Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome

Abstract

The long non-coding RNA Xist exploits numerous effector proteins to progressively induce gene silencing across the X chromosome and form the inactive X (Xi)-compartment. The mechanism underlying formation of the chromosome-wide Xi-compartment is poorly understood. Here, we find that formation of the Xi-compartment is induced by ∼50 locally confined granules, where two Xist RNA molecules nucleate supra-molecular complexes (SMCs) of interacting proteins. Xist-SMCs are transient structures that concentrate rapidly recycling proteins in the X by increasing protein binding affinity. We find that gene silencing originates at Xist-SMCs and propagates across the entire chromosome over time, achieved by Polycomb-mediated coalescence of chromatin regions and aggregation, via its intrinsically disordered domains, of the critical silencing factor SPEN. Our results suggest a new model for X chromosome inactivation, in which Xist RNA induces macromolecular crowding of heterochromatinizing proteins near distinct sites which ultimately increases their density throughout the chromosome. This mechanism enables deterministic gene silencing without the need for Xist ribonucleoprotein complex-chromatin interactions at each target gene.

RNA polymerase II depletion from the inactive X chromosome territory is not mediated by physical compartmentalization

“…Taken together, our results suggest that the exclusion of RNAPII from the inactive X chromosome is not caused by a homogenous biophysical compartment-sequestering function, but rather by a modification of its chromatin that protects/represses RNAPII binding events. This is in agreement with the fact that the multivalent protein complexes involved in X inactivation decorate small chromatin domains in a manner not compatible with the formation of a large phase separated whole chromosome domains ( 13 ).…”

“…The inactive X chromosome has previously been shown to be a heterogeneous structure (27), formed of distinct, tightly packed heterochromatin domains (28). In addition, Xist-associated proteins have been shown to form supra-molecular complexes through protein-protein interactions, which was proposed to increase molecular crowding and participate in chromatin compaction (Markaki et al 2020). Indeed, molecular condensates have been the subject of much attention recently.Phase separated protein domains are seen as regions where multivalent protein-protein interactions can modulate mass action, tweaking the "on rates" of transcription factors binding to DNA (29).…”

“…Protein-RNA and protein-protein interactions have been proposed to allow their accumulations through liquid-liquid phase separation, forming so-called membraneless organelles such as the nucleolus or stress granules. A number of Xist recruited factors, including SPEN, PRC1 and PTBP1-MATR3, form low affinity protein-protein interactions ( 12, 13 ), suggested to increase molecular crowding in the Xist compartment and participate in the compaction of chromatin ( 13 ). Accordingly, phase separation has been proposed to underlie the compartmentalization of the inactive X ( 14 ).…”

“…The inactive X has previously been shown to be more compact than its active counterpart (~ 1.2 fold compaction) ( 25, 26 ). In addition, Xist -recruited proteins such as SPEN, Ciz1 and PRC1 have recently been shown to form supra-molecular complexes upon their recruitment on the inactive X ( 13 ). Increased molecular crowding, due to protein complexes and/or higher chromatin density, could constrain the diffusion of RNAPII ( Fig 4A ).…”

“…Furthermore, the inactive X chromosome has been shown to occupy a smaller volume (~1.2 fold) than its active counterpart in differentiated cells in culture and in vivo blastocyst stage embryos ( 33 ) ( 25 ) ( 26 ). Indeed, recent reports suggest that this compaction occurs only after several days of differentiation ( 13 ), probably through the late recruitment of the SMCHD1 protein ( 34 ) ( 35 ). These factors could affect RNAPII diffusion and lead to the formation of a biophysical compartment.…”