CIZ1 forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner and is required for accurate maintenance of polycomb targets genome-wide. Here we address requirements for assembly formation and show that CIZ1 undergoes two direct interactions with Xist, via independent N- and C-terminal domains. Interaction with Xist, assembly at Xi, and complexity of self-assemblies formed in vitro are modulated by two alternatively spliced glutamine-rich prion-like domains (PLD1 and 2). PLD2 is dispensable for accumulation at existing CIZ1–Xi assemblies in wild-type cells but is required in CIZ1-null cells where targeting, assembly, and enrichment for H3K27me3 and H2AK119ub occur de novo. In contrast, PLD1 is required for both de novo assembly and accumulation at preexisting assemblies and, in vitro, drives formation of a stable fibrillar network. Together they impart affinity for RNA and a complex relationship with repeat E of Xist. These data show that alternative splicing of two PLDs modulates CIZ1’s ability to build large RNA–protein assemblies.

SummaryCIP1-interacting zinc finger protein 1 (CIZ1) forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner. Here we address the requirements for assembly formation, and show that CIZ1 interacts directly with Xist via two independent domains in its N- and C-terminus. Interaction with Xist repeat E, assembly at Xi in cells, and the complexity of self-assemblies formed in vitro, are all modulated by alternatively-spliced exons that include two glutamine-rich prion-like domains (PLD1 and PLD2), both conditionally excluded from the N-terminal domain. Exclusion of PLD1 alone is sufficient to abrogate de novo establishment of new CIZ1 assemblies and Xi territories enriched for H3K27me3 in CIZ1-null fibroblasts. Together the data suggest that PLD1-driven CIZ1 assemblies form at Xi, are nucleated by interaction with Xist and amplified by multivalent interaction with RNA, so implicating a polyglutamine tract in the maintenance of epigenetic state.