elongation factors such as P-TEFb182 or SPT6186, and chromatin remodelers187 that can undergo local recycling to mediate efficient gene activation188 (see also Section 1.2).
Transcriptional downregulation of genes involved in metabolism, protein synthesis and cell cycle is the prevalent consequence of heat stress, and by far outnumbers the upregulated genes162, 175, 189. It is accompanied by an enhanced recruitment of negative elongation factors such as NELF to chromatin that accumulate near repressed gene promoters175. Consistently, paused Pol II becomes stabilized within the promoter-proximal region of these genes upon stress, resulting in increased pause duration162, 163, 189 (Fig. 1.3b). Since the presence of paused Pol II prevents new transcription initiation, enhanced Pol II pausing can facilitate swift transcriptional repression89, 190. But at the same time, it might keep the transcriptional machinery in a competent state that allows rapid reactivation after the heat stress ceases176. In contrast to stress-induced activation, the molecular mechanisms that cause genome-wide transcriptional downregulation upon heat shock are far less well understood175.
1.2 Spatiotemporal organization of Pol II transcription
Each human diploid cell contains 23 pairs of chromosomes that encompass together about six billion base pairs DNA with a total length of ~2 m. To accommodate the genetic information in the cell nucleus that is about five orders of magnitude smaller, the DNA is highly packaged at multiple levels. This degree of compaction is equivalent to accommodating a DNA strand encircling the earth for >6000 times inside a chicken egg191. Given this highly crowded nuclear environment the question arises how the manifold factors involved in Pol II transcription can efficiently encounter each other in a spatiotemporally controlled manner. In the middle of the 1990s this puzzling question was first addressed in pioneering studies by Peter Cook and colleagues who observed that Bromo-UTP labelled nascent transcripts in fixed human cells were not evenly distributed throughout the entire nucleus, but localized to distinct focal sites that they termed ‘transcription factories’192. Several follow-up studies reported similar observations using different nucleotide analogs and electron microscopy, and detected the co-localization of Pol II with labelled nascent transcripts in foci193-195. About 2400 of such foci were detected, each estimated to contain on average about 30 engaged polymerases195. Using similar techniques, even distant genes spaced several megabases apart were observed to
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colocalize with Pol II foci, which in turn colocalized with fluorescence in situ hybridization signals from the produced transcripts196. Correspondingly, these and other197-199 early studies led to the concept that stable pre-assembled transcription factories, dedicated nuclear sites for RNA synthesis with high concentrations of transcriptional components such as Pol II, exist, to which genes must translocate in order to become transcribed200-203 (Fig. 1.4a). However, it was argued at the same time that results obtained in these studies could have been affected by intrinsic methodological limitations. For example, it was criticized that chemical cell fixation might have introduced artificial aggregation artefacts204. Similarly, the number of Pol II molecules might be overestimated through indirect Pol II immunolabeling with antibodies targeting the repetitive CTD as multiple antibody molecules bind a single Pol II enzyme205. Importantly, the dynamics of (dis-)assembly of the detected transcription factories could not be explored due to cell fixation, impeding conclusions regarding their stability. Subsequent attempts to detect stable clusters of transcriptionally active Pol II in living mammalian cells using GFP-tagged Pol II and confocal microscopy were not successful206, 207. Rather, initiating and elongating forms of Pol II were observed to possess a distinct but adjacent nuclear localization204. More recent single-molecule super-resolution microscopy approaches suggest that the majority of Pol II molecules are solitary and spaced on average >200 nm away from each other205, arguing against the predominant occurrence of Pol II in large stable transcription factories.
Using an elegant super-resolution microscopy approach that focuses on transiently (~50 ms) immobile Pol II molecules, Cisse et al. (2013) showed that a small fraction of Pol II molecules indeed forms transient clusters in live human cells208. For these experiments, the authors used a stable human cell line encoding RPB1 that was N-terminally tagged with the photo-switchable fluorescent protein Dendra2. Successive cycles of photoactivation and localization allowed time-resolved counting of detections used then for pair-correlation analysis208-210. Interestingly, the detected Pol II clusters possessed highly transient lifetimes of only few seconds (5.1 ± 0.4 s in208, 8.3 ± 0.2 s in211, and 12.9 ± 1.4 s in212) and average sizes below the diffraction limit, representing potential reasons why they could not be detected in previous studies.
As estimated in fixed cells, an average cluster contains ~80 Pol II molecules211. Live-cell two-color imaging of Pol II and mRNA produced from the β-actin locus revealed that transient Pol II clustering precedes mRNA synthesis211, consistent with a notable stabilization of cluster
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lifetimes after inhibition of transcription elongation208, 211. Together, these findings provided compelling evidence that small populations of Pol II transiently form high local concentrations in close proximity to gene promoters prior to transcription initiation (Fig. 1.4b).
Figure 1.4 | Models for the spatiotemporal organization of gene transcription.
a, Gene transcription requires the translocation into static pre-assembled transcription factories containing high concentrations of relevant factors (i.e. RNA Pol II). b, Nucleoplasmic pool of Pol II surrounds the gene and dynamically forms high concentration clusters upon transcriptional activation. Figure concept was adapted from Buckley & Lis (2016)200.
The rapid Pol II clustering kinetics also match residence times observed for several transcription factors (TFs) on their target sites remarkably well: FRAP and recent single-molecule tracking experiments showed that the large majority of TF single-molecules occupy fast-diffusing states213-216 and that just a small percentage of molecules is bound at specific target sites. At the same time each binding event persists for only few seconds216. Consistent with the kinetics of Pol II clustering, the coactivator complex Mediator also forms transient clusters at enhancer elements with average lifetimes of 11.1 ± 0.9 s212. In agreement with the transient assembly/disassembly of Pol II and co-activator clusters, recent analysis of transcription in single-cells revealed that transcription initiation is not a constant continuous process, but occurs in short ‘bursts’ followed by long periods of transcriptional inactivity126, 217-220. Transcriptional
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bursts generate convoys of closely spaced Pol IIs, which transcribe the gene body220. Bursts are triggered when enhancer elements come in close proximity to gene promoters through DNA looping116, 117, 120, 121.
Cellular stress such as heat shock causes the dynamic nuclear redistribution of the Pol II machinery. The heat shock response has been extensively studied on Drosophila polytene chromosomes, where heat shock stress causes local chromatin decondensation at transcriptionally active loci called puffs221. Because of the naturally amplified HSP70 gene cluster at polytene chromosomes it is possible to image transcriptional activation at high signal-to-noise using diffraction-limited fluorescence microscopy203, 222. Heat stress-induced transcriptional activation caused the sequential accumulation of heat shock factor HSF1, Pol II and other positive transcription elongation factors (i.e. P-TEFb, SPT6, and chromatin remodelers) at the HSP70 locus185, 188, 223. Prolonged gene activation resulted in sustained recruitment of Pol II and elongation factors beyond the amount that can bind to the transcription unit and the ADP-ribosylation-dependent compartmentalization of the locus185 that facilitated the local recycling of these factors over the time of activation185, 188.
Taken together, these insights into the spatiotemporal organization of transcription in living cells suggests a very dynamic regulation involving transient high local concentrations of Pol II and relevant cofactors during gene activation in steady state and upon stress. While these studies suggest the functional importance of transient macromolecular assemblies encompassing Pol II, a mechanistic understanding of the molecular principles that govern such factor concentration only begins to emerge.