S2 and and and and and and and rate constants (see and and Fig

S2 and and and and and and and rate constants (see and and Fig. promoter pausing is a widespread phenomenon occurring on most metazoan genes (5, 8). However, despite this prevalence, the dynamics of promoter-paused Pol II remain under debate. The currently prevailing model suggests that Pol II pauses at promoters with a half-life of 5C15 min (8C12), serving as an integrative hub to control pause release into productive elongation, while promoter-proximal termination is infrequent. However, conflicting studies have reported that promoter-paused Pol II is less stable due to repeated premature termination and chromatin release proximal to the promoter, which is accompanied by the release of short transcription start site-associated RNAs (13C16). Thus far, genome-wide dynamics of promoter-paused Pol II have been studied by Gro-Seq (8), ChIP-Seq (10, 11), or methyltransferase footprinting (15) after inhibiting Pol II initiation. While these techniques provide gene-specific snapshots of Pol II transcription, relative abundance, or position at a given time, they do not allow measurement of steady-state Pol II kinetics (i.e., chromatin binding times) in real time. Although these studies have gained insights into the turnover of paused Pol II, most experiments have been performed after inhibiting transcription initiation by Triptolide (8, 10C12). This covalent XPB inhibitor severely affects Pol II levels (17, 18) and has been recently shown to have a slow mode of action (16), which makes it less suitable to study a potentially rapid cellular process. To overcome these limitations, we developed photobleaching of endogenously expressed GFP-RPB1 followed by computational modeling to quantitatively assess the kinetics of Pol II in unperturbed living cells. Here we show that GFP-RPB1 knockin (KI) cells generated by CRISPR/Cas9-mediated gene targeting are fully functional and provide a promising tool to study the steady-state kinetics of endogenous Pol II. By photobleaching of GFP-RPB1, we identified three kinetically distinct fractions of chromatin-bound Pol II. Using Monte Carlo (MC) -based modeling of Pol II kinetics, we assessed the quantitative framework of the Pol II transcription cycle and elucidated its timeframe and quantitative set-up. Our findings are highly supportive of a model in which Pol II initiation and promoter pausing are highly dynamic events of iterative cycles of Pol II chromatin binding and release. Results Generation and Characterization of GFP-RPB1 Cells. To study the in vivo kinetics of endogenous Pol II, we generated a GFP-RPB1 (POLR2A) KI cell line (MRC-5 sv40) fluorescently labeling the largest subunit of Pol II. We transiently expressed a single-guide RNA (sgRNA) to induce a CRISPR-associated protein 9 (Cas9)-mediated double-strand break (DSB) downstream of the RPB1 transcriptional start site. A repair template containing GFP cDNA flanked by homology arms comprised of the genomic RPB1 sequence (19) (Fig. S1and Fig. S1= 60 cells, two independent experiments. (= 40 cells of two independent experiments. Because GFP-RPB1 is expressed from its endogenous gene loci and RPB1 only translocates to the nucleus as part of the fully assembled Pol II complex (20), nuclear GFP fluorescence can be used as a direct readout for endogenous Pol II localization and concentration in living cells. To estimate the number of Pol II complexes, we compared the nuclear GFP intensity of KI cells to the extracellular fluorescence Salvianolic acid A of known, increasing concentrations of recombinant fluorescent GFP added to the culture medium (Fig. 1= 20 cells Salvianolic acid A of two independent experiments. Image size is 15 18 m. (= 20 cells of two independent experiments. ( 16 cells per condition measured in two independent experiments. FI chart shows mean SD. (= 3 independent experiments. (= 20 cells of two.KI cells were generated from Sv40-immortalized MRC-5 fibroblasts, as described here (19). is infrequent. However, conflicting studies have reported that promoter-paused Pol II is less stable due to repeated premature termination and chromatin release proximal to the promoter, which is accompanied by the release of short transcription start site-associated RNAs (13C16). Thus far, genome-wide dynamics of promoter-paused Pol II have been studied by Gro-Seq (8), ChIP-Seq (10, 11), or methyltransferase footprinting (15) after inhibiting Pol II initiation. While these techniques provide gene-specific snapshots of Pol II transcription, relative abundance, or position at a given time, they do not allow measurement of steady-state Pol II kinetics (i.e., chromatin binding times) in real time. Although these studies have gained insights Salvianolic acid A into the turnover of paused Pol II, most experiments have been performed after inhibiting transcription initiation by Triptolide (8, 10C12). This covalent XPB inhibitor severely affects Pol II levels (17, 18) and has been recently shown to have a slow mode of action (16), which makes it less suitable to study a potentially rapid cellular process. To overcome these limitations, we developed photobleaching Salvianolic acid A of endogenously expressed GFP-RPB1 followed by computational modeling to quantitatively assess the kinetics of Pol II in unperturbed living cells. Here we show that GFP-RPB1 knockin (KI) cells generated by CRISPR/Cas9-mediated gene targeting are fully functional and provide a promising tool Salvianolic acid A to study the steady-state kinetics of endogenous Pol II. By photobleaching of GFP-RPB1, we identified three kinetically distinct fractions of chromatin-bound Pol II. Using Monte Carlo (MC) -based modeling of Pol II kinetics, we assessed the quantitative framework of the Pol II transcription cycle and elucidated its timeframe and quantitative set-up. Our findings are highly supportive of a model in which Pol II initiation and promoter pausing are highly dynamic events of iterative cycles of Pol II chromatin binding and release. Results Generation and Characterization of GFP-RPB1 Cells. To study the in vivo kinetics of endogenous Pol II, we generated a GFP-RPB1 (POLR2A) KI cell line (MRC-5 sv40) fluorescently labeling the largest subunit of Pol Rabbit Polyclonal to VRK3 II. We transiently expressed a single-guide RNA (sgRNA) to induce a CRISPR-associated protein 9 (Cas9)-mediated double-strand break (DSB) downstream of the RPB1 transcriptional start site. A repair template containing GFP cDNA flanked by homology arms comprised of the genomic RPB1 sequence (19) (Fig. S1and Fig. S1= 60 cells, two independent experiments. (= 40 cells of two independent experiments. Because GFP-RPB1 is expressed from its endogenous gene loci and RPB1 only translocates to the nucleus as part of the fully assembled Pol II complex (20), nuclear GFP fluorescence can be used as a direct readout for endogenous Pol II localization and concentration in living cells. To estimate the number of Pol II complexes, we compared the nuclear GFP intensity of KI cells to the extracellular fluorescence of known, increasing concentrations of recombinant fluorescent GFP added to the culture medium (Fig. 1= 20 cells of two independent experiments. Image size is 15 18 m. (= 20 cells of two independent experiments. ( 16 cells per condition measured in two independent experiments. FI chart shows mean SD. (= 3 independent experiments. (= 20 cells of two independent experiments. For a more detailed analysis of Pol II kinetics, we performed FRAP in a narrow strip spanning the nucleus (Strip-FRAP) (26), allowing fluorescence measurements every 0.4 s. In line with half-nucleus FRAP, Strip-FRAP of GFP-RPB1 in nontreated (NT) cells showed a long-term immobilization of a large fraction of Pol II (Fig. 2and and Fig. S2and Fig. S2and and Fig. S2 and and and Fig. S2 and and Fig. S2 and and and and and and and rate constants (see and and Fig. S5and = 20 cells of two independent experiments, FI chart shows mean SD. Modeled, FI-corrected Pol II fraction sizes in NT KI cells or after treatment with Triptolide (and Fig. S5polytene chromosomes (9), we found that on a genome-wide average 23% of nuclear Pol II is paused for merely 42 s in human cells (Fig. 2 and cells, that have revealed that most promoter-paused Pol II is lost within 2.5 min (the earliest time point assessed by the authors) after Triptolide (15). Rapid promoter-proximal termination was also found to be the most plausible explanation for the drastic increase of promoter-paused Pol II within a.