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Extensive cellular heterogeneity of X inactivation revealed by single-cell allele-specific expression in human fibroblasts

Marco Garieri, Georgios Stamoulis, Xavier Blanc, Emilie Falconnet, Pascale Ribaux, Christelle Borel, Federico Santoni, and Stylianos E. Antonarakis
PNAS December 18, 2018 115 (51) 13015-13020; published ahead of print December 3, 2018 https://doi.org/10.1073/pnas.1806811115
Marco Garieri
aDepartment of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, CH, Switzerland;
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Georgios Stamoulis
aDepartment of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, CH, Switzerland;
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Xavier Blanc
aDepartment of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, CH, Switzerland;
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Emilie Falconnet
aDepartment of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, CH, Switzerland;
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Pascale Ribaux
aDepartment of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, CH, Switzerland;
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Christelle Borel
aDepartment of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, CH, Switzerland;
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  • For correspondence: christelle.borel@unige.chfederico.santoni@chuv.chstylianos.antonarakis@unige.ch
Federico Santoni
aDepartment of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, CH, Switzerland;bService of Endocrinology, Diabetology, and Metabolism, University Hospitals of Lausanne, 1011 Lausanne, CH, Switzerland;eFaculty of Biology and Medicine, University of Lausanne, 1015 Lausanne, CH, Switzerland
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  • ORCID record for Federico Santoni
  • For correspondence: christelle.borel@unige.chfederico.santoni@chuv.chstylianos.antonarakis@unige.ch
Stylianos E. Antonarakis
aDepartment of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, CH, Switzerland;cUniversity Hospitals of Geneva, 1205 Geneva, CH, Switzerland;diGE3 Institute of Genetics and Genomics, University of Geneva, 1211 Geneva, CH, Switzerland;
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  • For correspondence: christelle.borel@unige.chfederico.santoni@chuv.chstylianos.antonarakis@unige.ch
  1. Edited by Adrian P. Bird, University of Edinburgh, Edinburgh, United Kingdom, and approved October 29, 2018 (received for review April 20, 2018)

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Significance

X-chromosome inactivation (XCI) is a female dosage compensation mechanism where one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). By means of single-cell transcriptome we show that XCI is variable among individuals and from cells from the same individual. We discovered that XCI is indeed a cellular phenotype and that the expression of escapees is regulated by cell-cycle phases and by the expression of XIST. Additionally, in this context, we identified a list of undescribed escapee genes, and we validate many already suggested escapee genes with a direct measurement on single cells.

Abstract

X-chromosome inactivation (XCI) provides a dosage compensation mechanism where, in each female cell, one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). We investigated XCI at single-cell resolution combining deep single-cell RNA sequencing with whole-genome sequencing to examine allelic-specific expression in 935 primary fibroblast and 48 lymphoblastoid single cells from five female individuals. In this framework we integrated an original method to identify and exclude doublets of cells. In fibroblast cells, we have identified 55 genes as escapees including five undescribed escapee genes. Moreover, we observed that all genes exhibit a variable propensity to escape XCI in each cell and cell type and that each cell displays a distinct expression profile of the escapee genes. A metric, the Inactivation Score—defined as the mean of the allelic expression profiles of the escapees per cell—enables us to discover a heterogeneous and continuous degree of cellular XCI with extremes represented by “inactive” cells, i.e., cells exclusively expressing the escaping genes from the active X chromosome and “escaping” cells expressing the escapees from both alleles. We found that this effect is associated with cell-cycle phases and, independently, with the XIST expression level, which is higher in the quiescent phase (G0). Single-cell allele-specific expression is a powerful tool to identify novel escapees in different tissues and provide evidence of an unexpected cellular heterogeneity of XCI.

  • single-cell transcriptomics
  • X inactivation
  • XIST
  • cell cycle
  • cell heterogeneity

Footnotes

  • ↵1M.G. and G.S. contributed equally to this work.

  • ↵2C.B., F.S., and S.E.A. contributed equally to this work.

  • ↵3To whom correspondence may be addressed. Email: christelle.borel{at}unige.ch, federico.santoni{at}chuv.ch, or stylianos.antonarakis{at}unige.ch.
  • Author contributions: C.B., F.S., and S.E.A. designed research; M.G., G.S., E.F., P.R., C.B., F.S., and S.E.A. performed research; M.G., C.B., and F.S. contributed new reagents/analytic tools; M.G., X.B., C.B., F.S., and S.E.A. analyzed data; and G.S., C.B., F.S., and S.E.A. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The newly generated RNA and DNA sequencing data have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE123028). Single-cell RNA sequences from limphoblasts and fibroblasts of individual 5 were obtained from the European Genome-phenome Archive, https://www.ebi.ac.uk/ega/studies/EGAS00001001009 (accession no. EGAS00001001009).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1806811115/-/DCSupplemental.

Published under the PNAS license.

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Extensive cellular heterogeneity of X inactivation revealed by single-cell allele-specific expression in human fibroblasts
Marco Garieri, Georgios Stamoulis, Xavier Blanc, Emilie Falconnet, Pascale Ribaux, Christelle Borel, Federico Santoni, Stylianos E. Antonarakis
Proceedings of the National Academy of Sciences Dec 2018, 115 (51) 13015-13020; DOI: 10.1073/pnas.1806811115

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Extensive cellular heterogeneity of X inactivation revealed by single-cell allele-specific expression in human fibroblasts
Marco Garieri, Georgios Stamoulis, Xavier Blanc, Emilie Falconnet, Pascale Ribaux, Christelle Borel, Federico Santoni, Stylianos E. Antonarakis
Proceedings of the National Academy of Sciences Dec 2018, 115 (51) 13015-13020; DOI: 10.1073/pnas.1806811115
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