De-erosion of X Chromosome Dosage Compensation by the Editing of Regulatory Regions Restores the Differentiation Potential in HPSCs

Overview

Journal Cell Rep Methods

Specialty Cell Biology

Date 2023 Jan 2

PMID 36590687

Authors

Nami Motosugi

Akiko Sugiyama

Chisa Okada

Asako Otomo

Akihiro Umezawa

Hidenori Akutsu

Shinji Hadano

Atsushi Fukuda

Affiliations

Soon will be listed here.

Abstract

Human pluripotent stem cells (hPSCs) regularly and irreversibly show the erosion of X chromosome inactivation (XCI) by long non-coding RNA (lncRNA) silencing, causing challenges in various applications of female hPSCs. Here, we report reliable methods to reactivate with monoallelic expression in female hPSCs. Surprisingly, we find that the editing of regulatory regions by Cas9-mediated non-homologous end joining is sufficient for the reactivation of by endogenous systems. Proliferated hPSCs with reactivation show XCI from an eroded X chromosome, suggesting that hPSCs with normal dosage compensation might lead to a growth advantage. Furthermore, the use of targeting vectors, including the regulatory region sequences and selection cassette, enables reactivation in hPSCs with high efficiency. -reactivated hPSCs can show the restoration of differentiation potential. Thus, our findings demonstrate that re-expression is a beneficial method to maximize the use of female hPSCs in various applications, such as proper disease modeling.

Citing Articles

Gain of 1q confers an MDM4-driven growth advantage to undifferentiated and differentiating hESC while altering their differentiation capacity.

Krivec N, Couvreu de Deckersberg E, Lei Y, Al Delbany D, Regin M, Verhulst S Cell Death Dis. 2024; 15(11):852.

PMID: 39572522 PMC: 11582570. DOI: 10.1038/s41419-024-07236-x.

X-chromosome inactivation in human iPSCs provides insight into X-regulated gene expression in autosomes.

Topa H, Benoit-Pilven C, Tukiainen T, Pietilainen O Genome Biol. 2024; 25(1):144.

PMID: 38822397 PMC: 11143737. DOI: 10.1186/s13059-024-03286-8.

Sex-biased gene expression during neural differentiation of human embryonic stem cells.

Pottmeier P, Nikolantonaki D, Lanner F, Peuckert C, Jazin E Front Cell Dev Biol. 2024; 12:1341373.

PMID: 38764741 PMC: 11101176. DOI: 10.3389/fcell.2024.1341373.

Effect of PCDH19 missense mutations on cell-to-cell proximity and neuronal development under heterotypic conditions.

Motosugi N, Sugiyama A, Otomo A, Sakata Y, Araki T, Hadano S PNAS Nexus. 2024; 3(3):pgae060.

PMID: 38516276 PMC: 10957236. DOI: 10.1093/pnasnexus/pgae060.

Inheritance of epigenetic transcriptional memory.

Ge T, Brickner J Curr Opin Genet Dev. 2024; 85:102174.

PMID: 38430840 PMC: 10947848. DOI: 10.1016/j.gde.2024.102174.

References

Perez-Pinera P, Kocak D, Vockley C, Adler A, Kabadi A, Polstein L . RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. 2013; 10(10):973-6. PMC: 3911785. DOI: 10.1038/nmeth.2600. View

Clemson C, McNeil J, Willard H, Lawrence J . XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol. 1996; 132(3):259-75. PMC: 2120729. DOI: 10.1083/jcb.132.3.259. View

Anguera M, Sadreyev R, Zhang Z, Szanto A, Payer B, Sheridan S . Molecular signatures of human induced pluripotent stem cells highlight sex differences and cancer genes. Cell Stem Cell. 2012; 11(1):75-90. PMC: 3587778. DOI: 10.1016/j.stem.2012.03.008. View

Liao J, Karnik R, Gu H, Ziller M, Clement K, Tsankov A . Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells. Nat Genet. 2015; 47(5):469-78. PMC: 4414868. DOI: 10.1038/ng.3258. View

Sahakyan A, Kim R, Chronis C, Sabri S, Bonora G, Theunissen T . Human Naive Pluripotent Stem Cells Model X Chromosome Dampening and X Inactivation. Cell Stem Cell. 2016; 20(1):87-101. PMC: 5218861. DOI: 10.1016/j.stem.2016.10.006. View

Ziller M, Ortega J, Quinlan K, Santos D, Gu H, Martin E . Dissecting the Functional Consequences of De Novo DNA Methylation Dynamics in Human Motor Neuron Differentiation and Physiology. Cell Stem Cell. 2018; 22(4):559-574.e9. PMC: 6535433. DOI: 10.1016/j.stem.2018.02.012. View

Patel S, Bonora G, Sahakyan A, Kim R, Chronis C, Langerman J . Human Embryonic Stem Cells Do Not Change Their X Inactivation Status during Differentiation. Cell Rep. 2016; 18(1):54-67. PMC: 5214931. DOI: 10.1016/j.celrep.2016.11.054. View

Yamanaka S . Pluripotent Stem Cell-Based Cell Therapy-Promise and Challenges. Cell Stem Cell. 2020; 27(4):523-531. DOI: 10.1016/j.stem.2020.09.014. View

Vallot C, Ouimette J, Makhlouf M, Feraud O, Pontis J, Come J . Erosion of X Chromosome Inactivation in Human Pluripotent Cells Initiates with XACT Coating and Depends on a Specific Heterochromatin Landscape. Cell Stem Cell. 2015; 16(5):533-46. DOI: 10.1016/j.stem.2015.03.016. View

10.

Salomonis N, Dexheimer P, Omberg L, Schroll R, Bush S, Huo J . Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consortium. Stem Cell Reports. 2016; 7(1):110-25. PMC: 4944587. DOI: 10.1016/j.stemcr.2016.05.006. View

11.

Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S . Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020; 581(7807):215-220. DOI: 10.1038/s41586-020-2180-5. View

12.

Tukiainen T, Villani A, Yen A, Rivas M, Marshall J, Satija R . Landscape of X chromosome inactivation across human tissues. Nature. 2017; 550(7675):244-248. PMC: 5685192. DOI: 10.1038/nature24265. View

13.

Guo G, von Meyenn F, Rostovskaya M, Clarke J, Dietmann S, Baker D . Epigenetic resetting of human pluripotency. Development. 2017; 144(15):2748-2763. PMC: 5560041. DOI: 10.1242/dev.146811. View

14.

Wang Z, Oron E, Nelson B, Razis S, Ivanova N . Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell. 2012; 10(4):440-54. DOI: 10.1016/j.stem.2012.02.016. View

15.

Han X, Zhou Z, Fei L, Sun H, Wang R, Chen Y . Construction of a human cell landscape at single-cell level. Nature. 2020; 581(7808):303-309. DOI: 10.1038/s41586-020-2157-4. View

16.

McCarthy D, Chen Y, Smyth G . Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012; 40(10):4288-97. PMC: 3378882. DOI: 10.1093/nar/gks042. View

17.

Stadtfeld M, Apostolou E, Akutsu H, Fukuda A, Follett P, Natesan S . Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature. 2010; 465(7295):175-81. PMC: 3987905. DOI: 10.1038/nature09017. View

18.

Thomson J, Itskovitz-Eldor J, Shapiro S, Waknitz M, Swiergiel J, Marshall V . Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282(5391):1145-7. DOI: 10.1126/science.282.5391.1145. View

19.

Ohashi M, Korsakova E, Allen D, Lee P, Fu K, Vargas B . Loss of MECP2 Leads to Activation of P53 and Neuronal Senescence. Stem Cell Reports. 2018; 10(5):1453-1463. PMC: 5995366. DOI: 10.1016/j.stemcr.2018.04.001. View

20.

Fukuda A, Hazelbaker D, Motosugi N, Hao J, Limone F, Beccard A . De novo DNA methyltransferases DNMT3A and DNMT3B are essential for XIST silencing for erosion of dosage compensation in pluripotent stem cells. Stem Cell Reports. 2021; 16(9):2138-2148. PMC: 8452533. DOI: 10.1016/j.stemcr.2021.07.015. View