Letter | Published:

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

Nature volume 523, pages 621625 (30 July 2015) | Download Citation

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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Accessions

Primary accessions

Gene Expression Omnibus

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

    • Lanfeng Wang
    • , Yu Zhou
    • , Liang Xu
    •  & Rui Xiao

    These authors contributed equally to this work.

Affiliations

  1. Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, 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, California 92093, 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, Illinois 60637, USA

    • Xingyu Lu
    •  & Chuan He

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Xiang-Dong Fu or Dong Wang.

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https://doi.org/10.1038/nature14482

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