Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Pol II directly recognizes 5caC during transcription.
Figure 2: Interaction between 5caC and epi-DNA recognition loop compromises GTP incorporation.
Figure 3: Similar ‘above-the-bridge-helix’ translocation intermediates captured in pausing/arrested Pol II ECs and a common 5caC-recognition mode shared by a variety of 5caC-recognition proteins.
Figure 4: Impact of 5fC/5caC on Pol II transcription elongation in mouse ES cells.

Similar content being viewed by others

Accession codes

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.

References

  1. Pastor, W. A., Aravind, L. & Rao, A. TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nature Rev. Mol. Cell Biol. 14, 341–356 (2013)

    Article  CAS  Google Scholar 

  2. Wu, H. & Zhang, Y. Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell 156, 45–68 (2014)

    Article  CAS  Google Scholar 

  3. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009)

    Article  ADS  CAS  Google Scholar 

  4. Pfaffeneder, T. et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew. Chem. Int. Ed. Engl. 50, 7008–7012 (2011)

    Article  CAS  Google Scholar 

  5. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011)

    Article  ADS  CAS  Google Scholar 

  6. He, Y. F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011)

    Article  ADS  CAS  Google Scholar 

  7. Song, C. X. et al. Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153, 678–691 (2013)

    Article  CAS  Google Scholar 

  8. Shen, L. et al. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153, 692–706 (2013)

    Article  CAS  Google Scholar 

  9. Klose, R. J. & Bird, A. P. Genomic DNA methylation: the mark and its mediators. Trends Biochem. Sci. 31, 89–97 (2006)

    Article  CAS  Google Scholar 

  10. Moore, L. D., Le, T. & Fan, G. DNA methylation and its basic function. Neuropsychopharmacology 38, 23–38 (2013)

    Article  CAS  Google Scholar 

  11. Spruijt, C. G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013)

    Article  CAS  Google Scholar 

  12. Iurlaro, M. et al. A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 14, R119 (2013)

    Article  Google Scholar 

  13. Hashimoto, H. et al. Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence. Genes Dev. 28, 2304–2313 (2014)

    Article  Google Scholar 

  14. Kellinger, M. W. et al. 5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription. Nature Struct. Mol. Biol. 19, 831–833 (2012)

    Article  CAS  Google Scholar 

  15. Huang, Y. & Rao, A. New functions for DNA modifications by TET-JBP. Nature Struct. Mol. Biol. 19, 1061–1064 (2012)

    Article  CAS  Google Scholar 

  16. Cramer, P., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: RNA polymerase II at 2.8 Ångstrom resolution. Science 292, 1863–1876 (2001)

    Article  ADS  CAS  Google Scholar 

  17. Wang, D., Bushnell, D. A., Westover, K. D., Kaplan, C. D. & Kornberg, R. D. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 127, 941–954 (2006)

    Article  CAS  Google Scholar 

  18. Brueckner, F. & Cramer, P. Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation. Nature Struct. Mol. Biol. 15, 811–818 (2008)

    Article  CAS  Google Scholar 

  19. van Luenen, H. G. et al. Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania. Cell 150, 909–921 (2012)

    Article  CAS  Google Scholar 

  20. Iyer, L. M. et al. Lineage-specific expansions of TET/JBP genes and a new class of DNA transposons shape fungal genomic and epigenetic landscapes. Proc. Natl Acad. Sci. USA 111, 1676–1683 (2014)

    Article  ADS  CAS  Google Scholar 

  21. Korzheva, N. et al. A structural model of transcription elongation. Science 289, 619–625 (2000)

    Article  ADS  CAS  Google Scholar 

  22. Silva, D. A. et al. Millisecond dynamics of RNA polymerase II translocation at atomic resolution. Proc. Natl Acad. Sci. USA 111, 7665–7670 (2014)

    Article  ADS  CAS  Google Scholar 

  23. Wang, D., Zhu, G. Y., Huang, X. H. & Lippard, S. J. X-ray structure and mechanism of RNA polymerase II stalled at an antineoplastic monofunctional platinum-DNA adduct. Proc. Natl Acad. Sci. USA 107, 9584–9589 (2010)

    Article  ADS  CAS  Google Scholar 

  24. Walmacq, C. et al. Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage. Mol. Cell 46, 18–29 (2012)

    Article  CAS  Google Scholar 

  25. Weixlbaumer, A., Leon, K., Landick, R. & Darst, S. A. Structural basis of transcriptional pausing in bacteria. Cell 152, 431–441 (2013)

    Article  CAS  Google Scholar 

  26. Lindsey-Boltz, L. A. & Sancar, A. RNA polymerase: the most specific damage recognition protein in cellular responses to DNA damage? Proc. Natl Acad. Sci. USA 104, 13213–13214 (2007)

    Article  ADS  CAS  Google Scholar 

  27. Hanawalt, P. C. & Spivak, G. Transcription-coupled DNA repair: two decades of progress and surprises. Nature Rev. Mol. Cell Biol. 9, 958–970 (2008)

    Article  CAS  Google Scholar 

  28. Zhang, L. et al. Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA. Nature Chem. Biol. 8, 328–330 (2012)

    Article  CAS  Google Scholar 

  29. Polymenidou, M. et al. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nature Neurosci. 14, 459–468 (2011)

    Article  CAS  Google Scholar 

  30. Sarker, A. H. et al. Recognition of RNA polymerase II and transcription bubbles by XPG, CSB, and TFIIH: insights for transcription-coupled repair and Cockayne syndrome. Mol. Cell 20, 187–198 (2005)

    Article  CAS  Google Scholar 

  31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  32. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  33. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  34. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, 2002)

    Google Scholar 

  35. Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008)

    Article  ADS  CAS  Google Scholar 

  36. Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011)

    Article  CAS  Google Scholar 

  37. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

    Article  Google Scholar 

  38. Karolchik, D. et al. The UCSC Genome Browser database: 2014 update. Nucleic Acids Res. 42, D764–D770 (2014)

    Article  CAS  Google Scholar 

Download references

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
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liang Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rui Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xingyu Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Liang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jenny Chong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hairi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chuan He
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xiang-Dong Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Dong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

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 to Xiang-Dong Fu or Dong Wang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

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

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

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 (kobs) 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
Full size table

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature14482

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing