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A lncRNA regulates alternative splicing via establishment of a splicing-specific chromatin signature

Nature Structural & Molecular Biology volume 22, pages 370–376 (2015)Cite this article

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Abstract

Alternative pre-mRNA splicing is a highly cell type–specific process essential to generating protein diversity. However, the mechanisms responsible for the establishment and maintenance of heritable cell-specific alternative-splicing programs are poorly understood. Recent observations point to a role of histone modifications in the regulation of alternative splicing. Here we report a new mechanism of chromatin-mediated splicing control involving a long noncoding RNA (lncRNA). We have identified an evolutionarily conserved nuclear antisense lncRNA, generated from within the human FGFR2 locus, that promotes epithelial-specific alternative splicing of FGFR2. The lncRNA acts through recruitment of Polycomb-group proteins and the histone demethylase KDM2a to create a chromatin environment that impairs binding of a repressive chromatin-splicing adaptor complex important for mesenchymal-specific splicing. Our results uncover a new function for lncRNAs in the establishment and maintenance of cell-specific alternative splicing via modulation of chromatin signatures.

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Figure 1: Polycomb-complex components in FGFR2 alternative-splicing regulation.
Figure 2: EZH2 promotes FGFR2 exon IIIb inclusion via KDM2a.
Figure 3: A lncRNA antisense to FGFR2 promotes exon IIIb inclusion.
Figure 4: asFGFR2 effect on FGFR2 splicing is independent of Argonaute and Dicer.
Figure 5: asFGFR2 induces an epithelial-specific chromatin signature in FGFR2.
Figure 6: asFGFR2's effect on alternative splicing is dependent on the IIIc region and KDM2a.

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Acknowledgements

We thank V.J. Bardwell (University of Minnesota), R. Carstens (University of Pennsylvania), C.S. Duckett (University of Michigan), M. Garcia-Blanco (Duke University), K. Helin (Biotech Research and Innovation Centre, University of Copenhagen), H. Kimura (Osaka University), H. Koseki (RIKEN Center for Integrative Medical Sciences), R. Klose (Oxford University), D. Reinberg (New York University School of Medicine), K. Tominaga (Jichi Medical School) and D. Haber (Massachusetts General Hospital) for reagents; A. Farcas (R. Klose laboratory) for developing the anti-KDM2b antibody; S. Martinez (R.F.L. laboratory) for establishing the epithelial-to-mesenchymal-transition system; and P. Scaffidi, N. Rascovan, K. Meaburn and R. Klose for discussions and critical reading of the manuscript. This work was supported by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research (T.M., R.F.L. and T.A.D.), the French Fondation de la Recherche Medical starting grant AJE201132 (R.F.L., I.G. and E.A.), the Fondation ARC pour la Recherche sur le Cancer under the program ATIP-AVENIR (I.G.), the European EpiGeneSys network of excellence (E.A.) and the Chilean Millennium Science Initiative grant P10/063-F (R.M. and K.G.).

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Author notes

  1. Tom Misteli and Reini F Luco: These authors contributed equally to this work.

Authors and Affiliations

  1. ATIP-AVENIR team, Institute of Human Genetics, CNRS UPR 1142, Montpellier, France

    Inma Gonzalez, Eneritz Agirre & Reini F Luco

  2. Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile

    Roberto Munita & Katia Gysling

  3. National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Travis A Dittmer & Tom Misteli

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  1. Inma Gonzalez
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Contributions

I.G. performed ChIP experiments, RNase protection assays and biotinylated-RNA pulldowns; R.M. performed RACE experiments under K.G.'s supervision; E.A. performed the bioinformatic analysis; T.A.D. performed coimmunoprecipitations and western blots; R.F.L. designed the study; R.F.L. and T.M. discussed and wrote the paper.

Corresponding authors

Correspondence to Tom Misteli or Reini F Luco.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 A chromatin signature characteristic of FGFR2 splicing pattern.

(a) Schematic representation of FGFR2 locus and the oligonucleotide pairs used in ChIP (see Supplementary Table 1 for details), together with general expression levels of FGFR2 exons 13-14 and the alternatively spliced exons IIIb and IIIc in epithelial PNT2 cells (in red) and mesenchymal hMSC cells (in black). qRT-PCR values (mean ± SEM, n=6) are normalized to Cyclophilin A. (b-q) Enrichment levels of H3K27me3, H3K9me3, H3K36me3, H3K36me2, HP1α, BCOR, BMI1 and RING1B along FGFR2 and control alternatively spliced CD44 or HMGA1 loci in PNT2 (red) and hMSC (black) cells. ChIP values (mean ± SEM of n=2-5) are either normalized to H3 or depicted as the percentage of input. No antibody ChIP is used as negative control in PNT2 (yellow) and hMSC (light grey) cells. The transcription start site of p15 is used as a positive control in BCOR, BMI1 and RING1B ChIPs. * p<0.05 and ** p<0.01 in two tailed Student’s t-test relative to hMSC values. (r) mRNA expression levels of EZH2, SUZ12, KDM2a, KDM2b, CBX8 and HP1α in PNT2 and hMSC cells. qRT-PCR values (mean ± SEM, n=3) are normalized to Cyclophilin A. (s) Western blot (WB) of KDM2a (upper panel) and CBX8 or SUZ12 (lower panels) as positive IP controls, after immunoprecipitation (IP) with CBX8 (lane 4) or SUZ12 (lane 5) in PNT2 cells. No antibody IP is used as a negative control (lane 3) and serial dilutions of the total input are used as a reference (lane 1 and 2).

Supplementary Figure 2 EZH2 overexpression promotes exon IIIb inclusion in mesenchymal cells.

(a-r) Inclusion levels of FGFR2 exons IIIb, IIIc constitutive e14 (a-d), alternatively spliced control CD44-v6 and HMGA1-e2 (e-f), PTB-dependent spliced PKM2-e9 and TPM2-e7 (g-h), FGFR2 splicing regulators ESRP1, ESRP2, TIA1, RBFOX2, hnRNPA1, hnRNPH and PTB (i-o) and KDM2a, EZH2 and PHF1 (p-r) after combined overexpression of the H3K27 methyltransferase EZH2 and its co-factor PHF1 for 72h in PNT2 (red) and hMSC (black) cells. qRT-PCR values (mean ± SEM, n=9) are normalized to Cyclophilin A or total mRNA of the corresponding gene. (s-v) Inclusion levels of FGFR2 exons IIIb, IIIc and control CD44-v6 in hMSC after overexpression for 72h of EZH2 alone. qRT-PCR values (mean ± SEM, n=6) are normalized to total mRNA of the corresponding gene and depicted as the x-fold change of transfection with empty vector (mock). * p<0.01 and ** p<0.001 in two tailed Student’s t-test relative to mock transfection. IIIb/IIIc: splicing ratio of exons IIIb and IIIc.

Supplementary Figure 3 KDM2a overexpression promotes exon IIIb inclusion in mesenchymal cells.

Inclusion levels of FGFR2 exons IIIb, IIIc constitutive e14 (a-d), alternatively spliced control CD44-v6 and HMGA1-e2 (e-f), PTB-dependent spliced PKM2-e9 and TPM2-e7 (g-h), FGFR2 splicing regulators ESRP1, ESRP2, TIA1, RBFOX2, hnRNPA1, hnRNPH and PTB (i-o) and MRG15 and KDM2a (p-q) after overexpression of KDM2a for 72h in PNT2 (red) and hMSC (black) cells. qRT-PCR values (mean ± SEM, n=6-9) are normalized to Cyclophilin A or total mRNA of the corresponding gene. * p<0.01 and ** p<0.001 in two tailed Student’s t-test relative to mock. IIIb/IIIc: splicing ratio of spliced exons IIIb and IIIc.

Supplementary Figure 4 EZH2 or KDM2a downregulation reduces exon IIIb inclusion in epithelial cells.

Expression levels of FGFR2 exons IIIb, IIIc, control CD44-v6 and HMGA1-e2, FGFR2 splicing regulators ESRP1, PTB, TIA1 and RBFOX2 and EZH1 and EZH2 or KDM2a after downregulation for 72h of EZH1 and EZH2 (a-k) or KDM2a (l-u) in PNT2 (red) and hMSC (black) cells. qRT-PCR values (mean ± SEM, n=6) are normalized to total mRNA of the corresponding gene and depicted as the x-fold change of transfection with a negative control siRNA. * p<0.05 and ** p<0.01 in two tailed Student’s t-test relative to negative siRNA. IIIb/IIIc: splicing ratio of exons IIIb and IIIc.

Supplementary Figure 5 EZH2 and KDM2a induce changes in FGFR2 chromatin.

Enrichment levels of H3K27me3, KDM2a, H3K36me3, H3K36me2 and MRG15 along FGFR2 and control HMGA1 loci after combined expression of EZH2+PHF1 (a-f) or KDM2a (g-j) for 72h in hMSCs. Note that the amplitude of reduction in H3K36me2 and K36me3 levels are comparable to the fold increase in H3K27me3 levels. ChIP values (mean ± SEM, n=3-4) are normalized to control H3. * p<0.05 and ** p<0.01 in two tailed Student’s t-test relative to mock.

Supplementary Figure 6 Expression of a nuclear lncRNA antisense to FGFR2 when exon IIIb is included.

(a-c) Enrichment levels of H3K4me3, using two different antibodies, and the transcription initiation complex RNA polymerase II phosphorylated in serine 5 along FGFR2 locus in PNT2 (red) and hMSC (black) cells. No antibody control is shown for PNT2 (yellow) and hMSC (grey) cells. ChIP values (mean ± SE, n=3) are normalized to H3 and total RNA Pol II, respectively. The transcription start site of H1 is used as a positive control in ser5 Pol II ChIP. * p<0.05 and ** p<0.01 in two tailed Student’s t-test relative to hMSCs. (d) UCSC browser mapping of FGFR2 locus (blue), asFGFR2 transcription start site (5’RACE, orange) and end (3’RACE, orange) and the full sequence of asFGFR2 (red). A human EST (CV310688.1) antisense to FGFR2 that shares 99.4% identity with asFGFR2 and three mouse ESTs (BI854242.1, BG975446.1, BG863185.1) with 89%, 91.4% and 89% homology, respectively, are shown as an evidence of evolutionary conservation of the lncRNA. Conservation values of FGFR2 alternative splicing region and asFGFR2 with 46 vertebrate species are depicted in green. (e) Representative Northern blots (n=4) in MCF7, PNT2 and hMSC cells detecting different regions of asFGFR2. The lncRNA was detected with probes complementary to the region identified by 5’RACE, antisense IIIc and antisense intron i8. Probes complementary to antisense exon IIIb, antisense e7 and sense intron i8 were used to verify that the detected band is not an artifact nor corresponds to sense FGFR2 RNA. As a loading control, we used a probe complementary to sense GAPDH (lower panel). The GAPDH panel corresponding to as(i8-IIIc) and 5’RACE loading control comes from the same gel. A band of ~1 kbp was detected in MCF7 and PNT2 cells, but not hMSCs, and it spans the 5’RACE region, antisense IIIc and stops along intron i8, before reaching antisense exon IIIb. A scheme of FGFR2 loci and the position of the probes used (colored arrows) is represented on top of the Northern blots for better comprehension. (f) Inclusion levels of FGFR2 exon IIIb and IIIc in the cell lines used for Northern blot. MCF7 and PNT2 include IIIb, CRL and hMSC include IIIc and FGFR2 is not expressed in PRC30. qRT-PCR values (mean ± SEM, n=3) are normalized to total FGFR2 mRNA levels. (g) Strand-specific RT-PCR for detecting asFGFR2 in MCF7 (dark red), PNT2 (red) and hMSC (black) cells. qRT-PCR values (mean ± SEM, n=6) are depicted as the x-fold change relative to an RT with no primer. The asFGFR2 is detected by amplification of the 5’RACE-IIIc region. As a negative control, we amplified a region upstream to the cDNA retrotranscribed by the strand-specific primer (FGFR2 e4-e5). (h) Nuclear and cytoplasmatic RNA was extracted and retrotranscribed for detection of asFGFR2 by strand-specific qRT-PCR. qRT-PCR values (mean ± SEM, n=5) are normalized to TBP or depicted as the x-fold change of an RT with no primer. Spliced HMGA1 (exons e4-e5) cDNA and unspliced Cyclophilin A (intron i1) pre-cDNA are used to detect cytoplasmatic and nuclear RNA, respectively. (i-l) Expression levels of the EMT-markers E-cadherin and Fibronectin, FGFR2 exons IIIb and IIIc, and asFGFR2 upon induction for 6 days (T6) of the epithelial-to-mesenchymal transition in MCF10a-Snail-ER cells. qRT-PCR values (mean ± SEM, n=4) are normalized to TBP or total FGFR2. Strand-specific RT is normalized to no primer RT. ** p<0.01 in two tailed Student’s t-test relative to T0. (m-n) Inclusion levels of FGFR2 exons IIIb, IIIc and constitutive e15 after downregulation of PTB in hMSC cells (grey) for 72h. The expression levels of PTB are shown as control. qRT-PCR values (mean ± SEM, n=5) are normalized to FGFR2 or Cyclophilin A and depicted as percentage of inclusion or the x-fold change of transfection with negative siRNA, respectively. * p<0.05 and ** p<0.01 in two tailed Student’s t-test relative to negative control siRNA.

Supplementary Figure 7 asFGFR2 lncRNA specifically affects FGFR2 splicing.

(a-k) Expression levels of FGFR2 exons IIIb, IIIc, control CD44-v6, constitutive FGFR2-e14 and FGFR2 splicing regulators ESRP1, ESRP2, TIA1, RBFOX2, hnRNPA1, hnRNPH and PTB after downregulation of asFGFR2 with LNA-modified antisense DNA oligos against antisense exon IIIc and the 5’UTR of asFGFR2 in PNT2 cells for 72h (siIIIc). qRT-PCR values (mean ± SEM, n=8) are normalized to Cyclophilin A, total FGFR2 or CD44 as percentage of inclusion. (l-o) Inclusion levels of FGFR2 exons IIIb, IIIc, control CD44-v6 and constitutive FGFR2-e14 after treatment of PNT2 cells with an siRNA against antisense exon IIIb for 72h (siIIIb). qRT-PCR values (mean ± SEM, n=3) are normalized to Cyclophilin, total FGFR2 or CD44 as percentage of inclusion. * p<0.05 and ** p<0.01 in two tailed Student’s t-test relative to negative siRNA. (p-f’) Expression levels of FGFR2 exons IIIb, IIIc and constitutive e14 (p-s), control CD44-v6 ( t), asFGFR2 (u), FGFR2 splicing regulators ESRP1, ESRP2, TIA1, RBFOX2, hnRNPA1, hnRNPH, PTB ( v-z) and FGFR2 homologs FGFR1 exons IIIb, IIIc and total (a’-c’) and FGFR3 exons IIIb, IIIc and total (d’-f’) after overexpression of asFGFR2 for 72h in PNT2 (red) and hMSC (black) cells. Despite the sequence complementarity with FGFR2 exons IIIb and IIIc, asFGFR2 does not affect the tissue-specific splicing of FGFR1 and FGFR3. qRT-PCR values (mean ± SEM, n=3-9) are normalized to Cyclophilin A or total FGFR2 or CD44 as percentage of inclusion. * p<0.01 and ** p<0.001 in two tailed Student’s t-test relative to mock transfection. IIIb/IIIc: splicing ratio of exons IIIb and IIIc.

Supplementary Figure 8 asFGFR2 lncRNA regulates splicing via recruitment of PRC2 and KDM2a to the FGFR2 locus.

(a-f) Levels of SUZ12, KDM2a, MRG15, RING1B and KDM2b along FGFR2 and control CD44 loci after ectopic expression of asFGFR2 in hMSC (grey). ChIP values (mean ± SEM, n=3-6) are depicted as the percentage of input. No antibody ChIP is used as negative control in asGAPDH (brown) and asFGFR2 (light brown) expressing hMSC cells. The transcription start site of p15 is used as a positive control in RING1B ChIPs. * p<0.05 and ** p<0.01 in two tailed Student’s t-test relative to asGAPDH (black). (k) UV crosslinked RNA immunoprecipitation of asFGFR2 (5’UTR) to SUZ12 or RING1B in hMSC cells ectopically expressing asFGFR2 (grey) or control asGAPDH (black). Exon e9 of FGFR2 pre-mRNA and PTENpg1 asRNA are used as negative controls. RIP values (mean ± SEM, n=2) are depicted as the percentage of input. (l) RNA expression levels of asFGFR2 5’UTR, FGFR2 exons IIIb and IIIc pre-mRNA and, as a positive control, the region of PTENpg1 RNA known to interact with a divergent antisense RNA after treatment with or without RNAse A for detection of double stranded RNA in PNT2 cells. qRT-PCR values (mean ± SEM, n=2) are depicted as the fold change in RNA levels in untreated (RNAse A -) relative to treated (RNAse A +) nuclear RNAs. Contrary to PTENpg1, neither FGFR2 pre-mRNA nor asFGFR2 lncRNA are protected upon RNAse A digestion, suggesting that there are no RNA:RNA hybrids. (m) Expression levels of KDM2a and asFGFR2 after combined ovexpression of asFGFR2 and downregulation of KDM2a for 72h in hMSC cells. qRT-PCR values (mean ± SEM, n=6) are normalized to Cyclophilin A and depicted as the x-fold change to transfection with a negative control siRNA and asGAPDH.

Supplementary information

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Supplementary Figures 1–8 and Supplementary Tables 1 and 2 (PDF 1224 kb)

Supplementary Data Set 1

Northern blot detection of asFGFR2 (PDF 172 kb)

Supplementary Data Set 2

Validation of antibodies and siRNAs (PDF 349 kb)

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Gonzalez, I., Munita, R., Agirre, E. et al. A lncRNA regulates alternative splicing via establishment of a splicing-specific chromatin signature. Nat Struct Mol Biol 22, 370–376 (2015). https://doi.org/10.1038/nsmb.3005

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