When it’s the dead of winter, and the world outside is frozen solid, there’s no better place to be than under a mountain of warm blankets, like an inactivated X chromosome under a cloud of Xist long-noncoding RNA. Although we’ve known for a while that Xist is necessary for X chromosome inactivation (XCI), the mechanisms behind its action have remained tightly under wraps.
Like any good blanket fort, XCI involves multiple layers of support, and a new study from the lab of Edith Heard (European Molecular Biology Laboratory, Heidelberg, Germany) uncovers how the transcriptional repressor SPEN connects multiple layers of epigenetic regulation. The well-rested researchers developed a line of mouse embryonic stem cells (ES cells) where they could induce Xist expression with doxycycline and get rid of endogenous SPEN with an auxin-inducible degron. By allowing Xist to be expressed, in the absence of SPEN, they found that:
- Xist still forms RNA clouds and localizes to the X chromosome but gene silencing is lost
- 80% of X linked genes need SPEN in order to be silenced by Xist, according to allele-specific RNAs-seq
- When HaloTagged SPEN is added to cells with fluorescence-labelled Xist, SPEN localizes with Xist immediately after Xist is induced
- In neural progenitor cells, where one X chromosome is already stably inactivated, degrading SPEN has no impact on Xist, and doesn’t alter X chromosome gene expression, except for the few genes that already escape XCI
Next, the talented team introduced their cells to cDNA fragments coding for specific domains of the SPEN protein to narrow down which regions are the most important for XCI. They found that:
- The RNA binding domains and the SPEN paralogue/orthologue C-terminal (SPOC) domain are necessary for SPEN to accumulate along the X chromosome
- A truncated SPOC domain allows SPEN to localize
to the X chromosome, but fails to rescue XCI
- Mutating the SPOC domain in the endogenous Spen gene in mouse ES cells has the same effect
- Guiding a BglG-tagged SPOC domain to Bgl stem-loops on Xist allows the peptide to inactivate genes on the underlying X chromosome
- The SPOC domain binds to members of NCoR and SMRT repressor complexes, the HDAC3 histone deacetylase, and the RNA m6A reader YTHDC1 according to mass spectrometry
Finally, to understand where it normally binds to the genome, the savvy scientists used CUT&RUN to map SPEN binding sites when Xist was expressed, and found:
- SPEN tends to bind to the promoters of actively expressed genes, 4 hours after Xist induction, and these sites overlap with RNAPII ChIP-seq peaks
- SPEN also binds to enhancers that are deacetylated during XCI, and overlaps with enhancers that are enriched in HDAC3 from separate ChIP-seq data
- Many promoters lose SPEN binding after 24 hours of Xist expression, especially those that belong to genes that are efficiently silenced
Underneath it all, it seems like SPEN’s job is to bring Xist and other epigenetic regulators together with the X chromosome to get cozy and wind things down for inactivation. Cuddle up with the original article in Nature, January 2020.