Abstract
The transcriptional machinery is thought to dissociate from DNA during replication. Certain proteins, termed epigenetic marks, must be transferred from parent to daughter DNA strands in order to maintain the memory of transcriptional states1,2. These proteins are believed to re-initiate rebuilding of chromatin structure, which ultimately recruits RNA polymerase II (Pol II) to the newly replicated daughter strands. It is believed that Pol II is recruited back to active genes only after chromatin is rebuilt3,4. However, there is little experimental evidence addressing the central questions of when and how Pol II is recruited back to the daughter strands and resumes transcription. Here we show that immediately after passage of the replication fork, Pol II in complex with other general transcription proteins and immature RNA re-associates with active genes on both leading and lagging strands of nascent DNA, and rapidly resumes transcription. This suggests that the transcriptionally active Pol II complex is retained in close proximity to DNA, with a Pol II–PCNA interaction potentially underlying this retention. These findings indicate that the Pol II machinery may not require epigenetic marks to be recruited to the newly synthesized DNA during the transition from DNA replication to resumption of transcription.
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Data availability
The data supporting the findings of this study are available from the corresponding author on reasonable request.
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Acknowledgements
The authors thank B. Calabretta and S. McMahon for feedback on experiments and the manuscript. This work was supported by the following grants: NIH R01GM075141 to A.M.; T.K.F. was supported by NIH F31GM128300 and NIH training grant T32GM100836. Leica SP8 STED microscope imaging was supported by the Bioimaging Shared Resource of the Sidney Kimmel Cancer Center (NCI 5 P30 CA-56036).
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T.K.F., S.P., H.W.B. and A.M. conceptualized the study. T.K.F. performed experiments presented in Figs. 1–5 and Extended Data Figs. 1–4, 6 and 7. S.P. performed experiments in Extended Data Fig. 5 and performed preliminary experiments pertaining to the results in Figs. 1 and 5 and Extended Data Fig. 4. S.K.K performed preliminary experiments pertaining to the results in Fig. 1. T.K.F. and S.P. designed and developed the RDIA assay. T.K.F. developed the multiplex CAA assay. T.K.F. wrote the manuscript with input from S.P., H.W.B. and A.M. S.P., H.W.B., T.K.F. and A.M. acquired the funding. A.M. provided overall supervision.
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Extended data figures and tables
Extended Data Fig. 1 CAA reactions with IgG control do not generate PLA signals.
Cells were labelled for 10 min and PLA was performed using the indicated antibodies and an IgG control. Red, PLA, Green, EdU, Blue, DAPI. Scale bar = 10 μm. Data shown are representative of 3 independent experiments.
Extended Data Fig. 2 Detection of RNA Pol II on nascent DNA is not the result of a new round of transcription.
a, Cells were treated for 30 min 2 h, or 4 h with the indicated inhibitors or left untreated as a control and then labelled with EdU for 10 min in the presence of the inhibitors and CAA was performed for Pol II S5P (left) or Pol II S2P (right). Data are presented as mean values ± s.d. (n = 3 independent experiments). For each of the following, the number of nuclei examined over 3 independent experiments are: 112 (S5P top), 153 (S5P bottom), 70 (S2P top), and 139 (S2P bottom). Statistical significance was determined by one-way ANOVA and p-values shown were determined with Tukey’s post-hoc. ns = not significant, **P < 0.01, ****P < 0.0001. b, Cells were treated for 30 min with either THZ-1 or DRB or left untreated as a control and then blotted for the indicated proteins. Data shown are representative of 3 independent experiments. For blot source data, see Supplementary Fig. 1. c, Cells were treated with either TPL or DRB or left untreated as a control and then labelled with EdU for 10 min in the presence of the inhibitors, and CAA was performed for PCNA. Data are presented as mean values ± s.d. (n = 3 independent experiments), and 117 nuclei were examined over 3 independent experiments, and statistical significance was determined by one-way ANOVA. ns = not significant. d, Cells were treated for 4 h with DRB or left untreated as a control and then labelled with EdU for 10 min in the presence of the inhibitors and CAA was performed for TBP. Data are presented as mean values ± s.d. (n = 3 independent experiments), 77 nuclei were examined over 3 independent experiments, and statistical significance was determined by two-way, unpaired t-test. ns = not significant. e, Cells were treated for 4 h with DRB or left untreated as a control and then labelled with EdU for 10 min in the presence of the inhibitors and CAA was performed for either SPT4 (top) or SPT5 (bottom). Data are presented as mean values ± s.d. (n = 3 independent experiments), and 77 (SPT4) or 88 (SPT5) nuclei were examined over 3 independent experiments. Statistical significance was determined by two-way, unpaired t-test. ****P < 0.0001.
Extended Data Fig. 3 Multiplex CAA detects proteins close to each other on labelled DNA.
a, Schematic representation of multiplex CAA illustrating that while 7.4 to 12 kb of DNA is labeled in 10 min, overlapping PLA signals are on average within 400 bp of one another. b, Quantification of the percent of overlaps following 10 min and 20 min EdU labelling for multiplex CAA with PCNA and either Pol II S5P (top) or Pol II S2P (bottom). Data are presented as mean values ± s.d. from n = 5 independent samples. Statistical significance was determined by a two-way, unpaired t-test. **P < 0.01, ****P < 0.0001. c, Direct PLA between replication proteins: Pol ε and PCNA, Pol ε and FEN1, and FEN1 and PCNA. Data are presented as mean values ± s.d. (n = 3 independent experiments), and 152 nuclei were examined over 3 independent experiments. Statistical significance was determined by one-way ANOVA and p-values shown were determined with Tukey’s post-hoc. ****P < 0.0001.
Extended Data Fig. 4 RNA polymerase II associates with target genes on nascent DNA.
a, Schematic representation of BrdU re-ChIP. DNA was labelled with BrdU (red circle) and then immunoprecipitated with anti-Pol II antibody. Following reverse crosslinking, DNA was denatured and immunoprecipitated with anti-BrdU antibody. Recovered DNA was analyzed by qPCR. Red ring is PCNA, replisome is in blue. b, Schematic representing locations of qPCR products within each analysed gene. Bent arrow represents the TSS and pA indicates the polyadenylation site. For each gene two sets of primers were designed: one close to the TSS and one farther downstream in the gene. c, Pol II ChIP. ChIP was performed for Pol II or IgG and subsequently qPCR was performed at upstream and downstream regions the active genes GAPDH, PPIA, and TNFAIP3 and at the repressed gene GFAP. d, BrdU re-ChIP following 25 min BrdU labelling. DNA was labelled with BrdU for 25 min or unlabelled as a control. ChIP was performed for Pol II or IgG. Following purification of DNA, Pol II ChIP samples were subjected to a second round of immunoprecipitation for BrdU. e, BrdU re-ChIP following 40 min BrdU labelling. DNA was labelled with BrdU for 40 min or unlabelled as a control. ChIP was performed for Pol II or IgG. Following purification of DNA, Pol II ChIP samples were subjected to a second round of immunoprecipitation for BrdU. Numbering represents primer sets shown in b. Data in c–e are presented as mean values and are representative of 2 independent experiments.
Extended Data Fig. 5 PCNA immunoprecipitates short fragments of nascent DNA.
a, Top: Primer map showing forward primer (at position zero) and reverse primers at increasing distances within the TNFAIP3 gene. Bottom: The percent of input for PCNA immunoprecipitated DNA either without S1 nuclease (left) or following digestion with 1 U S1 nuclease (right). b, Top: Primer map showing forward primer (at position zero) and reverse primers at increasing distances within the GFAP gene. Bottom: The percent of input for PCNA immunoprecipitated DNA either without S1 nuclease (left) or following digestion with 1 U S1 nuclease (right). c, m13mp18 ssDNA was treated with 1 U S1 nuclease for 10 min in order to ensure that single stranded DNA was completely digested under treatment conditions. Data in a–c are presented as mean values and are representative of 2 independent experiments.
Extended Data Fig. 6 PCNA is a mark of nascent chromatin throughout the genome.
a, PCNA ChIP. Cells were immunoprecipitated with an anti-PCNA antibody or with IgG and analysed by qPCR at upstream and downstream regions the active genes GAPDH, PPIA, and TNFAIP3 and at the repressed gene GFAP. Numbering represents primer sets shown in Extended Data Fig. 4b. Data are presented as mean values and are representative of 2 independent experiments. b, Cells were labelled for 5 min with EdU, chased for the indicated times, and then CAA was performed for PCNA. Data are presented as mean values ± s.d. (n = 3 independent experiments), and 208 nuclei were examined over 3 independent experiments. Statistical significance was determined by one-way ANOVA and p-values shown were determined with Tukey’s post-hoc. ns = not significant, ****P < 0.0001. c, Sequential ChIP with PCNA and SPT4. Upper panel: ChIP was performed for SPT4 or IgG and then assessed by PCR at the same genes indicated in Extended Data Fig. 4b. Lower panel: re-ChIP of samples first immunoprecipitated for SPT4. Following elution, samples were re-immunoprecipitated for PCNA or IgG. Following DNA purification, samples were analyzed by qPCR. Data are presented as mean values and are representative of 2 independent experiments. d, Western blotting validation of PCNA and FEN1 polyclonal antibodies generated in this study. Data shown are representative of 3 independent experiments. For blot source data, see Supplementary Fig. 1.
Extended Data Fig. 7 Pol II transfer to nascent DNA is likely not the result of LLPS and the interaction between PCNA and Pol II is DNA-independent.
a, Cells were labelled for 30 min with BrU and then labelled for 10 min with EdU in the presence of either THZ-1 or DRB or left untreated as a control. Subsequently, RDIA was performed to assess immature RNA retention post-replication. Data are presented as mean values ± s.d. (n = 3 independent experiments), and 84 nuclei were examined over 3 independent experiments. Statistical significance was determined by one-way ANOVA. ns = not significant. b, Cells were labelled for 15 min with EdU either in the presence of 2% hexanediol, 3% hexanediol, or without hexanediol as a control, then CAA was performed for Pol II S5P (top) or Pol II S2P (bottom). Data are presented as mean values ± s.d. (n = 3 independent experiments). 50 nuclei were examined over 3 independent experiments. Statistical significance was determined by one-way ANOVA. ns = not significant. c, Nuclear extracts were either treated or untreated with ethidium bromide prior to immunoprecipitation with anti-PCNA antibody and subsequent western blotting. Data shown are representative of 3 independent experiments. For blot source data, see Supplementary Fig. 1. d, Top: Cells were untreated or treated with 3 µM PCNA-I1 for the indicated times and then EdU labelled and fixed. Bottom: Cells were untreated or treated with 3 µM PCNA-I1 for 3 h and then the inhibitor was washed out for the indicated times. Cells were labelled with EdU and fixed. In both experiments, following fixation, biotin was conjugated to EdU using click chemistry and immunostained in order to assess DNA replication. Corrected cell total fluorescence (CTCF) was quantified as described in the methods. Data are presented as mean values ± s.d. (n = 3 independent experiments), and 140 (top) or 94 (bottom) nuclei were examined over 3 independent experiments. Statistical significance was determined by one-way ANOVA and p-values shown were determined by Tukey’s post-hoc. ns = not significant, *P < 0.05, **P < 0.01, ****P < 0.0001.
Supplementary information
Supplementary Information
This file contains Supplemental Table 1: List of PCR primers used in this study; and Supplementary Fig. 1: Source western blot images.
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Fenstermaker, T.K., Petruk, S., Kovermann, S.K. et al. RNA polymerase II associates with active genes during DNA replication. Nature 620, 426–433 (2023). https://doi.org/10.1038/s41586-023-06341-9
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DOI: https://doi.org/10.1038/s41586-023-06341-9
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