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

Journal name:
Nature
Volume:
479,
Pages:
74–79
Date published:
DOI:
doi:10.1038/nature10442
Received
Accepted
Published online
Corrected online

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.

At a glance

Figures

  1. Binding of CTCF to exon 5 of CD45 DNA is associated with inclusion of exon 5 in CD45 transcripts.
    Figure 1: Binding of CTCF to exon 5 of CD45 DNA is associated with inclusion of exon 5 in CD45 transcripts.

    a, CTCF ChIP in murine splenocytes and quantitative PCR (qPCR) relative to rabbit Ig control ChIP (n = 2). b, Cell-surface staining for CD45RA (exon 4-containing) and CD45RB (exon 5-containing) isoforms and total CD45 (pan) in parental BL41 B cells (RBhigh), cell-culture-derived CD45RB bimodal cells, and CD45RB low (RBlow) cells sorted from the bimodal BL41 population. c, GAPDH-normalized qRT–PCR data from RBhigh and RBlow cells using the indicated junction-spanning primers (n = 3). d, CTCF ChIP in BJAB, RBhigh and RBlow cells and qPCR for CD45 exons and introns (n = 3–6). All graphs show mean values ±standard deviation (s.d.). P = two-tailed Student’s t test comparing the indicated samples.

  2. CTCF depletion leads to reduced exon 5 inclusion in CD45 transcripts.
    Figure 2: CTCF depletion leads to reduced exon 5 inclusion in CD45 transcripts.

    a, Cell-surface CD45RB isoform and total CD45 expression in cells transduced with short hairpin RNA (shRNA)against CTCF (CTCF-sh3 and/or sh-4) or control shRNA against red fluorescent protein (RFP). b, c, qRT–PCR in CTCF-depleted RBlow (b) and BJAB cells (c) from a to detect CD45 (left) and CTCF (right) mRNA levels (n = 3). Graphs show mean values ±s.d. P, two-tailed Student’s t test.

  3. CTCF binding at CD45 exon 5 DNA facilitates exon 5 inclusion in CD45 transcripts through local pol II pausing.
    Figure 3: CTCF binding at CD45 exon 5 DNA facilitates exon 5 inclusion in CD45 transcripts through local pol II pausing.

    a, RNA pol II ChIP and qPCR relative to mouse Ig control IP (n = 3). b, CTCF ChIP in RBhigh cells transduced with shRNA against CTCF versus shRFP-transduced cells and qPCR relative to rabbit Ig control IP (n = 2). c, RNA pol II ChIP of RBhigh cells from b and qPCR relative to mouse Ig control IP (n = 2). d, In vitro transcription with a DNA oligo incorporating a CTCF binding site at position 26 relative to elongation complex assembly. Recombinant CTCF and TFIIS protein were introduced as indicated, with variable effects on pausing at adenine 21 (A21). e, Representation of CD45 minigenes with wild-type (I3-I7) or mutated exon 5 CTCF binding site (I3-I7*CTCF), used in fj. f, CTCF-ChIP in NIH3T3 and CHO cells transfected with the CD45 minigenes and qPCR relative to rabbit Ig control IP. Error bars represent standard error of the mean (s.e.m.) (n = 3). gi, qRT–PCR from minigene-transfected HEK293, NIH3T3 and CHO cells to detect the junctions of exons 4/5 (g), 5/6 (h) and 4/6 (i) relative to exon 6 (n = 3). j, RNA pol II ChIP in CHO cells transfected with the CD45 minigenes and qPCR relative to mouse Ig control IP (n = 3). Unless indicated otherwise, graphs show mean values ±s.d. P, two-tailed Student’s t test.

  4. 5-methylcytosine levels (5-mC) are inversely related to CTCF binding and exon 5 inclusion.
    Figure 4: 5-methylcytosine levels (5-mC) are inversely related to CTCF binding and exon 5 inclusion.

    a, Methylated DNA immunoprecipitation (MedIP) in B cell line genomic DNA and qPCR relative to input (n = 5). b, Representative CD45 isoform expression in primary peripheral human CD3+ T cells sorted on the basis of cell-surface CD45RB and CD45RO. c, MedIP and qPCR relative to input in sorted primary human CD3+ T cells (n = 6, compiled from two donors). d, CTCF-ChIP and qPCR relative to rabbit Ig control IP, in sorted primary CD3+ T cells (n = 2). e, MedIP and qPCR relative to input in BL41 RBlow cells transduced with shRNA against DNMT1 versus shRFP-transduced cells (n = 3). f, CTCF ChIP in cells from e and qPCR relative to rabbit Ig control IP (n = 3). g, Cell-surface CD45RB expression in cells from e. h, RNA pol II ChIP and qPCR in cells from e relative to mouse Ig control IP (n = 3). Unless indicated otherwise, graphs show mean values ±s.d. P, two-tailed Student’s t test.

  5. Global identification of CTCF-dependent exons.
    Figure 5: Global identification of CTCF-dependent exons.

    a, Alternative exons were classified on the basis of the relative location of an exclusive CTCF peak within 1kb of the exon. b, Difference in the mean exon inclusion level between bimodal BL41 cells transduced with shRNA against CTCF versus shRFP-transduced cells (from Fig. 2a) for exons with CTCF peak in upstream (blue) or downstream regions (red) but not in the exon body and for exons with no CTCF binding (black). The mean ±s.e.m. for each class of exons is plotted against increasing Bayes factor thresholds. *P<0.05, **P<0.01, ***P<0.001, Wilcoxon rank sum test for differences in exon inclusion at the different thresholds. c, Same as b for BJAB shCTCF compared with wild-type BJAB cells (from Fig. 2a). d, Normalized CD4+ T cell RNA pol II read signal centred on the alternative exon or the corresponding downstream CTCF peak summit.

Accession codes

Primary accessions

Gene Expression Omnibus

Change history

Corrected online 03 November 2011
Panel labelling in Fig. 3e was corrected.

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Author information

Affiliations

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

    • Masahiko Imashimizu &
    • Mikhail Kashlev

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

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

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Supplementary information

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  1. Supplementary Information (1.8M)

    The file contains Supplementary Figures 1-10 with legends and Supplementary Tables 1-3.

Excel files

  1. Supplementary Table 4 (1.3M)

    The table shows a list of exons with significantly different inclusion levels after CTCF knock-down.

Additional data