Experimental Analysis of Imprinted Mouse X-Chromosome Inactivation

Abstract

X-chromosome inactivation is a dosage compensation mechanism that equalizes X-linked gene expression between male and female mammals through the transcriptional silencing of most genes on one of the two X-chromosomes in females. With a few key exceptions, once the X-chromosome is inactivated replicated copies of that X-chromosome are maintained as inactive in all descendant cells. X-inactivation is therefore a paradigm of epigenetic inheritance. Imprinted X-inactivation is a specialized form of X-inactivation that results in the silencing of the paternally derived X-chromosome. Due to its parent-of-origin-specific pattern of inactivation, imprinted X-inactivation is a model of mitotic as well as meiotic, i.e., transgenerational, epigenetic inheritance. All cells of the early mouse embryo undergo imprinted X-inactivation, a pattern that is subsequently maintained in extraembryonic cell types in vivo and in vitro. Here, we describe both high- and low-throughput approaches to interrogate imprinted X-inactivation in the mouse embryo as well in cultured extraembryonic stem cells.

Keywords: Allele-specific gene expression analyses; Extraembryonic endoderm (XEN) stem cells; Preimplantation mouse embryogenesis; Trophoblast stem cells (TSCs); X-chromosome inactivation.

Fig. 1

Comparative morphology of TSCs, XEN, and embryonic stem cells (ESCs). Viewed at 20×, TSC and XEN cells can be seen as individual nuclei, compared to the more densely packed colonies of ESCs. TSCs also grow in colonies, while XEN can grow singly as well as in clusters

Fig. 2

Characterization of TSC and XEN and ESCs through immunofluorescence staining. TSCs are marked by CDX2; XEN cells are marked by GATA6; and ESCs are marked with NANOG [26]. Nuclei are stained blue with DAPI

Fig. 3

Sanger sequencing distinguishes genes expressed in a parent-of-origin-specific manner. Top, the two parental strains are characterized by a single nucleotide polymorphism (SNP) in the X-linked Xist gene marked by the blue arrow. Xist encodes a long noncoding (lnc) RNA that is only expressed from the inactive X-chromosome [27]. In female blastocysts, Sanger sequencing of the Xist cDNA shows that only the paternal allele of Xist is expressed

Fig. 4

Representative Pyrosequencing pyrograms for analysis of allele specific gene expression. (a) Pyrogram of Xist cDNA from a hybrid TSC line derived from a cross of the Mus musculus 129/Sv strain (dam) to the Mus molossinus JF1 strain (sire). Xist is only expressed from the paternal (JF1) X-chromosome in TSCs and marks the inactive X-chromosome [8] (“C” allele = 0% expression; “T” allele = 100% expression). The height of the peaks is proportional to the number of the indicated nucleotide at that position. (b) In TSCs from the reverse cross (sire = JF1; dam = 129/Sv), Xist is only expressed from the paternal 129/Sv X-chromosome (“C” allele = 100% expression; “T” allele = 0% expression)

Fig. 5

A summary of the steps in allele-specific RNA-Seq analysis of imprinted X-inactivation