Two-Step Imprinted X Inactivation: Repeat versus Genic Silencing in the Mouse

Genomic imprinting refers to a parent-of-origin effect on gene expression in the developing embryo (3, 57). The existence of imprinting in the mammal means that male and female gametes contribute significantly different information to the zygote. One important difference is illustrated by X chromosome inactivation (XCI), the mechanism of dosage compensation in the mammal that results in the silencing of one X chromosome in the female embryo (2, 33, 34, 49, 64). While the eutherian form of XCI occurs randomly in the soma, the marsupial form is imprinted to occur exclusively on the paternal X (XP) (54). Imprinted XCI also occurs in some eutherians but is restricted to the preimplantation embryo and the extraembryonic tissues (25, 37, 47, 60). Imprinted XCI precedes random XCI in the early mouse embryo and continues through the placental lineages. In the epiblast (embryo proper), transient X reactivation is followed by random XCI, which accounts for the mosaic pattern of inactivation seen in all somatic tissues of the eutherian. The mechanisms and developmental timing of imprinted XCI remain unclear and are much debated. In principle, the maternal or paternal germ line (or both) may differentially mark the X chromosomes, with the maternal imprint protecting the maternal X (XM) from inactivation and/or the paternal mark predestining XP for inactivation. The search for parent-specific regulators frequently has focused on the X inactivation center (Xic) (7), an X-linked region harboring several noncoding regulators for random XCI. Xist produces a 17-kb noncoding transcript whose accumulation on the X has been associated with the initiation of both random and imprinted XCI (6, 38, 50). In the preimplantation mouse embryo and in the extraembryonic lineages of the postimplantation embryo, Xist is imprinted to be paternally expressed in accordance with preferential XP inactivation (28). The randomization of Xist expression following X reactivation in the epiblast lineage results in mosaic XM and XP inactivation in the embryo proper. The Xic also harbors Tsix, the 40-kb noncoding transcript that is complementary to and negatively regulates Xist (31). In contrast to Xist, Tsix is imprinted to be maternally expressed and therefore may be a maternal factor that protects XM from silencing in the early embryo and extraembryonic tissues (30, 53). Tsix expression also becomes randomized following X reactivation in the epiblast and is expressed exclusively from the future active X (Xa) in the developing embryo proper. In the eutherian embryo, the importance of Xist/Tsix in the imprinting of the X has been borne out by genetic analyses: deleting Xist from XP causes a loss of XP silencing in the placental lineages (38), whereas deleting Xist from XM (on which it is normally silent) has no consequence; conversely, deleting Tsix from XP (on which it is normally silent) has no consequence, whereas deleting Tsix from XM results in ectopic XCI on XM in the placental lineages (30, 53). Thus, for both imprinted and random XCI, Xist designates the future inactive X (Xi), while Tsix designates the future active X (Xa). Although Xic clearly regulates imprinting in eutherians, XIC or an equivalent has yet to be identified in marsupials (11, 12, 22, 55). The absence of a marsupial Xist suggests that an alternative means of silencing the X must occur in mammals. Since the discovery of meiotic sex chromosome inactivation (MSCI) in the male germ line of both eutherian and marsupial mammals (14, 23, 32, 43, 58), several groups have hypothesized a link between MSCI and the imprinting of XP (10, 24, 26, 35, 39). Recent reports that XY silencing persists into the long postmeiotic period of spermatogenesis (16, 42, 62) support the idea that zygotic XP silencing is built in part on MSCI and its aftereffects in the paternal germ line. Because MSCI is Xist independent and Xist is not highly expressed during spermatogenesis (40, 61), germ line-driven silencing would provide an alternative imprinting mechanism that would not require an XIC in the marsupial and would dosage compensate the marsupial zygote from the time of conception. The probability of an XIST-independent mechanism in the marsupial raises intriguing questions for imprinted XCI in eutherians. Did eutherian XCI evolve completely independently, or do vestiges of a marsupial mechanism still exist in the eutherians of today? Although the placental form of imprinted XCI in the mouse clearly depends on Xist (38), the role of Xist in the preimplantation embryo currently is unclear. Indeed, embryos deleted for Xist on XP are normal in the preimplantation stages and perish only after uterine implantation and the outgrowth of a placenta (38), suggesting that the early mouse embryo does not require Xist. There also is debate as to whether mouse XP is inherited from the male germ line in a partially inactive state, further raising the question of whether Xist is required to initiate imprinted XCI in the early mouse embryo (25, 45, 46). Here, we investigate the mechanism of XP silencing in the earliest stages following the gamete-to-embryo transition. We discover that imprinted XP silencing takes place in two sequential steps, one involving repetitive elements and the other involving coding genes, and implicate repeats in the transmission of parental information to the early embryo.