Highlights d Repeat expansions in transcription factors (TFs) alter their phase separation capacity d Repeat expansions in TFs perturb the composition of TFcontaining condensates d Hoxd13 repeat expansions alter the transcriptional program in a synpolydactyly model d Features of intrinsically disordered regions in TFs are linked to condensation behavior

Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate ‘hijacking’ of transcriptional condensates in various developmental and disease contexts.

Endogenous retroviruses (ERVs) comprise ~10% of mammalian genomes, and ERVs are repressed by multiple cellular mechanisms including heterochromatin, DNA methylation, and methylation of their RNA transcripts 1-13. Although the vast majority of ERVs in mammals are incapable of retrotransposition, ERV de-repression during early development is associated with embryonic lethality 14-16, suggesting that ERV transcription or RNA transcripts may underlie essentiality of ERV repression. Here we report that rapid and selective degradation of the TRIM28 heterochromatin adapter protein in murine embryonic stem cells triggers dissociation of transcriptional condensates from loci encoding super-enhancer -driven pluripotency genes, and association of transcriptional condensates with transcribed ERV loci. Knockdown of ERV RNA or forced expression of super-enhancer -enriched transcription factors rescued condensate localization at super-enhancers in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated phase separation of RNA Polymerase II, and partitioning of NFY and Mediator into heterotypic droplets, suggesting a mechanistic basis for the association of transcriptional condensates with ERVs. Using a zygotic perturbation platform, we found that timing and amount of ERV transcription correlate with the onset of lethality, and that loss of TRIM28 leads to specific depletion of pluripotent lineages in mouse embryos. We propose that retrotransposons contribute to the genomic distribution of nuclear condensates, and that coding and non-coding nascent RNAs may facilitate “hijacking” of transcriptional condensates in various developmental and disease contexts.