Unusual chromatin status and organization of the inactive X chromosome in murine trophoblast giant cells

Abstract

SUMMARYMammalian X-chromosome inactivation (XCI) enables dosage compensation between XX females and XY males. It is an essential process and its absence in XX individuals results in early lethality due primarily to extra-embryonic defects. This sensitivity to X-linked gene dosage in extra-embryonic tissues is difficult to reconcile with the reported tendency of escape from XCI in these tissues. The precise transcriptional status of the inactive X chromosome in different lineages has mainly been examined using transgenes or in in vitro differentiated stem cells and the degree to which endogenous X-linked genes are silenced in embryonic and extra-embryonic lineages during early postimplantation stages is unclear. Here we investigate the precise temporal and lineage-specific X-inactivation status of several genes in postimplantation mouse embryos. We find stable gene silencing in most lineages, with significant levels of escape from XCI mainly in one extra-embryonic cell type: trophoblast giant cells (TGCs). To investigate the basis of this epigenetic instability, we examined the chromatin structure and organization of the inactive X chromosome in TGCs obtained from ectoplacental cone explants. We find that the Xist RNA-coated X chromosome has a highly unusual chromatin content in TGCs, presenting both heterochromatic marks such as H3K27me3 and euchromatic marks such as histone H4 acetylation and H3K4 methylation. Strikingly, Xist RNA does not form an overt silent nuclear compartment or Cot1 hole in these cells. This unusual combination of silent and active features is likely to reflect, and might underlie, the partial activity of the X chromosome in TGCs.

Transcriptional Analysis by Nascent RNA FISH of <em>In Vivo</em> Trophoblast Giant Cells or <em>In Vitro</em> Short-term Cultures of Ectoplacental Cone Explants

“…Defects in TGC differentiation cause embryonic lethality (for review see reference 15 ). Transcriptional activity of TGCs using RNA FISH allowed us to demonstrate an unusual X chromosome inactivation status of such cell type during mouse development 3 .…”

“…We have successfully used this method of RNA FISH on another type of TGCs, such as primary TGCs which appear at an earlier stage of mouse development, E3.0 blastocyts, which can be individually cultured for 4-5 days during which developed outgrowth, the ICM being surrounded by large primary TGCs 16,17 . Nascent transcripts for different genes as well as Xist could be visualized and quantified using the same RNA FISH approach 3 .…”

“…Combined immunofluorescence and RNA FISH can also be performed on cryostat sections as well as in vitro cultured TGCs 3 . This demonstrates that the method is quite robust, as primary transcripts are highly susceptible to degradation and there is an absolute requirement of RNase free compounds.…”

“…Indeed, TGCs are endoreplicating cells, able to undergo multiple rounds of DNA synthesis without any division 2 . In order to investigate the transcriptional status of genes in vivo, in TGCs compared to other embryonic and extraembryonic cell types, nascent RNA FISH can be performed in vivo on cryostat sections 3 at specific post-implantation stages. TGCs are easily recognizable on sections due to their large size and their transcriptional gene activity can be recorded but their number at early post-implantation stages is low.…”

“…RNA FISH to analyze nuclear primary transcripts has never been done at the level of the single cell level on secondary TGCs. This allows precise analysis of transcription and was used to show the epigenetic instability of TGCs at post-implantation stages 3 .…”