CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B leads to disruption of neural cell differentiation from NT2 cells.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
25 Jan 2024
25 Jan 2024
Historique:
received:
24
11
2023
accepted:
22
01
2024
medline:
26
1
2024
pubmed:
26
1
2024
entrez:
25
1
2024
Statut:
epublish
Résumé
The human neuron-specific gene, GPM6B (Glycoprotein membrane 6B), is considered a key gene in neural cell functionality. This gene contains an exceptionally long and strictly monomorphic short tandem repeat (STR) of 9-repeats, (GA)9. STRs in regulatory regions, may impact on the expression of nearby genes. We used CRISPR-based tool to delete this GA-repeat in NT2 cells, and analyzed the consequence of this deletion on GPM6B expression. Subsequently, the edited cells were induced to differentiate into neural cells, using retinoic acid (RA) treatment. Deletion of the GA-repeat significantly decreased the expression of GPM6B at the RNA (p < 0.05) and protein (40%) levels. Compared to the control cells, the edited cells showed dramatic decrease of the astrocyte and neural cell markers, including GFAP (0.77-fold), TUBB3 (0.57-fold), and MAP2 (0.2-fold). Subsequent sorting of the edited cells showed an increased number of NES (p < 0.01), but a decreased number of GFAP (p < 0.001), TUBB3 (p < 0.05), and MAP2 (p < 0.01), compared to the control cells. In conclusion, CRISPR/Cas9-mediated deletion of a GA-repeat in human GPM6B, led to decreased expression of this gene, which in turn, disrupted differentiation of NT2 cells into neural cells.
Identifiants
pubmed: 38273037
doi: 10.1038/s41598-024-52675-3
pii: 10.1038/s41598-024-52675-3
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
2136Informations de copyright
© 2024. The Author(s).
Références
Sanchez-Roige, S. et al. A mutant allele of glycoprotein M6-B (Gpm6b) facilitates behavioral flexibility but increases delay discounting. Genes Brain Behav. 21, e12800. https://doi.org/10.1111/gbb.12800 (2022).
doi: 10.1111/gbb.12800
pubmed: 35243767
pmcid: 9211103
Zhang, X. et al. Glycoprotein M6B interacts with TbetaRI to activate TGF-beta-Smad2/3 signaling and promote smooth muscle cell differentiation. Stem Cells 37, 190–201. https://doi.org/10.1002/stem.2938 (2019).
doi: 10.1002/stem.2938
pubmed: 30372567
Choi, K. M., Kim, J. Y. & Kim, Y. Distribution of the immunoreactivity for glycoprotein M6B in the neurogenic niche and reactive glia in the injury penumbra following traumatic brain injury in mice. Exp. Neurobiol. 22, 277–282. https://doi.org/10.5607/en.2013.22.4.277 (2013).
doi: 10.5607/en.2013.22.4.277
pubmed: 24465143
pmcid: 3897689
Mita, S. et al. Transcallosal projections require glycoprotein M6-dependent neurite growth and guidance. Cereb. Cortex 25, 4111–4125. https://doi.org/10.1093/cercor/bhu129 (2015).
doi: 10.1093/cercor/bhu129
pubmed: 24917275
Fernandez, M. E., Alfonso, J., Brocco, M. A. & Frasch, A. C. Conserved cellular function and stress-mediated regulation among members of the proteolipid protein family. J. Neurosci. Res. 88, 1298–1308. https://doi.org/10.1002/jnr.22298 (2010).
doi: 10.1002/jnr.22298
pubmed: 19937804
Drabek, K., van de Peppel, J., Eijken, M. & van Leeuwen, J. P. GPM6B regulates osteoblast function and induction of mineralization by controlling cytoskeleton and matrix vesicle release. J. Bone Miner. Res. 26, 2045–2051. https://doi.org/10.1002/jbmr.435 (2011).
doi: 10.1002/jbmr.435
pubmed: 21638316
Miao, Z. et al. Integrated analysis reveals prognostic value and mesenchymal identity suppression by glycoprotein M6B in glioma. Am. J. Transl. Res. 14, 3052–3065 (2022).
pubmed: 35702116
pmcid: 9185087
Nurk, S. et al. The complete sequence of a human genome. Science 376, 44–53. https://doi.org/10.1126/science.abj6987 (2022).
doi: 10.1126/science.abj6987
pubmed: 35357919
pmcid: 9186530
Gymrek, M. A genomic view of short tandem repeats. Curr. Opin. Genet. Dev. 44, 9–16. https://doi.org/10.1016/j.gde.2017.01.012 (2017).
doi: 10.1016/j.gde.2017.01.012
pubmed: 28213161
Ranathunge, C. et al. Transcribed microsatellite allele lengths are often correlated with gene expression in natural sunflower populations. Mol. Ecol. 29, 1704–1716. https://doi.org/10.1111/mec.15440 (2020).
doi: 10.1111/mec.15440
pubmed: 32285554
Valipour, E. et al. Polymorphic core promoter GA-repeats alter gene expression of the early embryonic developmental genes. Gene 531, 175–179. https://doi.org/10.1016/j.gene.2013.09.032 (2013).
doi: 10.1016/j.gene.2013.09.032
pubmed: 24055488
Arabfard, M., Kavousi, K., Delbari, A. & Ohadi, M. Link between short tandem repeats and translation initiation site selection. Hum. Genom. 12, 47. https://doi.org/10.1186/s40246-018-0181-3 (2018).
doi: 10.1186/s40246-018-0181-3
Bushehri, A., Barez, M. R., Mansouri, S. K., Biglarian, A. & Ohadi, M. Genome-wide identification of human- and primate-specific core promoter short tandem repeats. Gene 587, 83–90. https://doi.org/10.1016/j.gene.2016.04.041 (2016).
doi: 10.1016/j.gene.2016.04.041
pubmed: 27108803
Ohadi, M. et al. Core promoter short tandem repeats as evolutionary switch codes for primate speciation. Am. J. Primatol. 77, 34–43. https://doi.org/10.1002/ajp.22308 (2015).
doi: 10.1002/ajp.22308
pubmed: 25099915
Afshar, H. et al. Natural selection at the NHLH2 core promoter exceptionally long CA-repeat in human and disease-only genotypes in late-onset neurocognitive disorder. Gerontology 66, 514–522. https://doi.org/10.1159/000509471 (2020).
doi: 10.1159/000509471
pubmed: 32877896
Mohammadparast, S., Bayat, H., Biglarian, A. & Ohadi, M. Exceptional expansion and conservation of a CT-repeat complex in the core promoter of PAXBP1 in primates. Am. J. Primatol. 76, 747–756. https://doi.org/10.1002/ajp.22266 (2014).
doi: 10.1002/ajp.22266
pubmed: 24573656
Alizadeh, F. et al. Disease-only alleles at the extreme ends of the human ZMYM3 exceptionally long 5’ UTR short tandem repeat in bipolar disorder: A pilot study. J. Affect. Disord. 251, 86–90. https://doi.org/10.1016/j.jad.2019.03.056 (2019).
doi: 10.1016/j.jad.2019.03.056
pubmed: 30909162
Afshar, H. et al. Evolving evidence on a link between the ZMYM3 exceptionally long GA-STR and human cognition. Sci. Rep. 10, 19454. https://doi.org/10.1038/s41598-020-76461-z (2020).
doi: 10.1038/s41598-020-76461-z
pubmed: 33173136
pmcid: 7655811
Emamalizadeh, B. et al. The human RIT2 core promoter short tandem repeat predominant allele is species-specific in length: A selective advantage for human evolution?. Mol. Genet. Genom. 292, 611–617. https://doi.org/10.1007/s00438-017-1294-4 (2017).
doi: 10.1007/s00438-017-1294-4
Jakubosky, D. et al. Properties of structural variants and short tandem repeats associated with gene expression and complex traits. Nat. Commun. 11, 2927. https://doi.org/10.1038/s41467-020-16482-4 (2020).
doi: 10.1038/s41467-020-16482-4
pubmed: 32522982
pmcid: 7286898
Zhang, G. & Andersen, E. C. Interplay between polymorphic short tandem repeats and gene expression variation in Caenorhabditis elegans. Mol. Biol. Evol. 40, 4. https://doi.org/10.1093/molbev/msad067 (2023).
doi: 10.1093/molbev/msad067
Horton, C. A. et al. Short tandem repeats bind transcription factors to tune eukaryotic gene expression. Science 381, eaad1250. https://doi.org/10.1126/science.add1250 (2023).
doi: 10.1126/science.add1250
Lambert, S. A. et al. The human transcription factors. Cell 172, 650–665. https://doi.org/10.1016/j.cell.2018.01.029 (2018).
doi: 10.1016/j.cell.2018.01.029
pubmed: 29425488
Namdar-Aligoodarzi, P. et al. Exceptionally long 5’ UTR short tandem repeats specifically linked to primates. Gene 569, 88–94. https://doi.org/10.1016/j.gene.2015.05.053 (2015).
doi: 10.1016/j.gene.2015.05.053
pubmed: 26022613
Ohadi, M., Mohammadparast, S. & Darvish, H. Evolutionary trend of exceptionally long human core promoter short tandem repeats. Gene 507, 61–67. https://doi.org/10.1016/j.gene.2012.07.001 (2012).
doi: 10.1016/j.gene.2012.07.001
pubmed: 22796130
Khamse, S. et al. Predominant monomorphism of the RIT2 and GPM6B exceptionally long GA blocks in human and enriched divergent alleles in the disease compartment. Genetica 150, 27–40. https://doi.org/10.1007/s10709-021-00143-5 (2022).
doi: 10.1007/s10709-021-00143-5
pubmed: 34984576
Bayat, H., Omidi, M., Rajabibazl, M., Sabri, S. & Rahimpour, A. The CRISPR growth spurt: From bench to clinic on versatile small RNAs. J. Microbiol. Biotechnol. 27, 207–218. https://doi.org/10.4014/jmb.1607.07005 (2017).
doi: 10.4014/jmb.1607.07005
pubmed: 27840399
Yameogo, P., Gerard, C., Majeau, N. & Tremblay, J. P. Removal of the GAA repeat in the heart of a Friedreich’s ataxia mouse model using CjCas9. Gene Ther. 30, 612–619. https://doi.org/10.1038/s41434-023-00387-0 (2023).
doi: 10.1038/s41434-023-00387-0
pubmed: 36781946
Lo-Scrudato, M. et al. Genome editing of expanded CTG repeats within the human DMPK gene reduces nuclear RNA foci in the muscle of DM1 mice. Mol. Ther. 27, 1372–1388. https://doi.org/10.1016/j.ymthe.2019.05.021 (2019).
doi: 10.1016/j.ymthe.2019.05.021
pubmed: 31253581
pmcid: 6697452
Shams, F. et al. Advance trends in targeting homology-directed repair for accurate gene editing: An inclusive review of small molecules and modified CRISPR-Cas9 systems. Bioimpacts 12, 371–391. https://doi.org/10.34172/bi.2022.23871 (2022).
doi: 10.34172/bi.2022.23871
pubmed: 35975201
pmcid: 9376165
Baba, Y., Onishi-Sakamoto, S., Ide, K. & Nishifuji, K. Nestin is a marker of unipotent embryonic and adult progenitors differentiating into an epithelial cell lineage of the hair follicles. Sci. Rep. 12, 17820. https://doi.org/10.1038/s41598-022-22427-2 (2022).
doi: 10.1038/s41598-022-22427-2
pubmed: 36280775
pmcid: 9592581
Jurga, A. M., Paleczna, M., Kadluczka, J. & Kuter, K. Z. Beyond the GFAP-astrocyte protein markers in the brain. Biomolecules 11, 9. https://doi.org/10.3390/biom11091361 (2021).
doi: 10.3390/biom11091361
Fu, J. Q. et al. A single factor induces neuronal differentiation to suppress glioma cell growth. CNS Neurosci. Ther. 25, 486–495. https://doi.org/10.1111/cns.13066 (2019).
doi: 10.1111/cns.13066
pubmed: 30264483
Thierry-Mieg, D. & Thierry-Mieg, J. AceView: A comprehensive cDNA-supported gene and transcripts annotation. Genome Biol. 7(1), 11–14. https://doi.org/10.1186/gb-2006-7-s1-s12 (2006).
doi: 10.1186/gb-2006-7-s1-s12
Werner, H. B. et al. A critical role for the cholesterol-associated proteolipids PLP and M6B in myelination of the central nervous system. Glia 61, 567–586. https://doi.org/10.1002/glia.22456 (2013).
doi: 10.1002/glia.22456
pubmed: 23322581
Fjorback, A. W., Muller, H. K. & Wiborg, O. Membrane glycoprotein M6B interacts with the human serotonin transporter. J. Mol. Neurosci. 37, 191–200. https://doi.org/10.1007/s12031-008-9092-4 (2009).
doi: 10.1007/s12031-008-9092-4
pubmed: 18581270
Sanchez-Roige, S. et al. Genome-wide association study of delay discounting in 23,217 adult research participants of European ancestry. Nat. Neurosci. 21, 16–18. https://doi.org/10.1038/s41593-017-0032-x (2018).
doi: 10.1038/s41593-017-0032-x
pubmed: 29230059
Wright, S. E. & Todd, P. K. Native functions of short tandem repeats. Elife 12, 104. https://doi.org/10.7554/eLife.84043 (2023).
doi: 10.7554/eLife.84043
Marsh, S., Hanson, B., Wood, M. J. A., Varela, M. A. & Roberts, T. C. Application of CRISPR-Cas9-mediated genome editing for the treatment of myotonic dystrophy type 1. Mol. Ther. 28, 2527–2539. https://doi.org/10.1016/j.ymthe.2020.10.005 (2020).
doi: 10.1016/j.ymthe.2020.10.005
pubmed: 33171139
pmcid: 7704741
Batra, R. et al. The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1. Nat. Biomed. Eng. 5, 157–168. https://doi.org/10.1038/s41551-020-00607-7 (2021).
doi: 10.1038/s41551-020-00607-7
pubmed: 32929188
Mosbach, V. et al. Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. PLoS Genet. 16, e1008924. https://doi.org/10.1371/journal.pgen.1008924 (2020).
doi: 10.1371/journal.pgen.1008924
pubmed: 32673314
pmcid: 7413560
Rohrmoser, M. et al. MIR sequences recruit zinc finger protein ZNF768 to expressed genes. Nucleic Acids Res. 47, 700–715. https://doi.org/10.1093/nar/gky1148 (2019).
doi: 10.1093/nar/gky1148
pubmed: 30476274
Leszczynski, P. et al. Deletion of the Prdm3 gene causes a neuronal differentiation deficiency in P19 cells. Int. J. Mol. Sci. 21, 7192. https://doi.org/10.3390/ijms21197192 (2020).
doi: 10.3390/ijms21197192
pubmed: 33003409
pmcid: 7582457
Datta, T. K. et al. Requirement of the transcription factor USF1 in bovine oocyte and early embryonic development. Reproduction 149, 203–212. https://doi.org/10.1530/REP-14-0445 (2015).
doi: 10.1530/REP-14-0445
pubmed: 25385722
Di Pietro, A. et al. Targeting BMI-1 in B cells restores effective humoral immune responses and controls chronic viral infection. Nat. Immunol. 23, 86–98. https://doi.org/10.1038/s41590-021-01077-y (2022).
doi: 10.1038/s41590-021-01077-y
pubmed: 34845392
Li, X. et al. Initial activation of STAT2 induced by IAV infection is critical for innate antiviral immunity. Front. Immunol. 13, 960544. https://doi.org/10.3389/fimmu.2022.960544 (2022).
doi: 10.3389/fimmu.2022.960544
pubmed: 36148221
pmcid: 9486978
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10. https://doi.org/10.14806/ej.17.1.200 (2011).
doi: 10.14806/ej.17.1.200
Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37, 907–915. https://doi.org/10.1038/s41587-019-0201-4 (2019).
doi: 10.1038/s41587-019-0201-4
pubmed: 31375807
pmcid: 7605509
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930. https://doi.org/10.1093/bioinformatics/btt656 (2014).
doi: 10.1093/bioinformatics/btt656
pubmed: 24227677
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47. https://doi.org/10.1093/nar/gkv007 (2015).
doi: 10.1093/nar/gkv007
pubmed: 25605792
pmcid: 4402510
Leek, J. T., Johnson, W. E., Parker, H. S., Jaffe, A. E. & Storey, J. D. The sva package for removing batch effects and other unwanted variation in high-throughput experiments. Bioinformatics 28, 882–883. https://doi.org/10.1093/bioinformatics/bts034 (2012).
doi: 10.1093/bioinformatics/bts034
pubmed: 22257669
pmcid: 3307112
Wickham, H. ggplot2: Elegant Graphics for Data Analysis 2nd edn. https://doi.org/10.1007/978-3-319-24277-4 (Springer, New York, 2016).
doi: 10.1007/978-3-319-24277-4
Concordet, J. P. & Haeussler, M. CRISPOR: Intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245. https://doi.org/10.1093/nar/gky354 (2018).
doi: 10.1093/nar/gky354
pubmed: 29762716
pmcid: 6030908
Bayat, H., Modarressi, M. H. & Rahimpour, A. The conspicuity of CRISPR-Cpf1 system as a significant breakthrough in genome editing. Curr. Microbiol. 75, 107–115. https://doi.org/10.1007/s00284-017-1406-8 (2018).
doi: 10.1007/s00284-017-1406-8
pubmed: 29189942
Bayat, H., Naderi, F., Khan, A. H., Memarnejadian, A. & Rahimpour, A. The impact of CRISPR-cas system on antiviral therapy. Adv. Pharm. Bull. 8, 591–597. https://doi.org/10.15171/apb.2018.067 (2018).
doi: 10.15171/apb.2018.067
pubmed: 30607331
pmcid: 6311650
Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308. https://doi.org/10.1038/nprot.2013.143 (2013).
doi: 10.1038/nprot.2013.143
pubmed: 24157548
pmcid: 3969860
Darbinian, N. Cultured cell line models of neuronal differentiation: NT2, PC12, and SK-N-MC. Methods Mol. Biol. 2311, 25–38. https://doi.org/10.1007/978-1-0716-1437-2_3 (2021).
doi: 10.1007/978-1-0716-1437-2_3
pubmed: 34033075
Andrews, P. W. Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro. Dev. Biol. 103, 285–293. https://doi.org/10.1016/0012-1606(84)90316-6 (1984).
doi: 10.1016/0012-1606(84)90316-6
pubmed: 6144603
Guschin, D. Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256. https://doi.org/10.1007/978-1-60761-753-2_15 (2010).
doi: 10.1007/978-1-60761-753-2_15
pubmed: 20680839
Bayat, H., Pourgholami, M. H., Rahmani, S., Pournajaf, S. & Mowla, S. J. Synthetic miR-21 decoy circularized by tRNA splicing mechanism inhibited tumorigenesis in glioblastoma in vitro and in vivo models. Mol. Ther. Nucleic Acids 32, 432–444. https://doi.org/10.1016/j.omtn.2023.04.001 (2023).
doi: 10.1016/j.omtn.2023.04.001
pubmed: 37181451
pmcid: 10173299