of transcriptional regulation.
染色质凝聚水平通常与转录水平呈负相关。染色质凝聚与RNA聚合酶II活性之间的机制联系仍有待阐明。在本研究中，我们试图通过实验确定操纵染色质凝聚水平是否会对转录活性产生直接影响。
我们构建了一个U-2-OS细胞系，在该细胞系中，报告基因的新生转录可以与活细胞中的染色质压缩水平一起成像。转录本在其5'端用PP7茎环标记，当与绿色荧光蛋白融合的PP7衣壳蛋白表达时可以检测到。在成像过程中，通过灌注不同渗透压的培养基来进行全局染色质超凝聚和去凝聚循环。我们使用光漂白后荧光恢复技术来分析两种条件下的转录动力学。令人惊讶的是，我们发现，尽管信号强度下降，但在超凝聚染色质中新生转录似乎仍以相同速率继续。此外，转录谱的定量分析表明，染色质去凝聚伴随着转录输出的短暂且瞬时的峰值。
我们提出了一个模型，即转录起始在凝聚染色质中不受损害，但在这些条件下低效的延伸导致RNA聚合酶II在转录位点积累。在染色质去凝聚时，RNA聚合酶II停滞的解除触发了一波转录，我们将其检测为活性的瞬时峰值。
本文给出的结果揭示了RNA聚合酶II在染色质凝聚和去凝聚过程中的活性。因此，它们指出了转录调控的一个新水平。
