Design of iPSC-based cell model to study the functions of the UBE2A gene

Abstract

BACKGROUND: The UBE2A protein belongs to the E2 family of ubiquitin-binding enzymes involved in the ubiquitination of substrate proteins. UBE2A mutations lead to congenital X-linked mental retardation syndrome-type Nascimento. How UBE2A participates in the central nervous system development is still unknown.

AIM: To establish a cell model based on induced pluripotent stem cells (iPSCs) to study the molecular and cellular functions of UBE2A in neurogenesis.

METHODS: Using genomic CRISPR-Cas9 editing and lentiviral transduction, a cell model based on iPSCs from two healthy donors was designed. This cell model includes isogenic iPSCs with knockout and inducible hyperexpression of UBE2A. In addition, iPSCs were obtained by reprogramming peripheral blood mononuclear cells of a patient diagnosed with X-linked mental retardation of Nascimento type, which has a deletion spanning the whole UBE2A locus.

RESULTS: The obtained iPSCs demonstrate an ESC-like morphology. They express pluripotent cell markers OCT4, SOX2, SSEA-4, and TRA-1-81 and have normal karyotypes. iPSCs with UBE2A knockout or hyperexpression had significantly increased nuclei size compared with the isogenic control.

CONCLUSION: The developed iPSC-based cell model can be used for fundamental studies of the functions of UBE2A in neurogenesis.

Keywords

iPSC / ubiquitin-conjugating enzyme E2A (RAD6 homolog), human / X-linked mental retardation / gene knockout

References

[1] Haddad DM, Vilain S, Vos M, et al. Mutations in the intellectual disability gene Ube2a cause neuronal dysfunction and impair parkin-dependent mitophagy. Mol Cell. 2013;50(6):831–843. doi: 10.1016/j.molcel.2013.04.012

[2] Haddad D.M., Vilain S., Vos M., et al. Mutations in the intellectual disability gene Ube2a cause neuronal dysfunction and impair parkin-dependent mitophagy // Mol Cell. 2013. Vol. 50, N 6. P. 831–843. doi: 10.1016/j.molcel.2013.04.012

[3] Fu J, Liao L, Balaji KS, et al. Epigenetic modification and a role for the E3 ligase RNF40 in cancer development and metastasis. Oncogene. 2021;40(3):465–474. doi: 10.1038/s41388-020-01556-w

[4] Fu J., Liao L., Balaji K.S., et al. Epigenetic modification and a role for the E3 ligase RNF40 in cancer development and metastasis // Oncogene. 2021. Vol. 40, N 3. P. 465–474. doi: 10.1038/s41388-020-01556-w

[5] Yamada T, Imamachi N, Imamura K, et al. Systematic analysis of targets of pumilio-mediated mRNA decay reveals that PUM1 repression by DNA damage activates translesion synthesis. Cell Rep. 2020;31(5):107542. doi: 10.1016/j.celrep.2020.107542

[6] Yamada T., Imamachi N., Imamura K., et al. Systematic analysis of targets of pumilio-mediated mRNA decay reveals that PUM1 repression by DNA damage activates translesion synthesis // Cell Rep. 2020. Vol. 31, N 5. P. 107542. doi: 10.1016/j.celrep.2020.107542

[7] Barnsby-Greer L, Mabbitt PD, Dery MA, et al. An atypical E3 ligase module in UBR4 mediates destabilization of N-degron substrates. bioRxiv. 2023. doi: 10.1101/2023.05.08.539884

[8] Barnsby-Greer L., Mabbitt P.D., Dery M.A., et al. An atypical E3 ligase module in UBR4 mediates destabilization of N-degron substrates // bioRxiv. 2023. doi: 10.1101/2023.05.08.539884

[9] Nascimento RM, Otto PA, de Brouwer AP, Vianna-Morgante AM. UBE2A, which encodes a ubiquitin-conjugating enzyme, is mutated in a novel X-linked mental retardation syndrome. Am J Hum Genet. 2006;79(3):549–555. doi: 10.1086/507047

[10] Nascimento R.M., Otto P.A., de Brouwer A.P., Vianna-Morgante A.M. UBE2A, which encodes a ubiquitin-conjugating enzyme, is mutated in a novel X-linked mental retardation syndrome // Am J Hum Genet. 2006. Vol. 79, N 3. P. 549–555. doi: 10.1086/507047

[11] Czeschik JC, Bauer P, Buiting K, et al. X-linked intellectual disability type Nascimento is a clinically distinct, probably underdiagnosed entity. Orphanet J Rare Dis. 2013;8:146. doi: 10.1186/1750-1172-8-146

[12] Czeschik J.C., Bauer P., Buiting K., et al. X-linked intellectual disability type Nascimento is a clinically distinct, probably underdiagnosed entity // Orphanet J Rare Dis. 2013. Vol. 8. P. 146. doi: 10.1186/1750-1172-8-146

[13] Kopanos C, Tsiolkas V, Kouris A, et al. VarSome: the human genomic variant search engine. Bioinformatics. 2019;35(11):1978–1980. doi: 10.1093/bioinformatics/bty897

[14] Kopanos C., Tsiolkas V., Kouris A., et al. VarSome: the human genomic variant search engine // Bioinformatics. 2019. V. 35, N 11. P. 1978–1980. doi: 10.1093/bioinformatics/bty897

[15] Tolmacheva EN, Kashevarova AA, Nazarenko LP, et al. Delineation of clinical manifestations of the inherited Xq24 microdeletion segregating with sXCI in mothers: two novel cases with distinct phenotypes ranging from UBE2A deficiency syndrome to recurrent pregnancy loss. Cytogenet Genome Res. 2020;160(5):245–254. doi: 10.1159/000508050

[16] Tolmacheva E.N., Kashevarova A.A., Nazarenko L.P., et al. Delineation of clinical manifestations of the inherited Xq24 microdeletion segregating with sXCI in mothers: two novel cases with distinct phenotypes ranging from UBE2A deficiency syndrome to recurrent pregnancy loss // Cytogenet Genome Res. 2020. Vol. 160, N 5. P. 245–254. doi: 10.1159/000508050

[17] Roest HP, Baarends WM, de Wit J, et al. The ubiquitin-conjugating DNA repair enzyme HR6A is a maternal factor essential for early embryonic development in mice. Mol Cell Biol. 2004;24(12):5485–5495. doi: 10.1128/MCB.24.12.5485-5495.2004

[18] Roest H.P., Baarends W.M., de Wit J., et al. The ubiquitin-conjugating DNA repair enzyme HR6A is a maternal factor essential for early embryonic development in mice // Mol Cell Biol. 2004. Vol. 24, N 12. P. 5485–5495. doi: 10.1128/MCB.24.12.5485-5495.2004

[19] Foglizzo M, Day CL. E2 enzymes: lessons in ubiquitin transfer from XLID patients. Nat Chem Biol. 2019;15(1):6–7. doi: 10.1038/s41589-018-0191-4

[20] Foglizzo M., Day C.L. E2 enzymes: lessons in ubiquitin transfer from XLID patients // Nat Chem Biol. 2019. Vol. 15, N 1. P. 6–7. doi: 10.1038/s41589-018-0191-4

[21] Park IH, Arora N, Huo H, et al. Disease-specific induced pluripotent stem cells. Cell. 2008;134(5):877–886. doi: 10.1016/j.cell.2008.07.041

[22] Park I.H., Arora N., Huo H., et al. Disease-specific induced pluripotent stem cells // Cell. 2008. Vol. 134, N 5. P. 877–886. doi: 10.1016/j.cell.2008.07.041

[23] Eremeev AV, Lebedeva OS, Bogomiakova ME, et al. Cerebral organoids-challenges to establish a brain prototype. Cells. 2021;10(7):1790. doi: 10.3390/cells10071790

[24] Eremeev A.V., Lebedeva O.S., Bogomiakova M.E., et al. Cerebral organoids — challenges to establish a brain prototype // Cells. 2021. Vol. 10, N 7. P. 1790. doi: 10.3390/cells10071790

[25] Crook JM, Wallace G, Tomaskovic-Crook E. The potential of induced pluripotent stem cells in models of neurological disorders: implications on future therapy. Expert Rev Neurother. 2015;15(3):295–304. doi: 10.1586/14737175.2015.1013096

[26] Crook J.M., Wallace G., Tomaskovic-Crook E. The potential of induced pluripotent stem cells in models of neurological disorders: implications on future therapy // Expert Rev Neurother. 2015. Vol. 15, N 3. P. 295–304. doi: 10.1586/14737175.2015.1013096

[27] Deshpande A, Yadav S, Dao DQ, et al. Cellular phenotypes in human iPSC-derived neurons from a genetic model of autism spectrum disorder. Cell Rep. 2017;21(10):2678–2687. doi: 10.1016/j.celrep.2017.11.037

[28] Deshpande A., Yadav S., Dao D.Q., et al. Cellular phenotypes in human iPSC-derived neurons from a genetic model of autism spectrum disorder // Cell Rep. 2017. Vol. 21, N 10. P. 2678–2687. doi: 10.1016/j.celrep.2017.11.037

[29] Khomyakova EA, Fedorenko AV, Surdina AV, et al. Derivation of induced pluripotent stem cells line (RCPCMI009-A-1) with knockout of the UBE2A gene by CRISPR/CAS9 genome editing. Ontogenez. 2024. (In press). doi: 10.31857/S0475145023060046

[30] Хомякова E.A., Федоренко А.В., Сурдина А.В., и др. Создание линии индуцированных плюрипотентных стволовых клеток RCPMi009-A-1 с нокаутом гена UBE2A с помощью технологии редактирования генома CRISPR/Cas9 // Онтогенез. 2024. (In press). doi: 10.31857/S0475145023060046

[31] van Kuppeveld FJ, van der Logt JT, Angulo AF, et al. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol. 1992;58(8):2606–2615. Corrected and republished from: Appl Environ Microbiol. 1993;59(2):655. doi: 10.1128/aem.58.8.2606-2615.1992

[32] van Kuppeveld F.J., van der Logt J.T., Angulo A.F., et al. Genus-and species-specific identification of mycoplasmas by 16S rRNA amplification // Appl Environ Microbiol. 1992. Vol. 58, N 8. P. 2606–2615. Corrected and republished from: Appl Environ Microbiol. 1993. Vol. 59, N 2. P. 655. doi: 10.1128/aem.58.8.2606-2615.1992

[33] Concordet JP, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018;46(W1):W242–W245. doi: 10.1093/nar/gky354

[34] Concordet J.P., Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens // Nucleic Acids Res. 2018. Vol. 46, W1. P. W242–W245. doi: 10.1093/nar/gky354

[35] Pliatsika V, Rigoutsos I. “Off-Spotter”: very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs. Biol Direct. 2015;10:4. doi: 10.1186/s13062-015-0035-z

[36] Pliatsika V., Rigoutsos I. “Off-Spotter”: very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs // Biol Direct. 2015. Vol. 10. P. 4. doi: 10.1186/s13062-015-0035-z

[37] Okita K, Yamakawa T, Matsumura Y, et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2013;31(3):458–466. doi: 10.1002/stem.1293

[38] Okita K., Yamakawa T., Matsumura Y., et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells // Stem Cells. 2013. Vol. 31, N 3. P. 458–466. doi: 10.1002/stem.1293

[39] Gridina MM, Nurislamov AR, Minina JM, et al. Generation of iPS cell line (ICGi040-A) from skin fibroblasts of a patient with ring small supernumerary marker chromosome 4. Stem Cell Res. 2022;61:102740. doi: 10.1016/j.scr.2022.102740

[40] Gridina M.M., Nurislamov A.R., Minina J.M., et al. Generation of iPS cell line (ICGi040-A) from skin fibroblasts of a patient with ring small supernumerary marker chromosome 4 // Stem Cell Res. 2022. Vol. 61. P. 102740. doi: 10.1016/j.scr.2022.102740

[41] Holmqvist S, Lehtonen Š, Chumarina M, et al. Creation of a library of induced pluripotent stem cells from Parkinsonian patients. NPJ Parkinsons Dis. 2016;2:16009. doi: 10.1038/npjparkd.2016.9

[42] Holmqvist S., Lehtonen Š., Chumarina M., et al. Creation of a library of induced pluripotent stem cells from Parkinsonian patients // NPJ Parkinsons Dis. 2016. Vol. 2. P. 16009. doi: 10.1038/npjparkd.2016.9

[43] Benedetti MC, D’Andrea T, Colantoni A, et al. Cortical neurons obtained from patients-derived GNAO1 iPSCs show altered differentiation and functional properties. bioRxiv. 2023. doi: 10.1101/2023.06.15.545051

[44] Benedetti M.C., D’Andrea T., Colantoni A., et al. Cortical neurons obtained from patients-derived GNAO1 iPSCs show altered differentiation and functional properties // bioRxiv. 2023. doi: 10.1101/2023.06.15.545051

[45] Shi Y, Inoue H, Wu JC, Yamanaka S. Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov. 2017;16(2):115–130. doi: 10.1038/nrd.2016.245

[46] Shi Y., Inoue H., Wu J.C., Yamanaka S. Induced pluripotent stem cell technology: a decade of progress // Nat Rev Drug Discov. 2017. Vol. 16, N 2. P. 115–130. doi: 10.1038/nrd.2016.245

[47] Ramos-Mejía V, Montes R, Bueno C, et al. Residual expression of the reprogramming factors prevents differentiation of iPSC generated from human fibroblasts and cord blood CD34+ progenitors. PLoS One. 2012;7(4):e35824. doi: 10.1371/journal.pone.0035824

[48] Ramos-Mejía V., Montes R., Bueno C., et al. Residual expression of the reprogramming factors prevents differentiation of iPSC generated from human fibroblasts and cord blood CD34+ progenitors // PLoS One. 2012. Vol. 7, N 4. P. e35824. doi: 10.1371/journal.pone.0035824

[49] International Stem Cell Initiative; Amps K, Andrews PW, et al. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nat Biotechnol. 2011;29(12):1132–1144. doi: 10.1038/nbt.2051

[50] International Stem Cell Initiative; Amps K., Andrews P.W., et al. 2011. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage // Nat Biotechnol. 2011. Vol. 29, N 12. P. 1132–1144. doi: 10.1038/nbt.2051

[51] Uhlén M, Bjorling E, Agaton C, et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol Cell Proteomics. 2005;4(12):1920–1932. doi: 10.1074/mcp.M500279-MCP200

[52] Uhlén M., Björling E., Agaton C., et al. A human protein atlas for normal and cancer tissues based on antibody proteomics // Mol Cell Proteomics. 2005. Vol. 4, N 12. P. 1920–1932. doi: 10.1074/mcp.M500279-MCP200

[53] Shekhar MP, Lyakhovich A, Visscher DW, et al. Rad6 overexpression induces multinucleation, centrosome amplification, abnormal mitosis, aneuploidy, and transformation. Cancer Res. 2002;62(7):2115–2124.

[54] Shekhar M.P., Lyakhovich A., Visscher D.W., et al. Rad6 overexpression induces multinucleation, centrosome amplification, abnormal mitosis, aneuploidy, and transformation. // Cancer Res. 2002. Vol. 62, N 7. P. 2115–2124.

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