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Live-Cell Analysis of Endogenous GFP-RPB1 Uncovers Rapid Turnover of Initiating and Promoter-Paused RNA Polymerase II

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Live-Cell Analysis of Endogenous GFP-RPB1 Uncovers Rapid Turnover of Initiating and Promoter-Paused RNA Polymerase II Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II Barbara Steurera,b, Roel C. Janssensa,b, Bart Gevertsc, Marit E. Geijera,b, Franziska Wienholza, Arjan F. Theila, Jiang Changa, Shannon Dealya, Joris Pothofa, Wiggert A. van Cappellenc, Adriaan B. Houtsmullerc, and Jurgen A. Marteijna,b,1 aDepartment of Molecular Genetics, Erasmus Medical Center, 3015 AA Rotterdam, The Netherlands; bOncode Institute, Erasmus Medical Center, 3015 AA Rotterdam, The Netherlands; and cDepartment of Pathology, Optical Imaging Centre, Erasmus Medical Center, 3015 AA Rotterdam, The Netherlands Edited by Richard A. Young, Massachusetts Institute of Technology, Cambridge, MA, and approved March 12, 2018 (received for review October 17, 2017) Initiation and promoter-proximal pausing are key regulatory steps However, conflicting studies have reported that promoter- of RNA Polymerase II (Pol II) transcription. To study the in vivo paused Pol II is less stable due to repeated premature termi- dynamics of endogenous Pol II during these steps, we generated nation and chromatin release proximal to the promoter, which fully functional GFP-RPB1 knockin cells. GFP-RPB1 photobleaching is accompanied by the release of short transcription start site- combined with computational modeling revealed four kinetically associated RNAs (13–16). distinct Pol II fractions and showed that on average 7% of Pol II are Thus far, genome-wide dynamics of promoter-paused Pol II freely diffusing, while 10% are chromatin-bound for 2.4 seconds have been studied by Gro-Seq (8), ChIP-Seq (10, 11), or meth- during initiation, and 23% are promoter-paused for only 42 sec- yltransferase footprinting (15) after inhibiting Pol II initiation. onds. This unexpectedly high turnover of Pol II at promoters is While these techniques provide gene-specific snapshots of Pol II most likely caused by premature termination of initiating and transcription, relative abundance, or position at a given time, promoter-paused Pol II and is in sharp contrast to the 23 minutes they do not allow measurement of steady-state Pol II kinetics – that elongating Pol II resides on chromatin. Our live-cell imaging (i.e., chromatin binding times) in real time. Although these CELL BIOLOGY approach provides insights into Pol II dynamics and suggests that studies have gained insights into the turnover of paused Pol II, the continuous release and reinitiation of promoter-bound Pol II is most experiments have been performed after inhibiting tran- an important component of transcriptional regulation. scription initiation by Triptolide (8, 10–12). This covalent XPB inhibitor severely affects Pol II levels (17, 18) and has been re- RNA Polymerase II | transcription | transcription dynamics | cently shown to have a slow mode of action (16), which makes it promoter-proximal pausing | live-cell imaging less suitable to study a potentially rapid cellular process. To overcome these limitations, we developed photobleaching of ukaryotic gene expression is a highly regulated process ini- endogenously expressed GFP-RPB1 followed by computational Etiated by the sequential binding of transcription factors that modeling to quantitatively assess the kinetics of Pol II in un- facilitate the recruitment of RNA polymerase II (Pol II) and the perturbed living cells. assembly of the preinitiation complex (PIC) (1). During initia- Here we show that GFP-RPB1 knockin (KI) cells generated by tion the CDK7 subunit of transcription factor (TF) IIH phos- CRISPR/Cas9-mediated gene targeting are fully functional and phorylates the serine (Ser) 5 of the C-terminal domain (CTD) of RPB1, the core catalytic subunit of Pol II, allowing Pol II to Significance engage the DNA template and to start transcribing a short stretch of RNA (2, 3). This early elongation complex is paused Transcription by RNA Polymerase II (Pol II) is a highly dynamic 30–60 nucleotides downstream of the transcription start site by process that is tightly regulated at each step of the transcrip- negative elongation factor (NELF) and DRB sensitivity-inducing tion cycle. We generated GFP-RPB1 knockin cells and developed factor (DSIF) (4, 5). The subsequent pause release into pro- photobleaching of endogenous Pol II combined with compu- ductive elongation is mediated by the positive transcription tational modeling to study the in vivo dynamics of Pol II in real elongation factor b (P-TEFb), whose Cdk9 kinase converts DSIF time. This approach allowed us to dissect promoter-paused Pol into a positive elongation factor, facilitates the eviction of NELF, II from initiating and elongating Pol II and showed that initia- and phosphorylates the RPB1 CTD on Ser2 (2). tion and promoter proximal pausing are surprisingly dynamic Traditionally it was thought that transcription is primarily events, due to premature termination of Pol II. Our study regulated by Pol II recruitment and initiation, but because of the provides new insights into Pol II dynamics and suggests that advances in genome-wide sequencing technologies, we know the iterative release and reinitiation of promoter-bound Pol II is today that mRNA output is also controlled by the tight co- an important component of transcriptional regulation. ordination of postinitiation steps (2, 5). For example, promoter- proximal pausing is a key rate-limiting step of RNA synthesis ′ Author contributions: J.A.M. designed research; B.S., R.C.J., and M.E.G. performed re- that serves as a checkpoint for 5 capping of the nascent RNA search; B.S., R.C.J., B.G., F.W., A.F.T., S.D., J.P., W.A.v.C., and A.B.H. contributed new and maintains an open chromatin structure near promoters (6). reagents/analytic tools; B.S., R.C.J., B.G., M.E.G., J.C., S.D., J.P., W.A.v.C., A.B.H., and Originally discovered as a regulatory switch of stimulus-responsive J.A.M. analyzed data; and B.S. and J.A.M. wrote the paper. genes in Drosophila (7), recent genome-wide studies have The authors declare no conflict of interest. revealed that promoter pausing is a widespread phenomenon This article is a PNAS Direct Submission. occurring on most metazoan genes (5, 8). However, despite this This open access article is distributed under Creative Commons Attribution-NonCommercial- prevalence, the dynamics of promoter-paused Pol II remain NoDerivatives License 4.0 (CC BY-NC-ND). under debate. The currently prevailing model suggests that Pol 1To whom correspondence should be addressed. Email: [email protected]. II pauses at promoters with a half-life of 5–15 min (8–12), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. serving as an integrative hub to control pause release into productive 1073/pnas.1717920115/-/DCSupplemental. elongation, while promoter-proximal termination is infrequent. www.pnas.org/cgi/doi/10.1073/pnas.1717920115 PNAS Latest Articles | 1of9 Downloaded by guest on September 28, 2021 provide a promising tool to study the steady-state kinetics of GFP cDNA flanked by homology arms comprised of the geno- endogenous Pol II. By photobleaching of GFP-RPB1, we iden- mic RPB1 sequence (19) (Fig. S1A) was cotransfected to allow tified three kinetically distinct fractions of chromatin-bound Pol repair of the DSB by homologous recombination. GFP expressing II. Using Monte Carlo (MC) -based modeling of Pol II kinetics, cells were isolated by FACS and single-cell clones of homo- we assessed the quantitative framework of the Pol II transcrip- zygous KI cells were selected (Fig. S1B)inwhichWTRPB1 tion cycle and elucidated its timeframe and quantitative set-up. was replaced by GFP-RPB1, as shown by Western blot (Fig. Our findings are highly supportive of a model in which Pol II 1A) and genotyping (Fig. S1C). Importantly, the GFP-RPB1 initiation and promoter pausing are highly dynamic events of expression level and phosphorylation status in KI cells were iterative cycles of Pol II chromatin binding and release. similar to WT cells (Fig. 1A), the GFP tag did not compro- mise the basal transcription rate (Fig. 1B and Fig. S1D), and did Results not alter gene-expression profiles (Fig. S1E). Live-cell con- Generation and Characterization of GFP-RPB1 Cells. To study the in focal imaging of KI cells revealed a nonhomogenous, nuclear vivo kinetics of endogenous Pol II, we generated a GFP-RPB1 distribution of GFP-RPB1 with multiple bright foci and ex- (POLR2A) KI cell line (MRC-5 sv40) fluorescently labeling the clusion from nucleoli (Fig. 1C). Localization of GFP-RPB1 was largest subunit of Pol II. We transiently expressed a single-guide similar to endogenous RPB1, shown by immunofluorescent RNA (sgRNA) to induce a CRISPR-associated protein 9 (Cas9)- staining of the RPB1 CTD and N-terminal domain (NTD) (Fig. mediated double-strand break (DSB) downstream of the 1D). Taken together, these data illustrate that GFP-RPB1 is RPB1 transcriptional start site. A repair template containing fully functional. ACB WT KI 200 αGFP Pol IIO αRPB1-NTD 150 Pol IIA αRPB1-CTD 100 αP-Ser2 EU intensity 50 αP-Ser5 αP-Ser7 0 αXPC (loading ctrl) WT KI 60μm 6μm 1.5 μm D E WT KI WT KI μM GFP 0.0 0.1 2.0 3.0 .0 4 DAPI DAPI brighield 10 μm 10 μm 10 μm 10 μm 25 μm 25 μm 25 μm 25 μm 25 μm GFP GFP-RPB1 GFP-RPB1 free GFP standard curve 200 Nuclear GFP fluorescence (A.U.) 79.1 ± 8.7 y = 492.73x Nuclear GFP concentraon (μM) 0.18 ± 0.02 150 R² = 0.9945 3 RPB1-NTD Nuclear volume (μm ) 733.5 ± 181.5 αRPB1-CTD α 100 FI Pol II in MRC5 nucleus 78471 ± 2140 50 Chromosomes MRC5 cell 74.9 ± 2.2 0 Pol II in diploid cell 50302 ± 1372 00.10.20.30.4 GFP (μM) GFP-RPB1 GFP-RPB1 RPB1-NTD + + αRPB1-CTD α Fig.
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