Modification of internal RNA domains from the long non-coding RNA XIST refines roles in silencing and heterochromatin recruitment.

Abstract

XIST is a paradigmatic long non-coding RNA required for the initiation of the epigenetic silencing of X chromosomes, leading to dosage compensation in mammalian females. XIST includes tandem repeat domains (A-F) that serve as binding platforms for protein complexes involved in silencing, localization, and heterochromatinization of the inactive X chromosome. XIST/Xist transgenes can silence autosomes as well as the X in both early development and somatic cells. In our quest to create a minimal but highly functional XIST transgene, we have tested both removal of non-repeat regions of XIST and duplication of repeat regions previously established as critical for XIST function. Targeting inducible XIST transgenes to an autosomal FRT site in somatic cells allows us to compare the activity of induced constructs on silencing and chromatin recruitment. We have previously identified that the CCC-dense mouse polycomb-interaction domain (PID) could restore functionality to a minimal human XIST construct lacking the B and D repeats. We now demonstrate that the density of the CCC motifs is not the sole contributor to PID functionality in the human transgene. Surprisingly, a duplication of repeat A reduces the expression from the exogenous promoter, as does removal of the region around the F repeats including six YY1 binding sites. In this context of an inducible XIST in somatic cells the internal small exons were not required; however, duplication of the E repeat region augmented functionality. Overall, the strongest correlations we observed across our diverse set of constructs were between chromatin deposition and silencing.