Expression-based assay of an X-linked gene to examine effects of the X-controlling element (Xce) locus.

Through an incompletely understood process, one of the two X chromosomes (Chrs) in mammalian XX female cells is inactivated, thereby achieving dosage equivalence with XY males (Lyon 1961). Studies of X;autosome translocations and of chromosomally abnormal embryos suggest that trans-acting autosomal factors interact with cis-acting X-linked elements to “choose” one X to remain active; all additional X Chrs subsequently undergo X inactivation (Russell 1963; Lyon 1971; Rastan 1983). Despite the expected complexity of these events, thus far only two genetic loci, the X-controlling element (Xce) locus (Cattanach and Isaacson 1967) and the Xist gene (Brockdorff et al. 1991; Brown et al. 1991), are known to influence this process (reviewed in Brockdorff and Duthie 1998; Carrel and Willard 1998; Goto and Monk 1998; Panning and Jaenisch 1998). The Xce locus influences the choice process such that there is an equal probability that either parental X Chr will be inactivated in Xce homozygotes, whereas one parental X is preferentially inactivated in Xce heterozygotes (Cattanach and Isaacson 1967). The relative strengths of the known Xce alleles are Xce < Xce < Xce, with an X carrying the stronger allele demonstrating an increased probability of being the active X (Cattanach et al. 1969; Cattanach and Williams 1972; Johnston and Cattanach 1981). Xce allele strength was originally defined and has primarily been assayed by counting vibrissae in mice heterozygous for an X-linked mutation, Tabby, which affects vibrissae number (Cattanach et al. 1969). Alternative assays have examined protein polymorphisms (West and Chapman 1978), chromosomal markers (Nesbitt and Gartler 1970; Kanda 1973), gene expression (Singer-Sam et al. 1992; Buzin et al. 1994; Avner et al. 1998), or DNA methylation patterns (Courtier et al. 1995; Avner et al. 1998) as a measure or correlate of Xce allele strength. Using these assays, the Xce alleles of several inbred strains have been reported as Xce (CBA/H, C3H/HeH, and BALB/cH), Xce (C57BL/6H and DBA/2H), and Xce-like (CAST/Ei) (Cattanach et al. 1969; West and Chapman 1978; Johnston and Cattanach 1981; Cattanach and Rasberry 1991, 1994). The AKR/H Xce allele has been reported to be intermediate between Xce and Xce (Fowlis et al. 1991), although rigorous Xce typing has not been performed in this strain. Xist is a key gene in the X inactivation pathway. Both knockout and transgenic studies have shown that Xist expression, in the correct developmental context, is both necessary and sufficient for X inactivation (Lee et al. 1996; Penny et al. 1996; Herzing et al. 1997; Marahrens et al. 1997; Clerc and Avner 1998). While Xist itself may be the inactivation signal, additional developmentally regulated factors appear to be required for X inactivation (Panning et al. 1997; Sheardown et al. 1997; Clemson et al. 1998; Hansen et al. 1998; Tinker and Brown 1998). Mutations in or including the Xist gene cause preferential inactivation of one parental X Chr in both the mouse (Clerc and Avner 1998; Marahrens et al. 1998) and human (Plenge et al. 1997), implying that it is involved in the choice process (Carrel and Willard 1998). Xce appears to map close to, but downstream of, the Xist gene itself (Simmler et al. 1993). It is unclear how (or whether) the two loci interact to control the choice process, although recent work has focused on a series of differentially methylated repeated sequences downstream of the Xist gene as a possible candidate for Xce (Courtier et al. 1995; Avner et al. 1998). Further, an antisense transcript, termed Tsix, which initiates in this region and continues through the Xist locus on the opposite strand, has been proposed to be an antisense regulator of Xist (Lee and Lu 1999). Targeted deletions downstream of Xist suggest that this region is also involved in the choice process (Clerc and Avner 1998; Lee and Lu 1999). As a necessary prelude to attempts to define further the role of these loci and to search for additional factors in the X inactivation pathway, a robust assay is needed to measure and quantify X inactivation patterns early in embryogenesis. One useful approach is to measure the proportion of cells expressing alleles from one or the other X Chr (i.e., the X Chr inactivation pattern) in adult somatic cells. Current assays, however, are limited by their inability to detect subtle differences in X inactivation patterns or by insufficient throughput necessary for large-scale screens (Cattanach and Isaacson 1967; Cattanach et al. 1969; Nesbitt and Gartler 1970; Cattanach and Williams 1972; Kanda 1973; West and Chapman 1978; Singer-Sam et al. 1992). Although several expression-based assays have been developed (e.g., Singer-Sam et al. 1992; Buzin et al. 1994; Avner et al. 1998), limited characterization of relative Xce allele strength has been performed at the level of transcription or RNA abundance. To overcome these limitations and to further characterize the Xce locus, we have developed a quantitative assay that measures allele-specific RNA levels of a ubiquitously expressed gene subject to X Chr inactivation, Pctk (Carrel et al. 1996). Our data establish the Pctk X inactivation assay as a quantitative expression-based assay to measure X inactivation patterns in the mouse and provide the foundation for future experiments to search for additional X inactivation genes.