Abstract

Xist is the master regulator of X-Chromosome Inactivation (XCI), the mammalian dosage compensation mechanism that silences one of the two X chromosomes in a female cell. XCI is established during early embryonic development. Xist transgene (Tg) integrated into an autosome can induce transcriptional silencing of flanking genes; however, the effect and mechanism of Xist RNA on autosomal sequence silencing remain elusive. In this study, we investigate an autosomal integration of Xist Tg that is compatible with mouse viability but causes male sterility in homozygous transgenic mice. We observed ectopic Xist expression in the transgenic male cells along with a transcriptional reduction of genes clustered in four segments on the mouse chromosome 1 (Chr 1). RNA/DNA Fluorescent in situ Hybridization (FISH) and chromosome painting confirmed that Xist Tg is associated with chromosome 1. To determine the spreading mechanism of autosomal silencing induced by Xist Tg on Chr 1, we analyzed the positions of the transcriptionally repressed chromosomal sequences relative to the Xist Tg location inside the cell nucleus. Our results show that the transcriptionally repressed chromosomal segments are closely proximal to Xist Tg in the three-dimensional nucleus space. Our findings therefore support a model that Xist directs and maintains long-range transcriptional silencing facilitated by the three-dimensional chromosome organization.

Highlights

  • In 1961, Marie France Lyon proposed that in mammals, one of the two X chromosomes in females is inactivated to offset the difference in X-linked gene dose between male and female (Lyon, 1961)

  • We analyzed the transcriptional consequence of the ectopic Xist expression in the transgenic mouse line Tg2087, which carries two copies of the X inactivation center (Xic) sequence integrated in a single autosomal locus

  • Experimental models incorporating transgenes of the Xic sequence are foundational in defining the functional elements and mechanisms of X-chromosome inactivation (XCI) as previous studies have shown that the integration of Xic in an autosomal region can lead to Xist RNA expression (Heard et al, 1996; Lee and Jaenisch, 1997; Sun et al, 2015; Loda et al, 2017)

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Summary

Introduction

In 1961, Marie France Lyon proposed that in mammals, one of the two X chromosomes in females is inactivated to offset the difference in X-linked gene dose between male and female (Lyon, 1961) Such a dosage-compensation strategy is called X-chromosome inactivation (XCI). Xist is located on the X chromosome in the X inactivation center (Xic) locus that contains a cluster of five non-coding RNA genes, Ftx, Jpx, Xist, Tsix, and Tsx, which are involved in regulating XCI in eutherian mammals (Brockdorff et al, 1992; Brown et al, 1992; Wutz et al, 2002; Payer and Lee, 2008; Sun et al, 2013; Furlan et al, 2018). Xist RNA is Autosomal Silencing by Transgenic Xist capable of inducing chromosomal silencing when it is ectopically transcribed from a transgene in an autosome, recapitulating the XCI effects outside the X chromosome (Lee and Jaenisch, 1997; Okamoto et al, 2005; Sun et al, 2015)

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