Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex (original) (raw)

Nature volume 523, pages 621–625 (2015)Cite this article

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Abstract

DNA methylation at selective cytosine residues (5-methylcytosine (5mC)) and their removal by TET-mediated DNA demethylation are critical for setting up pluripotent states in early embryonic development1,2. TET enzymes successively convert 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), with 5fC and 5caC subject to removal by thymine DNA glycosylase (TDG) in conjunction with base excision repair1,2,3,4,5,6. Early reports indicate that 5fC and 5caC could be stably detected on enhancers, promoters and gene bodies, with distinct effects on gene expression, but the mechanisms have remained elusive7,8. Here we determined the X-ray crystal structure of yeast elongating RNA polymerase II (Pol II) in complex with a DNA template containing oxidized 5mCs, revealing specific hydrogen bonds between the 5-carboxyl group of 5caC and the conserved epi-DNA recognition loop in the polymerase. This causes a positional shift for incoming nucleoside 5′-triphosphate (NTP), thus compromising nucleotide addition. To test the implication of this structural insight in vivo, we determined the global effect of increased 5fC/5caC levels on transcription, finding that such DNA modifications indeed retarded Pol II elongation on gene bodies. These results demonstrate the functional impact of oxidized 5mCs on gene expression and suggest a novel role for Pol II as a specific and direct epigenetic sensor during transcription elongation.

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Primary accessions

Gene Expression Omnibus

Protein Data Bank

Data deposits

GRO-seq data have been deposited in the Gene Expression Omnibus database under accession GSE64748. Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accessions 4Y52 and 4Y7N for EC-I and EC-II, respectively.

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Acknowledgements

D.W. acknowledges the National Institutes of Health (NIH) (GM102362), a Kimmel Scholars award from the Sidney Kimmel Foundation for Cancer Research, and start-up funds from the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego. This work was also supported by NIH grant HG006827 and the Howard Hughes Medical Institute to C.H., and NIH grants GM052872 and HG004659 to X.-D. F. We are grateful to C. Kaplan for providing Saccharomyces cerevisiae Pol II Rpb2 Q531H and Q531A mutant strains.

Author information

Author notes

  1. Lanfeng Wang, Yu Zhou, Liang Xu and Rui Xiao: These authors contributed equally to this work.

Authors and Affiliations

  1. Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA
    Lanfeng Wang, Liang Xu, Jenny Chong & Dong Wang
  2. Department of Cellular and Molecular Medicine, School of Medicine, The University of California, San Diego, 9500 Gilman Drive, La Jolla, 92093, California, USA
    Yu Zhou, Rui Xiao, Liang Chen, Hairi Li & Xiang-Dong Fu
  3. Department of Chemistry, Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, 60637, Illinois, USA
    Xingyu Lu & Chuan He

Authors

  1. Lanfeng Wang
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  2. Yu Zhou
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  3. Liang Xu
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  4. Rui Xiao
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  5. Xingyu Lu
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  6. Liang Chen
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  7. Jenny Chong
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  8. Hairi Li
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  9. Chuan He
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  10. Xiang-Dong Fu
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  11. Dong Wang
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Contributions

D.W. conceived the original idea and, together with X.-D.F., designed the experiments. X.L. carried out synthesis of DNA templates. J.C., L.W. and D.W. purified Pol II. L.W. and D.W. performed crystallization, data collection and structural refinement. L.X. performed the in vitro transcription assay. Y.Z., R.X., L.C. and H.L. performed the in vivo GRO-seq assay. L.W., Y.Z., L.X., R.X., X.L., J.C., C.H., X.-D.F. and D.W. wrote the paper.

Corresponding authors

Correspondence toXiang-Dong Fu or Dong Wang.

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Extended data figures and tables

Extended Data Figure 1 Electron density maps of Pol II EC-I and EC-II.

a, 2_F_o − F_c map (blue) of Rpb2 Q531 in epi-DNA recognition loop and the opposite 5caC in Pol II EC-I, contoured at 1.0_σ. b, _F_o − F_c omit map (green) of Pol II EC-I (with 5caC omission), contoured at 3.0_σ. c, 2_F_o − F_c map (blue) of GMPCPP paired with 5caC in Pol II EC-II, contoured at 1.0_σ. d, _F_o − F_c omit map (green) of Pol II EC-II (with GMPCPP and 5caC omission), contoured at 3.0_σ.

Extended Data Figure 2 Structural comparison between Pol II EC-I, EC-II and Pol II EC containing unmodified C template and a matched GTP.

a, Superimposition of Pol II EC-I and EC-II structures. Rpb2 Q531 and 5caC in EC-II are in magenta to differentiate between those counterparts in EC-I. These two structures are aligned using the bridge helix (BH) region (Rpb1 822–840). b, Superposition of Pol II EC-II containing 5caC template and GMPCPP with Pol II EC with closed trigger loop (TL; containing unmodified C template and GTP; PDB accession 2E2H). The two structures are aligned using the bridge helix region (Rpb1 822–840).

Extended Data Figure 3 Kinetic study of GTP incorporation opposite 5caC template by purified Pol II proteins.

a–c, Representative kinetic parameter fitting curves from three independent experiments for GTP incorporation opposite 5caC template for Pol II wild type (WT; a), Pol II Q531H (b) and Pol II Q531A (c). d, Purified Pol II wild-type, Pol II Q531H and Pol II Q531A proteins used in the in vitro transcription experiments.

Extended Data Figure 4 Modelling potentially similar interactions for recognition of 5fC and 5caC templates, but not for 5hmC, 5mC and C templates.

a, Hydrogen bonds (black dotted lines) between Rpb2 Q531, 5caC and GMPCPP in EC-II. b, Model of the interaction between Pol II EC with 5fC template through the same hydrogen-bond interaction network. c, Model of Pol II EC with 5hmC template reveals no obvious hydrogen bonding between Q531 and 5hmC. The 5hmC nucleotide structure was based on PDB accession 4R2C. d, Model of Pol II EC with 5mC template. e, Model of Pol II EC with unmodified C template. The above models were derived from the Pol II EC-II structure.

Extended Data Figure 5 Sequence alignment of Pol II epi-DNA recognition loop across different species.

a, Pol II epi-DNA recognition loop (Rpb2 521–541) is conserved from fungi to human and strictly conserved among several fungal species, highlighted with magenta dotted rectangle, which contain active TET/JBP enzymes18. Key residues in the loop are highlighted in the green box. b, Hydrogen bonds (black dotted lines) between yeast Pol II Rpb2 Q531, 5caC and GMPCPP in EC-II. c, Model of human Pol II with the functionally equivalent His substitution based on EC-II structure. d, Comparison between Q531 and H531 substitution reveals the similar hydrogen-bonding interaction.

Extended Data Figure 6 Human Pol II slows down at 5caC template in comparison with unmodified template in the context of HeLa nuclear extract.

The relative transcription elongation rate is normalized by the transcription elongation rate (_k_obs) from unmodified template. The relative rates from unmodified template and 5caC template are coloured in black and grey, respectively. The error bars are standard deviations derived from three independent experiments.

Extended Data Figure 7 Comparison transcription on 5caC template with unmodified template using purified yeast Pol II and E. coli RNAP.

Top, comparison of yeast Pol II; bottom, comparison of E. coli RNAP. Time points are 0, 5 s, 15 s, 30 s, 1 min, 5 min, 20 min, and 1 h (left to right). The top panel is identical to Fig. 1c and is placed here for direct comparison. nt, nucleotides.

Extended Data Figure 8 Correlation between two replicates of GRO-seq data sets at different assay points.

GRO-seq replicates (−1 and −2) were pairwise compared gene by gene on the normalized number of reads for wild-type (WT; left) and TDG-knockout (KO; right) samples. The colours show the density of points or genes. The Pearson correlation coefficients were calculated from the points and are shown on the top of each subfigure. rpm, reads per million total reads.

Extended Data Table 1 Data collection and refinement statistics

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Wang, L., Zhou, Y., Xu, L. et al. Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex.Nature 523, 621–625 (2015). https://doi.org/10.1038/nature14482

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Editorial Summary

Pol II as a DNA methylation sensor

Epigenetic DNA methylation — to produce 5-methylcytosine (5mC) residues — is an important gene transcription regulator recognized by various protein readers. 5mC can be oxidized by TET enzymes to produce 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). This study of the structure and biochemistry of RNA polymerase II (Pol II) assembled on DNA containing 5caC suggests that Pol II is able to function as an epigenetic DNA modification reader by specifically recognizing 5caC and 5fC during transcription elongation. Pol II can sense the oxidized methylation state of DNA and transiently slows down during transcription. The authors propose that Pol II may act as a direct sensor for a variety of DNA modification and damage events to instruct distinct downstream pathways.