Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice

Abstract

In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.

Imprinted X‐chromosome inactivation impacts primitive endoderm differentiation in mouse blastocysts

“…S2c). These results indicate that although loss of iXCI causes developmental disorder in each developmental condition , the pathways influenced differ between the conditions.…”

“…In contrast to PrE development, we found no differences in EPI development in Xist KO females compared to WT females. Although the inactive X‐chromosome is reactivated in EPI , Xist ‐dependent X‐linked gene silencing occurs before X‐chromosome reactivation . Thus, these results indicate that having both X‐chromosomes continuously active is dispensable for EPI specification in blastocysts.…”

“…DNA FISH experiments were performed as described previously . Briefly, after washing with PBS, the samples were incubated with PBS (−) containing RNase A for 1 h at 37 °C.…”

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice

“…Overexpression of Rnf12 has also been shown to facilitate erasure of the repressive imprint on the maternally inherited Xist allele in XmXm parthenogenetic embryos 25 . This effect was eliminated by collective knockdown of Rnf12 and Rex1 , indicating that REX1 is normally involved in maintenance of Xist repression on the Xm.…”

What makes the maternal X chromosome resistant to undergoing imprinted X inactivation?

“…This experiment suggests that decondensation of the maternal genome by demethylation of H3K9me3 facilitates the expression of Xist on the maternally derived X chromosomes. They further showed by DNA-FISH that the chromosome region spanning the Xist locus becomes condensed during the growth of oocytes at the prophase of meiosis I, when the maternal imprint against X inactivation is established [32,33]. Although the condensed maternal Xist locus relaxes during early preimplantation development, the extent of the decondensation across the Xist loci derived from normally developed oocytes is significantly smaller than the extents across the Xist loci derived from nongrowing oocytes as well as those on the paternal X in androgenetic embryos.…”