In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

Institute of Molecular Genetics of the ASCR v v i Vídeňská 1083 142 20 Prague 4 Czech Republic

Max Planck Institute of Molecular Cell Biology and Genetics 01307 Dresden Germany

Zobrazit více v PubMed

Brugiolo M., Herzel L. & Neugebauer K. M. Counting on co-transcriptional splicing. F1000prime reports 5, 9, 10.12703/P5-9 (2013). PubMed DOI PMC

Carrillo Oesterreich F., Preibisch S. & Neugebauer K. M. Global analysis of nascent RNA reveals transcriptional pausing in terminal exons. Mol. Cell 40, 571–581, 10.1016/j.molcel.2010.11.004 (2010). PubMed DOI

Tilgner H. et al.. Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs. Genome Res. 22, 1616–1625, 10.1101/gr.134445.111 (2012). PubMed DOI PMC

Carrillo Oesterreich F., Bieberstein N. & Neugebauer K. M. Pause locally, splice globally. Trends in Cell Biology 21, 328–335, 10.1016/j.tcb.2011.03.002 (2011). PubMed DOI

Schwartz S., Meshorer E. & Ast G. Chromatin organization marks exon-intron structure. Nat Struct Mol Biol 16, 990–995, 10.1038/nsmb.1659 (2009). PubMed DOI

Tilgner H. et al.. Nucleosome positioning as a determinant of exon recognition. Nat Struct Mol Biol 16, 996–1001, 10.1038/nsmb.1658 (2009). PubMed DOI

Bieberstein N. I., Oesterreich F. C., Straube K. & Neugebauer K. M. First exon length controls active chromatin signatures and transcription. Cell reports 2, 62–68, 10.1016/j.celrep.2012.05.019 (2012). PubMed DOI

de Almeida S. F. et al.. Splicing enhances recruitment of methyltransferase HYPB/Setd2 and methylation of histone H3 Lys36. Nat Struct Mol Biol 18, 977–983, 10.1038/nsmb.2123 (2011). PubMed DOI

Kim S., Kim H., Fong N., Erickson B. & Bentley D. L. Pre-mRNA splicing is a determinant of histone H3K36 methylation. Proceedings of the National Academy of Sciences 108, 13564–13569, 10.1073/pnas.1109475108 (2011). PubMed DOI PMC

Hnilicova J. et al.. The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing. Mol Biol Cell 24, 3557–3568, 10.1091/mbc.E13-06-0303 (2013). PubMed DOI PMC

Luco R. et al.. Regulation of Alternative Splicing by Histone Modifications. Science 327, 996–1000, 10.1126/science.1184208 (2010). PubMed DOI PMC

Guo R. et al.. BS69/ZMYND11 reads and connects histone H3.3 lysine 36 trimethylation-decorated chromatin to regulated pre-mRNA processing. Mol. Cell 56, 298–310, 10.1016/j.molcel.2014.08.022 (2014). PubMed DOI PMC

Hnilicová J. et al.. Histone deacetylase activity modulates alternative splicing. PLoS One 6, e16727, 10.1371/journal.pone.0016727 (2011). PubMed DOI PMC

Sims R. J. et al.. Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing. Mol. Cell 28, 665–676, 10.1016/j.molcel.2007.11.010 (2007). PubMed DOI PMC

Salton M., Voss T. C. & Misteli T. Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing. Nucleic Acids Res. 42, 13662–13673, 10.1093/nar/gku1226 (2014). PubMed DOI PMC

Cermak T., Starker C. G. & Voytas D. F. Efficient design and assembly of custom TALENs using the Golden Gate platform. Methods Mol. Biol. 1239, 133–159, 10.1007/978-1-4939-1862-1_7 (2015). PubMed DOI

Cermak T. et al.. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82, 10.1093/nar/gkr218 (2011). PubMed DOI PMC

Miller J. C. et al.. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148, 10.1038/nbt.1755 (2011). PubMed DOI

Mendenhall E. M. et al.. Locus-specific editing of histone modifications at endogenous enhancers. Nature biotechnology 31, 1133–1136, 10.1038/nbt.2701 (2013). PubMed DOI PMC

Pradeepa M. M., Sutherland H. G., Ule J., Grimes G. R. & Bickmore W. A. Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing. PLoS genetics 8, e1002717, 10.1371/journal.pgen.1002717 (2012). PubMed DOI PMC

Saint-André V., Batsché E., Rachez C. & Muchardt C. Histone H3 lysine 9 trimethylation and HP1γ favor inclusion of alternative exons. Nat Struct Mol Biol 18, 337–344, 10.1038/nsmb.1995 (2011). PubMed DOI

Alló M. et al.. Control of alternative splicing through siRNA-mediated transcriptional gene silencing. Nat Struct Mol Biol 16, 717–724, 10.1038/nsmb.1620 (2009). PubMed DOI

Duskova E., Hnilicova J. & Stanek D. CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation. Rna Biol 11, 865–874, 10.4161/Rna.29441 (2014). PubMed DOI PMC

Pagani F., Stuani C., Zuccato E., Kornblihtt A. R. & Baralle F. E. Promoter architecture modulates CFTR exon 9 skipping. The Journal of biological chemistry 278, 1511–1517, 10.1074/jbc.M209676200 (2003). PubMed DOI

Doyle E. L. et al.. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 40, W117–122, 10.1093/nar/gks608 (2012). PubMed DOI PMC

Tachibana M., Sugimoto K., Fukushima T. & Shinkai Y. Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. The Journal of biological chemistry 276, 25309–25317, 10.1074/jbc.M101914200 (2001). PubMed DOI

Wu H. et al.. Structural biology of human H3K9 methyltransferases. PLoS One 5, e8570, 10.1371/journal.pone.0008570 (2010). PubMed DOI PMC

Rea S. et al.. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593–599, 10.1038/35020506 (2000). PubMed DOI

Whetstine J. R. et al.. Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell 125, 467–481, 10.1016/j.cell.2006.03.028 (2006). PubMed DOI

Sun X. J. et al.. Identification and characterization of a novel human histone H3 lysine 36-specific methyltransferase. The Journal of biological chemistry 280, 35261–35271, 10.1074/jbc.M504012200 (2005). PubMed DOI

An S., Yeo K. J., Jeon Y. H. & Song J. J. Crystal structure of the human histone methyltransferase ASH1L catalytic domain and its implications for the regulatory mechanism. The Journal of biological chemistry 286, 8369–8374, 10.1074/jbc.M110.203380 (2011). PubMed DOI PMC

Talbert P. B. & Henikoff S. Spreading of silent chromatin: inaction at a distance. Nature reviews. Genetics 7, 793–803, 10.1038/nrg1920 (2006). PubMed DOI

Schor I. E., Fiszbein A., Petrillo E. & Kornblihtt A. R. Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation. EMBO J. 32, 2264–2274 (2013). PubMed PMC

ENCODE. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74, 10.1038/nature11247 (2012). PubMed DOI PMC

Pandya-Jones A. & Black D. L. Co-transcriptional splicing of constitutive and alternative exons. RNA 15, 1896–1908, rna.1714509/10.1261/rna.1714509 (2009). PubMed PMC

Hnilicova J. & Stanek D. Where splicing joins chromatin. Nucleus 2, 182–188 (2011). PubMed PMC

Schor I. E., Rascovan N., Pelisch F., Allo M. & Kornblihtt A. R. Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing. Proc. Natl. Acad. Sci. USA 106, 4325–4330 (2009). PubMed PMC

Nogues G., Kadener S., Cramer P., Bentley D. & Kornblihtt A. R. Transcriptional activators differ in their abilities to control alternative splicing. The Journal of biological chemistry 277, 43110–43114, 10.1074/jbc.M208418200 (2002). PubMed DOI

Malik R. & Svoboda P. In Toxicology and Epigenetics (ed Saura C. Sahu) Ch. 15, (John Wiley & Sons, Ltd, 2012).

Kwak H., Fuda N. J., Core L. J. & Lis J. T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953, 10.1126/science.1229386 (2013). PubMed DOI PMC

Sanjana N. E. et al.. A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7, 171–192, 10.1038/nprot.2011.431 (2012). PubMed DOI PMC

Zoabi M. et al.. RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation. Nucleic Acids Res 42, 13026–13038, 10.1093/nar/gku1021 (2014). PubMed DOI PMC

Harrow J. et al.. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1774, 10.1101/gr.135350.111 (2012). PubMed DOI PMC

Shin H., Liu T., Manrai A. K. & Liu X. S. CEAS: cis-regulatory element annotation system. Bioinformatics 25, 2605–2606, 10.1093/bioinformatics/btp479 (2009). PubMed DOI

Splicing of long non-coding RNAs primarily depends on polypyrimidine tract and 5' splice-site sequences due to weak interactions with SR proteins