Summary A new level of chromosome organization, Topologically Associating Domains (TADs), was recently uncovered by chromosome-confirmation-capture (3C) techniques. To explore TAD structure and function, we developed a polymer model that can extract the full repertoire of chromatin conformations within TADs from population-based 3C data. This model predicts actual physical distances and to what extent chromosomal contacts vary between cells. It also identifies interactions within single TADs that stabilize boundaries between TADs and allows us to identify and genetically validate key structural elements within TADs. Combining the model’s predictions with high-resolution DNA FISH and quantitative RNA FISH for TADs within the X-inactivation center (Xic), we dissect the relationship between transcription and spatial proximity to cis-regulatory elements. We demonstrate that contacts between potential regulatory elements occur in the context of fluctuating structures rather than stable loops and propose that such fluctuations may contribute to asymmetric expression in the Xic during X inactivation.

The long non-coding RNA Xist is only expressed from the paternal X chromosome in mouse preimplantation female embryos and leads to its transcriptional silencing. In females, absence of Xist leads to post-implantation lethality. Here we report that the initiation of imprinted XCI absolutely requires Xist using single-cell RNA-sequencing of early pre-implantation mouse embryos. Lack of paternal Xist leads to genome-wide transcriptional misregulation in the early blastocyst, with failure to activate the extra-embryonic pathway that is essential for post-implantation development. We also demonstrate that the expression dynamics of X-linked genes depends both on strain and parent-of-origin, as well as on location along the X chromosome, particularly at Xist's first "entry" sites. This study demonstrates that dosage compensation failure has an impact as early as the blastocyst stage and reveals genetic and epigenetic contributions in orchestrating the transcriptional silencing of the X chromosome during early embryogenesis.