Histone acetylation modulates alternative splicing of several hundred genes. Here, we tested the role of the histone acetyltransferase p300 in alternative splicing and showed that knockdown of p300 promotes inclusion of the fibronectin (FN1) alternative EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as p300 knockdown. Next we showed that p300 controls histone H4 acetylation along the FN1 gene. Consistently, p300 depletion and CRE deletion/mutation both reduced histone H4 acetylation on mini-gene reporters. Finally, we provide evidence that the effect of CRE inactivation on H4 acetylation and alternative splicing is counteracted by the inhibition of histone deacetylases. Together, these data suggest that histone acetylation could be one of the mechanisms how promoter and promoter binding proteins influence alternative splicing.

Department of RNA Biology Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

Department of RNA Biology Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic; Faculty of Science Charles University Prague Prague Czech Republic

Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Croft L, Schandorff S, Clark F, Burrage K, Arctander P, Mattick JS. ISIS, the intron information system, reveals the high frequency of alternative splicing in the human genome. Nat Genet. 2000;24:340–1. doi: 10.1038/74153. PubMed DOI

Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008;40:1413–5. doi: 10.1038/ng.259. PubMed DOI

Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB. Alternative isoform regulation in human tissue transcriptomes. Nature. 2008;456:470–6. doi: 10.1038/nature07509. PubMed DOI PMC

Black DL. Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem. 2003;72:291–336. doi: 10.1146/annurev.biochem.72.121801.161720. PubMed DOI

Blencowe BJ. Alternative splicing: new insights from global analyses. Cell. 2006;126:37–47. doi: 10.1016/j.cell.2006.06.023. PubMed DOI

Brody Y, Neufeld N, Bieberstein N, Causse SZ, Böhnlein EM, Neugebauer KM, Darzacq X, Shav-Tal Y. The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing. PLoS Biol. 2011;9:e1000573. doi: 10.1371/journal.pbio.1000573. PubMed DOI PMC

Darzacq X, Shav-Tal Y, de Turris V, Brody Y, Shenoy SM, Phair RD, Singer RH. In vivo dynamics of RNA polymerase II transcription. Nat Struct Mol Biol. 2007;14:796–806. doi: 10.1038/nsmb1280. PubMed DOI PMC

Singh J, Padgett RA. Rates of in situ transcription and splicing in large human genes. Nat Struct Mol Biol. 2009;16:1128–33. doi: 10.1038/nsmb.1666. PubMed DOI PMC

Huranová M, Ivani I, Benda A, Poser I, Brody Y, Hof M, Shav-Tal Y, Neugebauer KM, Stanek D. The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. J Cell Biol. 2010;191:75–86. doi: 10.1083/jcb.201004030. PubMed DOI PMC

Schmidt U, Basyuk E, Robert MC, Yoshida M, Villemin JP, Auboeuf D, Aitken S, Bertrand E. Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation. J Cell Biol. 2011;193:819–29. doi: 10.1083/jcb.201009012. PubMed DOI PMC

Martin RM, Rino J, Carvalho C, Kirchhausen T, Carmo-Fonseca M. Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity. Cell Rep. 2013;4:1144–55. doi: 10.1016/j.celrep.2013.08.013. PubMed DOI PMC

Lacadie SA, Rosbash M. Cotranscriptional spliceosome assembly dynamics and the role of U1 snRNA:5’ss base pairing in yeast. Mol Cell. 2005;19:65–75. doi: 10.1016/j.molcel.2005.05.006. PubMed DOI

Listerman I, Sapra AK, Neugebauer KM. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells. Nat Struct Mol Biol. 2006;13:815–22. doi: 10.1038/nsmb1135. PubMed DOI

Sapra AK, Ankö ML, Grishina I, Lorenz M, Pabis M, Poser I, Rollins J, Weiland EM, Neugebauer KM. SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo. Mol Cell. 2009;34:179–90. doi: 10.1016/j.molcel.2009.02.031. PubMed DOI

Girard C, Will CL, Peng J, Makarov EM, Kastner B, Lemm I, Urlaub H, Hartmuth K, Lührmann R. Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion. Nat Commun. 2012;3:994. doi: 10.1038/ncomms1998. PubMed DOI

Das R, Dufu K, Romney B, Feldt M, Elenko M, Reed R. Functional coupling of RNAP II transcription to spliceosome assembly. Genes Dev. 2006;20:1100–9. doi: 10.1101/gad.1397406. PubMed DOI PMC

Gromak N, Talotti G, Proudfoot NJ, Pagani F. Modulating alternative splicing by cotranscriptional cleavage of nascent intronic RNA. RNA. 2008;14:359–66. doi: 10.1261/rna.615508. PubMed DOI PMC

Kornblihtt AR. Coupling transcription and alternative splicing. Adv Exp Med Biol. 2007;623:175–89. doi: 10.1007/978-0-387-77374-2_11. PubMed DOI

Perales R, Bentley D. “Cotranscriptionality”: the transcription elongation complex as a nexus for nuclear transactions. Mol Cell. 2009;36:178–91. doi: 10.1016/j.molcel.2009.09.018. PubMed DOI PMC

Neugebauer KM. On the importance of being co-transcriptional. J Cell Sci. 2002;115:3865–71. doi: 10.1242/jcs.00073. PubMed DOI

Bird G, Zorio DA, Bentley DL. RNA polymerase II carboxy-terminal domain phosphorylation is required for cotranscriptional pre-mRNA splicing and 3′-end formation. Mol Cell Biol. 2004;24:8963–9. doi: 10.1128/MCB.24.20.8963-8969.2004. PubMed DOI PMC

Dower K, Rosbash M. T7 RNA polymerase-directed transcripts are processed in yeast and link 3′ end formation to mRNA nuclear export. RNA. 2002;8:686–97. doi: 10.1017/S1355838202024068. PubMed DOI PMC

McCracken S, Rosonina E, Fong N, Sikes M, Beyer A, O’Hare K, Shuman S, Bentley D. Role of RNA polymerase II carboxy-terminal domain in coordinating transcription with RNA processing. Cold Spring Harb Symp Quant Biol. 1998;63:301–9. doi: 10.1101/sqb.1998.63.301. PubMed DOI

Sisodia SS, Sollner-Webb B, Cleveland DW. Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. Mol Cell Biol. 1987;7:3602–12. PubMed PMC

de la Mata M, Alonso CR, Kadener S, Fededa JP, Blaustein M, Pelisch F, Cramer P, Bentley D, Kornblihtt AR. A slow RNA polymerase II affects alternative splicing in vivo. Mol Cell. 2003;12:525–32. doi: 10.1016/j.molcel.2003.08.001. PubMed DOI

Eperon LP, Graham IR, Griffiths AD, Eperon IC. Effects of RNA secondary structure on alternative splicing of pre-mRNA: is folding limited to a region behind the transcribing RNA polymerase? Cell. 1988;54:393–401. doi: 10.1016/0092-8674(88)90202-4. PubMed DOI

Kadener S, Fededa JP, Rosbash M, Kornblihtt AR. Regulation of alternative splicing by a transcriptional enhancer through RNA pol II elongation. Proc Natl Acad Sci U S A. 2002;99:8185–90. doi: 10.1073/pnas.122246099. PubMed DOI PMC

Nogués G, Muñoz MJ, Kornblihtt AR. Influence of polymerase II processivity on alternative splicing depends on splice site strength. J Biol Chem. 2003;278:52166–71. doi: 10.1074/jbc.M309156200. PubMed DOI

Roberts GC, Gooding C, Mak HY, Proudfoot NJ, Smith CW. Co-transcriptional commitment to alternative splice site selection. Nucleic Acids Res. 1998;26:5568–72. doi: 10.1093/nar/26.24.5568. PubMed DOI PMC

Braunschweig U, Gueroussov S, Plocik AM, Graveley BR, Blencowe BJ. Dynamic integration of splicing within gene regulatory pathways. Cell. 2013;152:1252–69. doi: 10.1016/j.cell.2013.02.034. PubMed DOI PMC

Carrillo Oesterreich F, Bieberstein N, Neugebauer KM. Pause locally, splice globally. Trends Cell Biol. 2011;21:328–35. doi: 10.1016/j.tcb.2011.03.002. PubMed DOI

Hnilicová J, Staněk D. Where splicing joins chromatin. Nucleus. 2011;2:182–8. doi: 10.4161/nucl.2.3.15876. PubMed DOI PMC

Kornblihtt AR, Schor IE, Allo M, Blencowe BJ. When chromatin meets splicing. Nat Struct Mol Biol. 2009;16:902–3. doi: 10.1038/nsmb0909-902. PubMed DOI

Kornblihtt AR, Schor IE, Alló M, Dujardin G, Petrillo E, Muñoz MJ. Alternative splicing: a pivotal step between eukaryotic transcription and translation. Nat Rev Mol Cell Biol. 2013;14:153–65. doi: 10.1038/nrm3525. PubMed DOI

Luco RF, Allo M, Schor IE, Kornblihtt AR, Misteli T. Epigenetics in alternative pre-mRNA splicing. Cell. 2011;144:16–26. doi: 10.1016/j.cell.2010.11.056. PubMed DOI PMC

Luco RF, Pan Q, Tominaga K, Blencowe BJ, Pereira-Smith OM, Misteli T. Regulation of alternative splicing by histone modifications. Science. 2010;327:996–1000. doi: 10.1126/science.1184208. 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. 2011;18:337–44. doi: 10.1038/nsmb.1995. PubMed DOI

Sims RJ, 3rd, Millhouse S, Chen CF, Lewis BA, Erdjument-Bromage H, Tempst P, Manley JL, Reinberg D. Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing. Mol Cell. 2007;28:665–76. doi: 10.1016/j.molcel.2007.11.010. PubMed DOI PMC

de Almeida SF, Grosso AR, Koch F, Fenouil R, Carvalho S, Andrade J, Levezinho H, Gut M, Eick D, Gut I, et al. Splicing enhances recruitment of methyltransferase HYPB/Setd2 and methylation of histone H3 Lys36. Nat Struct Mol Biol. 2011;18:977–83. doi: 10.1038/nsmb.2123. PubMed DOI

Kim S, Kim H, Fong N, Erickson B, Bentley DL. Pre-mRNA splicing is a determinant of histone H3K36 methylation. Proc Natl Acad Sci U S A. 2011;108:13564–9. doi: 10.1073/pnas.1109475108. PubMed DOI PMC

Bieberstein NI, Carrillo Oesterreich F, Straube K, Neugebauer KM. First exon length controls active chromatin signatures and transcription. Cell Rep. 2012;2:62–8. doi: 10.1016/j.celrep.2012.05.019. PubMed DOI

Hnilicová J, Hozeifi S, Dušková E, Icha J, Tománková T, Staněk D. Histone deacetylase activity modulates alternative splicing. PLoS One. 2011;6:e16727. doi: 10.1371/journal.pone.0016727. PubMed DOI PMC

Zhou HL, Hinman MN, Barron VA, Geng C, Zhou G, Luo G, Siegel RE, Lou H. Hu proteins regulate alternative splicing by inducing localized histone hyperacetylation in an RNA-dependent manner. Proc Natl Acad Sci U S A. 2011;108:E627–35. doi: 10.1073/pnas.1103344108. PubMed DOI PMC

Schor IE, Rascovan N, Pelisch F, Alló M, Kornblihtt AR. Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing. Proc Natl Acad Sci U S A. 2009;106:4325–30. doi: 10.1073/pnas.0810666106. PubMed DOI PMC

Cramer P, Cáceres JF, Cazalla D, Kadener S, Muro AF, Baralle FE, Kornblihtt AR. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. Mol Cell. 1999;4:251–8. doi: 10.1016/S1097-2765(00)80372-X. PubMed DOI

Cramer P, Pesce CG, Baralle FE, Kornblihtt AR. Functional association between promoter structure and transcript alternative splicing. Proc Natl Acad Sci U S A. 1997;94:11456–60. doi: 10.1073/pnas.94.21.11456. PubMed DOI PMC

Pagani F, Stuani C, Zuccato E, Kornblihtt AR, Baralle FE. Promoter architecture modulates CFTR exon 9 skipping. J Biol Chem. 2003;278:1511–7. doi: 10.1074/jbc.M209676200. PubMed DOI

Auboeuf D, Hönig A, Berget SM, O’Malley BW. Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science. 2002;298:416–9. doi: 10.1126/science.1073734. PubMed DOI

Kadener S, Cramer P, Nogués G, Cazalla D, de la Mata M, Fededa JP, Werbajh SE, Srebrow A, Kornblihtt AR. Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing. EMBO J. 2001;20:5759–68. doi: 10.1093/emboj/20.20.5759. PubMed DOI PMC

Monsalve M, Wu Z, Adelmant G, Puigserver P, Fan M, Spiegelman BM. Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1. Mol Cell. 2000;6:307–16. doi: 10.1016/S1097-2765(00)00031-9. PubMed DOI

Nogues G, Kadener S, Cramer P, Bentley D, Kornblihtt AR. Transcriptional activators differ in their abilities to control alternative splicing. J Biol Chem. 2002;277:43110–4. doi: 10.1074/jbc.M208418200. PubMed DOI

Hnilicová J, Hozeifi S, Stejskalová E, Dušková E, Poser I, Humpolíčková J, Hof M, Staněk D. The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing. Mol Biol Cell. 2013;24:3557–68. doi: 10.1091/mbc.E13-06-0303. PubMed DOI PMC

Shaywitz AJ, Greenberg ME. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem. 1999;68:821–61. doi: 10.1146/annurev.biochem.68.1.821. PubMed DOI

Du K, Peng Y, Greenbaum LE, Haber BA, Taub R. HRS/SRp40-mediated inclusion of the fibronectin EIIIB exon, a possible cause of increased EIIIB expression in proliferating liver. Mol Cell Biol. 1997;17:4096–104. PubMed PMC

Muro AF, Bernath VA, Kornblihtt AR. Interaction of the -170 cyclic AMP response element with the adjacent CCAAT box in the human fibronectin gene promoter. J Biol Chem. 1992;267:12767–74. PubMed

Alonso CR, Pesce CG, Kornblihtt AR. The CCAAT-binding proteins CP1 and NF-I cooperate with ATF-2 in the transcription of the fibronectin gene. J Biol Chem. 1996;271:22271–9. doi: 10.1074/jbc.271.36.22271. PubMed DOI

Nejepinska J, Malik R, Moravec M, Svoboda P. Deep sequencing reveals complex spurious transcription from transiently transfected plasmids. PLoS One. 2012;7:e43283. doi: 10.1371/journal.pone.0043283. PubMed DOI PMC

LeRoy G, Rickards B, Flint SJ. The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription. Mol Cell. 2008;30:51–60. doi: 10.1016/j.molcel.2008.01.018. PubMed DOI PMC

Kornblihtt AR. Promoter usage and alternative splicing. Curr Opin Cell Biol. 2005;17:262–8. doi: 10.1016/j.ceb.2005.04.014. PubMed DOI

Perales R, Erickson B, Zhang L, Kim H, Valiquett E, Bentley D. Gene promoters dictate histone occupancy within genes. EMBO J. 2013;32:2645–56. doi: 10.1038/emboj.2013.194. PubMed DOI PMC

Struhl K. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev. 1998;12:599–606. doi: 10.1101/gad.12.5.599. PubMed DOI

Bannister AJ, Zegerman P, Partridge JF, Miska EA, Thomas JO, Allshire RC, Kouzarides T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature. 2001;410:120–4. doi: 10.1038/35065138. PubMed DOI

Mercer TR, Edwards SL, Clark MB, Neph SJ, Wang H, Stergachis AB, John S, Sandstrom R, Li G, Sandhu KS, et al. DNase I-hypersensitive exons colocalize with promoters and distal regulatory elements. Nat Genet. 2013;45:852–9. doi: 10.1038/ng.2677. PubMed DOI PMC

Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, et al. Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes. Genome Res. 2006;16:55–65. doi: 10.1101/gr.4039406. PubMed DOI PMC

Xin D, Hu L, Kong X. Alternative promoters influence alternative splicing at the genomic level. PLoS One. 2008;3:e2377. doi: 10.1371/journal.pone.0002377. PubMed DOI PMC

Shav-Tal Y, Darzacq X, Shenoy SM, Fusco D, Janicki SM, Spector DL, Singer RH. Dynamics of single mRNPs in nuclei of living cells. Science. 2004;304:1797–800. doi: 10.1126/science.1099754. PubMed DOI PMC

O’Neill LP, Turner BM. Immunoprecipitation of native chromatin: NChIP. Methods. 2003;31:76–82. doi: 10.1016/S1046-2023(03)00090-2. PubMed DOI

Huranová M, Hnilicová J, Fleischer B, Cvacková Z, Stanek D. A mutation linked to retinitis pigmentosa in HPRP31 causes protein instability and impairs its interactions with spliceosomal snRNPs. Hum Mol Genet. 2009;18:2014–23. doi: 10.1093/hmg/ddp125. 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

TALE-directed local modulation of H3K9 methylation shapes exon recognition