SUMMARY
Mammalian chromosomes are partitioned into A/B compartments and
topologically associated domains (TADs). The inactive X (Xi) chromosome,
however, adopts a distinct conformation without evident compartments or TADs.
Here, through exploration of an architectural protein, SMCHD1, we probe how the
Xi is reconfigured during X-chromosome inactivation. A/B compartments are first
fused into “S1” and “S2” compartments, coinciding
with Xist spreading into gene-rich domains. SMCHD1 then binds S1/S2 compartments
and merges them to create a compartment-less architecture. Contrary to current
views, TADs remain on the Xi but in an attenuated state. Ablating SMCHD1 results
in a persistent S1/S2 organization and strengthening of TADs. Furthermore, loss
of SMCHD1 causes regional defects in Xist spreading and erosion of
heterochromatic silencing. We present a step-wise model for Xi folding, where
SMCHD1 attenuates a hidden layer of Xi architecture to facilitate Xist
spreading.
During X-inactivation, Xist RNA spreads along an entire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct binding partner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topological Xi reconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/ Polycomb spreading, silencing, and changes in chromosome architecture.
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