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CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing

Nature volume 479, pages 74–79 (2011)Cite this article

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Abstract

Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.

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Figure 1: Binding of CTCF to exon 5 of CD45 DNA is associated with inclusion of exon 5 in CD45 transcripts.
Figure 2: CTCF depletion leads to reduced exon 5 inclusion in CD45 transcripts.
Figure 3: CTCF binding at CD45 exon 5 DNA facilitates exon 5 inclusion in CD45 transcripts through local pol II pausing.
Figure 4: 5-methylcytosine levels (5-mC) are inversely related to CTCF binding and exon 5 inclusion.
Figure 5: Global identification of CTCF-dependent exons.

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Primary accessions

Gene Expression Omnibus

Data deposits

All data sets in this publication are available in the NCBI Gene Expression Omnibus accession number GSE31278.

Change history

  • 03 November 2011

    Panel labelling in Fig. 3e was corrected.

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Acknowledgements

We thank A. Rao, C. Burge and K. Lynch for critical reading of this manuscript. We also thank A. Rao for reagents and K. Nyswaner and M. Prigge for technical assistance. This work is supported by the Intramural Research Program of NIH, the National Cancer Institute, The Center for Cancer Research (S.O., P.O., M.K.), and the Swedish Research Council Foundation and the Foundation for Strategic Research (R.S.).

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Authors and Affiliations

  1. Center for Cancer Research, Mouse Cancer Genetics Program, National Cancer Institute at Frederick, Frederick, 21702, Maryland, USA

    Sanjeev Shukla, Melissa Gregory, Bojan Shutinoski, Philipp Oberdoerffer & Shalini Oberdoerffer

  2. Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden,

    Ersen Kavak & Rickard Sandberg

  3. Ludwig Institute for Cancer Research, SE-171 77, Stockholm, Sweden ,

    Ersen Kavak & Rickard Sandberg

  4. Center for Cancer Research, Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute at Frederick, Frederick, 21702, Maryland, USA

    Masahiko Imashimizu & Mikhail Kashlev

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Contributions

S.S. performed ChIP, MedIP and EMSA. M.G. and S.S. performed lentiviral transductions, transfections, flow cytometry, and qPCR. E.K. analysed ChIP and RNA-seq data. M.I. performed in vitro transcription. S.S. and B.S. cloned the minigenes. All authors designed experiments and M.K., P.O., R.S. and S.O. supervised the project. S.O. and R.S. wrote the text. P.O. edited the text.

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Correspondence to Shalini Oberdoerffer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-10 with legends and Supplementary Tables 1-3. (PDF 1904 kb)

Supplementary Table 4

The table shows a list of exons with significantly different inclusion levels after CTCF knock-down. (XLS 1395 kb)

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Shukla, S., Kavak, E., Gregory, M. et al. CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing. Nature 479, 74–79 (2011). https://doi.org/10.1038/nature10442

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