The nuclear pore complex (NPC) has emerged as a hub for the transcriptional regulation of a subset of genes, and this type of regulation plays an important role during differentiation. Nucleoporin TPR forms the nuclear basket of the NPC and is crucial for the enrichment of open chromatin around NPCs. TPR has been implicated in the regulation of transcription; however, the role of TPR in gene expression and cell differentiation has not been described. Here we show that depletion of TPR results in an aberrant morphology of murine proliferating C2C12 myoblasts (MBs) and differentiated C2C12 myotubes (MTs). The ChIP-Seq data revealed that TPR binds to genes linked to muscle formation and function, such as myosin heavy chain (Myh4), myocyte enhancer factor 2C (Mef2C) and a majority of olfactory receptor (Olfr) genes. We further show that TPR, possibly via lysine-specific demethylase 1 (LSD1), promotes the expression of Myh4 and Olfr376, but not Mef2C. This provides a novel insight into the mechanism of myogenesis; however, more evidence is needed to fully elucidate the mechanism by which TPR affects specific myogenic genes.

1st Faculty of Medicine Department of Cell Biology Charles University 121 08 Prague Czech Republic

CLIP Childhood Leukaemia Investigation Prague Department of Pediatric Hematology Oncology 2nd Faculty of Medicine of the Charles University and University Hospital Motol 150 00 Prague Czech Republic

Department of Biology of the Cell Nucleus Institute of Molecular Genetics of the CAS 142 20 Prague Czech Republic

Department of Cybernetics Faculty of Electrical Engineering Czech Technical University Prague 121 35 Prague Czech Republic

Faculty of Science Department of Cell Biology Charles University 128 00 Prague Czech Republic

Microscopy Center LM and EM Institute of Molecular Genetics of the CAS 142 20 Prague Czech Republic

Zobrazit více v PubMed

Kuhn T.M., Pascual-Garcia P., Gozalo A., Little S.C., Capelson M. Chromatin targeting of nuclear pore proteins induces chromatin decondensation. J. Cell Biol. 2019;218:2945–2961. doi: 10.1083/jcb.201807139. PubMed DOI PMC

Pascual-Garcia P., Debo B., Aleman J.R., Talamas J.A., Lan Y., Nguyen N.H., Won K.J., Capelson M. Metazoan Nuclear Pores Provide a Scaffold for Poised Genes and Mediate Induced Enhancer-Promoter Contacts. Mol. Cell. 2017;66:63–76. doi: 10.1016/j.molcel.2017.02.020. PubMed DOI PMC

Raices M., Bukata L., Sakuma S., Borlido J., Hernandez L.S., Hart D.O., D’Angelo M.A. Nuclear Pores Regulate Muscle Development and Maintenance by Assembling a Localized Mef2C Complex. Dev. Cell. 2017;41:540–554.e7. doi: 10.1016/j.devcel.2017.05.007. PubMed DOI PMC

Su Y., Pelz C., Huang T., Torkenczy K., Wang X., Cherry A., Daniel C.J., Liang J., Nan X., Dai M.-S., et al. Post-translational modification localizes MYC to the nuclear pore basket to regulate a subset of target genes involved in cellular responses to environmental signals. Genes Dev. 2018;32:1398–1419. doi: 10.1101/gad.314377.118. PubMed DOI PMC

Ibarra A., Benner C., Tyagi S., Cool J., Hetzer M.W. Nucleoporin-mediated regulation of cell identity genes. Genes Dev. 2016;30:2253–2258. doi: 10.1101/gad.287417.116. PubMed DOI PMC

Krull S., Dörries J., Boysen B., Reidenbach S., Magnius L., Norder H., Thyberg J., Cordes V.C. Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion. EMBO J. 2010;29:1659–1673. doi: 10.1038/emboj.2010.54. PubMed DOI PMC

Buchwalter A.L., Liang Y., Hetzer M.W. Nup50 is required for cell differentiation and exhibits transcription-dependent dynamics. Mol. Biol. Cell. 2014;25:2472–2484. doi: 10.1091/mbc.e14-04-0865. PubMed DOI PMC

Kalverda B., Pickersgill H., Shloma V.V., Fornerod M. Nucleoporins directly stimulate expression of developmental and cell-cycle genes inside the nucleoplasm. Cell. 2010;140:360–371. doi: 10.1016/j.cell.2010.01.011. PubMed DOI

Capelson M., Liang Y., Schulte R., Mair W., Wagner U., Hetzer M.W. Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes. Cell. 2010;140:372. doi: 10.1016/j.cell.2009.12.054. PubMed DOI PMC

Frosst P., Guan T., Subauste C., Hahn K., Gerace L. Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export. J. Cell Biol. 2002;156:617–630. doi: 10.1083/jcb.200106046. PubMed DOI PMC

Krull S., Thyberg J., Björkroth B., Rackwitz H.-R., Cordes V.C. Nucleoporins as Components of the Nuclear Pore Complex Core Structure and Tpr as the Architectural Element of the Nuclear Basket. Mol. Biol. Cell. 2004;15:4261–4277. doi: 10.1091/mbc.e04-03-0165. PubMed DOI PMC

Aksenova V., Lee H.N., Smith A., Chen S., Bhat P., Iben J., Echeverria C., Fontoura B., Arnaoutov A., Dasso M. Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway. bioRxiv. 2019:685263. doi: 10.1101/685263. DOI

Lee E.S., Wolf E.J., Ihn S.S.J., Smith H.W., Emili A., Palazzo A.F. TPR is required for the efficient nuclear export of mRNAs and lncRNAs from short and intron-poor genes. Nucleic Acids Res. 2020;48:11645–11663. doi: 10.1093/nar/gkaa919. PubMed DOI PMC

Vomastek T., Iwanicki M.P., Burack W.R., Tiwari D., Kumar D., Parsons J.T., Weber M.J., Nandicoori V.K. Extracellular Signal-Regulated Kinase 2 (ERK2) Phosphorylation Sites and Docking Domain on the Nuclear Pore Complex Protein Tpr Cooperatively Regulate ERK2-Tpr Interaction. Mol. Cell. Biol. 2008;28:6954–6966. doi: 10.1128/MCB.00925-08. PubMed DOI PMC

Fontoura B.M., Dales S., Blobel G., Zhong H. The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR. Proc. Natl. Acad. Sci. USA. 2001;98:3208–3213. doi: 10.1073/pnas.061014698. PubMed DOI PMC

Agarwal S., Yadav S.K., Dixit A. Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus. Protein Expr. Purif. 2011;77:112–117. doi: 10.1016/j.pep.2011.01.001. PubMed DOI

Jin W., Liu M., Peng J., Jiang S. Function analysis of Mef2c promoter in muscle differentiation. Biotechnol. Appl. Biochem. 2017;64:647–656. doi: 10.1002/bab.1524. PubMed DOI

Stuart C.A., Stone W.L., Howell M.E.A., Brannon M.F., Hall H.K., Gibson A.L., Stone M.H. Myosin content of individual human muscle fibers isolated by laser capture microdissection. Am. J. Physiol. Cell Physiol. 2016;310:C381–C389. doi: 10.1152/ajpcell.00317.2015. PubMed DOI PMC

Shi Y., Lan F., Matson C., Mulligan P., Whetstine J.R., Cole P.A., Casero R.A., Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell. 2004;119:941–953. doi: 10.1016/j.cell.2004.12.012. PubMed DOI

Wang J., Telese F., Tan Y., Li W., Jin C., He X., Basnet H., Ma Q., Merkurjev D., Zhu X., et al. LSD1n is an H4K20 demethylase regulating memory formation via transcriptional elongation control. Nat. Neurosci. 2015;18:1256–1264. doi: 10.1038/nn.4069. PubMed DOI PMC

Metzger E., Wissmann M., Yin N., Müller J.M., Schneider R., Peters A.H.F.M., Günther T., Buettner R., Schüle R. LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature. 2005;437:436–439. doi: 10.1038/nature04020. PubMed DOI

Majello B., Gorini F., Saccà C.D., Amente S. Expanding the Role of the Histone Lysine-Specific Demethylase LSD1 in Cancer. Cancers. 2019;11:324. doi: 10.3390/cancers11030324. PubMed DOI PMC

Choi J., Jang H., Kim H., Kim S.-T., Cho E.-J., Youn H.-D. Histone demethylase LSD1 is required to induce skeletal muscle differentiation by regulating myogenic factors. Biochem. Biophys. Res. Commun. 2010;401:327–332. doi: 10.1016/j.bbrc.2010.09.014. PubMed DOI

Choi J., Jang H., Kim H., Lee J.-H., Kim S.-T., Cho E.-J., Youn H.-D. Modulation of lysine methylation in myocyte enhancer factor 2 during skeletal muscle cell differentiation. Nucleic Acids Res. 2014;42:224–234. doi: 10.1093/nar/gkt873. PubMed DOI PMC

Scionti I., Hayashi S., Mouradian S., Girard E., Esteves de Lima J., Morel V., Simonet T., Wurmser M., Maire P., Ancelin K., et al. LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription. Cell Rep. 2017;18:1996–2006. doi: 10.1016/j.celrep.2017.01.078. PubMed DOI

Munehira Y., Yang Z., Gozani O. Systematic Analysis of Known and Candidate Lysine Demethylases in the Regulation of Myoblast Differentiation. J. Mol. Biol. 2017;429:2055–2065. doi: 10.1016/j.jmb.2016.10.004. PubMed DOI PMC

Anan K., Hino S., Shimizu N., Sakamoto A., Nagaoka K., Takase R., Kohrogi K., Araki H., Hino Y., Usuki S., et al. LSD1 mediates metabolic reprogramming by glucocorticoids during myogenic differentiation. Nucleic Acids Res. 2018;46:5441–5454. doi: 10.1093/nar/gky234. PubMed DOI PMC

Tosic M., Allen A., Willmann D., Lepper C., Kim J., Duteil D., Schüle R. Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells. Nat. Commun. 2018;9:366. doi: 10.1038/s41467-017-02740-5. PubMed DOI PMC

Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012;9:357–359. doi: 10.1038/nmeth.1923. PubMed DOI PMC

Andrews S. FastQC: A Quality Control Tool for High Throughput Sequence Data. [(accessed on 15 February 2017)];2010 Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

García-Alcalde F., Okonechnikov K., Carbonell J., Cruz L.M., Götz S., Tarazona S., Dopazo J., Meyer T.F., Conesa A. Qualimap: Evaluating next-generation sequencing alignment data. Bioinformatics. 2012;28:2678–2679. doi: 10.1093/bioinformatics/bts503. PubMed DOI

Zhang Y., Liu T., Meyer C.A., Eeckhoute J., Johnson D.S., Bernstein B.E., Nusbaum C., Myers R.M., Brown M., Li W., et al. Model-based analysis of ChIP-Seq (MACS) Genome Biol. 2008;9:R137. doi: 10.1186/gb-2008-9-9-r137. PubMed DOI PMC

Love M.I., Anders S., Kim V., Huber W. RNA-Seq workflow: Gene-level exploratory analysis and differential expression. F1000Research. 2015;4:1070. doi: 10.12688/f1000research.7035.1. PubMed DOI PMC

Anders S., Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11:R106. doi: 10.1186/gb-2010-11-10-r106. PubMed DOI PMC

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M.Y., Geiger T., Mann M., Cox J. The Perseus computational platform for comprehensive analysis of proteomics data. Nat. Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI

Cox J., Mann M. 1D and 2D annotation enrichment: A statistical method integrating quantitative proteomics with complementary high-throughput data. BMC Bioinform. 2012;13(Suppl. 16):S12. doi: 10.1186/1471-2105-13-S16-S12. PubMed DOI PMC

Kanehisa M., Sato Y., Furumichi M., Morishima K., Tanabe M. New approach for understanding genome variations in KEGG. Nucleic Acids Res. 2019;47:D590–D595. doi: 10.1093/nar/gky962. PubMed DOI PMC

Kanehisa M., Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30. doi: 10.1093/nar/28.1.27. PubMed DOI PMC

Gough J., Karplus K., Hughey R., Chothia C. Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. J. Mol. Biol. 2001;313:903–919. doi: 10.1006/jmbi.2001.5080. PubMed DOI

Jacinto F.V., Benner C., Hetzer M.W. The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing. Genes Dev. 2015;29:1224–1238. doi: 10.1101/gad.260919.115. PubMed DOI PMC

Fišerová J., Maninová M., Sieger T., Uhlířová J., Šebestová L., Efenberková M., Čapek M., Fišer K., Hozák P. Nuclear pore protein TPR associates with lamin B1 and affects nuclear lamina organization and nuclear pore distribution. Cell. Mol. Life Sci. 2019 doi: 10.1007/s00018-019-03037-0. PubMed DOI PMC

Chal J., Pourquié O. Making muscle: Skeletal myogenesis in vivo and in vitro. Development. 2017;144:2104–2122. doi: 10.1242/dev.151035. PubMed DOI

Chen B., You W., Wang Y., Shan T. The regulatory role of Myomaker and Myomixer-Myomerger-Minion in muscle development and regeneration. Cell. Mol. Life Sci. 2020;77:1551–1569. doi: 10.1007/s00018-019-03341-9. PubMed DOI PMC

Zhang P., Wong C., Liu D., Finegold M. Harper JW and Elledge SJ: p21(CIP1) and p57(KIP2) control muscle differentiation at the myogenin step. Genes Dev. 1999;13:213–224. doi: 10.1101/gad.13.2.213. PubMed DOI PMC

Asp P., Blum R., Vethantham V., Parisi F., Micsinai M., Cheng J., Bowman C., Kluger Y., Dynlacht B.D. Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc. Natl. Acad. Sci. USA. 2011;108:E149–E158. doi: 10.1073/pnas.1102223108. PubMed DOI PMC

Wu F., Yao J. Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping. BMC Genom. 2013;14:591. doi: 10.1186/1471-2164-14-591. PubMed DOI PMC

Aisenberg W.H., Huang J., Zhu W., Rajkumar P., Cruz R., Santhanam L., Natarajan N., Yong H.M., De Santiago B., Oh J.J., et al. Defining an olfactory receptor function in airway smooth muscle cells. Sci. Rep. 2016;6:38231. doi: 10.1038/srep38231. PubMed DOI PMC

Pavlath G.K. A new function for odorant receptors. Cell Adhes. Migr. 2010;4:502–506. doi: 10.4161/cam.4.4.12291. PubMed DOI PMC

David-Watine B. Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells. PLoS ONE. 2011;6:e22423. doi: 10.1371/journal.pone.0022423. PubMed DOI PMC

Funasaka T., Tsuka E., Wong R.W. Regulation of autophagy by nucleoporin Tpr. Sci. Rep. 2012;2:878. doi: 10.1038/srep00878. PubMed DOI PMC

Liang Y., Franks T.M., Marchetto M.C., Gage F.H., Hetzer M.W. Dynamic association of NUP98 with the human genome. PLoS Genet. 2013;9:e1003308. doi: 10.1371/journal.pgen.1003308. PubMed DOI PMC

Mattout A., Cabianca D.S., Gasser S.M. Chromatin states and nuclear organization in development—A view from the nuclear lamina. Genome Biol. 2015;16:1–15. doi: 10.1186/s13059-015-0747-5. PubMed DOI PMC

Acakpo-Satchivi L.J.R., Edelmann W., Sartorius C., Lu B.D., Wahr P.A., Watkins S.C., Metzger J.M., Leinwand L., Kucherlapati R. Growth and Muscle Defects in Mice Lacking Adult Myosin Heavy Chain Genes. J. Cell Biol. 1997;139:1219–1229. doi: 10.1083/jcb.139.5.1219. PubMed DOI PMC

Dalesio N.M., Barreto Ortiz S.F., Pluznick J.L., Berkowitz D.E. Olfactory, Taste, and Photo Sensory Receptors in Non-sensory Organs: It Just Makes Sense. Front. Physiol. 2018;9:1673. doi: 10.3389/fphys.2018.01673. PubMed DOI PMC

Zhang H., Wen J., Bigot A., Chen J., Shang R., Mouly V., Bi P. Human myotube formation is determined by MyoD–Myomixer/Myomaker axis. Sci. Adv. 2020;6:eabc4062. doi: 10.1126/sciadv.abc4062. PubMed DOI PMC

Ganassi M., Badodi S., Ortuste Quiroga H.P., Zammit P.S., Hinits Y., Hughes S.M. Myogenin promotes myocyte fusion to balance fibre number and size. Nat. Commun. 2018;9:4232. doi: 10.1038/s41467-018-06583-6. PubMed DOI PMC

Milano-Foster J., Ray S., Home P., Ganguly A., Bhattacharya B., Bajpai S., Pal A., Mason C.W., Paul S. Regulation of human trophoblast syncytialization by histone demethylase LSD1. J. Biol. Chem. 2019;294:17301–17313. doi: 10.1074/jbc.RA119.010518. PubMed DOI PMC