Linking Chromosomal Silencing With Xist Expression From Autosomal Integrated Transgenes

Abstract

<i>Xist</i> 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. <i>Xist</i> transgene (Tg) integrated into an autosome can induce transcriptional silencing of flanking genes; however, the effect and mechanism of <i>Xist</i> RNA on autosomal sequence silencing remain elusive. In this study, we investigate an autosomal integration of <i>Xist</i> Tg that is compatible with mouse viability but causes male sterility in homozygous transgenic mice. We observed ectopic <i>Xist</i> 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 <i>in situ</i> Hybridization (FISH) and chromosome painting confirmed that <i>Xist</i> Tg is associated with chromosome 1. To determine the spreading mechanism of autosomal silencing induced by <i>Xist</i> Tg on Chr 1, we analyzed the positions of the transcriptionally repressed chromosomal sequences relative to the <i>Xist</i> Tg location inside the cell nucleus. Our results show that the transcriptionally repressed chromosomal segments are closely proximal to <i>Xist</i> Tg in the three-dimensional nucleus space. Our findings therefore support a model that <i>Xist</i> directs and maintains long-range transcriptional silencing facilitated by the three-dimensional chromosome organization.