Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions ^1–3 . Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus ^4,5 . This suggests that mutations in disordered proteins may alter condensate properties and function ^6–8 . Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Biomolecular condensates are thought to create subcellular microenvironments that have different physicochemical properties compared with their surrounding nucleoplasm or cytoplasm^1–5. However, probing the microenvironments of condensates and their relationship to biological function is a major challenge because tools to selectively manipulate specific condensates in living cells are limited^6–9. Here, we develop a non-natural micropeptide (that is, the killswitch) and a nanobody-based recruitment system as a universal approach to probe endogenous condensates, and demonstrate direct links between condensate microenvironments and function in cells. The killswitch is a hydrophobic, aromatic-rich sequence with the ability to self-associate, and has no homology to human proteins. When recruited to endogenous and disease-specific condensates in human cells, the killswitch immobilized condensate-forming proteins, leading to both predicted and unexpected effects. Targeting the killswitch to the nucleolar protein NPM1 altered nucleolar composition and reduced the mobility of a ribosomal protein in nucleoli. Targeting the killswitch to fusion oncoprotein condensates altered condensate compositions and inhibited the proliferation of condensate-driven leukaemia cells. In adenoviral nuclear condensates, the killswitch inhibited partitioning of capsid proteins into condensates and suppressed viral particle assembly. The results suggest that the microenvironment within cellular condensates has an essential contribution to non-stoichiometric enrichment and mobility of effector proteins. The killswitch is a widely applicable tool to alter the material properties of endogenous condensates and, as a consequence, to probe functions of condensates linked to diverse physiological and pathological processes.

Quercus suber (cork oak) is a West Mediterranean species of key economic interest, being extensively explored for its ability to generate cork. Like other Mediterranean plants, Q. suber is significantly threatened by climatic changes, imposing the need to quickly understand its physiological and molecular adaptability to drought stress imposition. In the present report, we uncovered the differential transcriptome of Q. suber roots exposed to long-term drought, using an RNA-Seq approach. 454 sequencing reads were used to de novo assemble a reference transcriptome, and mapping of reads allowed the identification of 546 differentially expressed unigenes. These were enriched in both effector genes (e.g. LEA, chaperones, transporters) as well as regulatory genes, including transcription factors (TFs) belonging to various different classes, and genes associated with protein turnover. To further extend functional characterization, we identified the orthologs of differentially expressed unigenes in the model species Arabidopsis thaliana, which then allowed us to perform in silico functional inference, including gene network analysis for protein function, protein subcellular localization and gene co-expression, and in silico enrichment analysis for TFs and cis-elements. Results indicated the existence of extensive transcriptional regulatory events, including activation of ABA-responsive genes and ABF-dependent signaling. We were then able to establish that all components of a core ABA-signaling pathway involving PP2C-SnRK2-ABF components was induced in stressed Q. suber roots, identifying a key mechanism in this species’ response to drought.

Transcription factors (TFs) control specificity and activity of gene transcription, but whether a relationship between these two features exists is unclear. Here we provide evidence for an evolutionary trade-off between the activity and specificity in human TFs encoded as submaximal dispersion of aromatic residues in their intrinsically disordered protein regions. We identified approximately 500 human TFs that encode short periodic blocks of aromatic residues in their intrinsically disordered regions, resembling imperfect prion-like sequences. Mutation of periodic aromatic residues reduced transcriptional activity, whereas increasing the aromatic dispersion of multiple human TFs enhanced transcriptional activity and reprogramming efficiency, promoted liquid–liquid phase separation in vitro and more promiscuous DNA binding in cells. Together with recent work on enhancer elements, these results suggest an important evolutionary role of suboptimal features in transcriptional control. We propose that rational engineering of amino acid features that alter phase separation may be a strategy to optimize TF-dependent processes, including cellular reprogramming.

Modifications of DNA and histones, including methylation and acetylation, are critical for the epigenetic regulation of gene expression during plant development, particularly during environmental adaptation processes. However, information on the enzymes catalyzing all these modifications in trees, such as Quercus suber L., is still not available. In this study, eight DNA methyltransferases (DNA Mtases) and three DNA demethylases (DDMEs) were identified in Q. suber. Histone modifiers involved in methylation (35), demethylation (26), acetylation (8), and deacetylation (22) were also identified in Q. suber. In silico analysis showed that some Q. suber DNA Mtases, DDMEs and histone modifiers have the typical domains found in the plant model Arabidopsis, which might suggest a conserved functional role. Additional phylogenetic analyses of the DNA and histone modifier proteins were performed using several plant species homologs, enabling the classification of the Q. suber proteins. A link between the expression levels of each gene in different Q. suber tissues (buds, flowers, acorns, embryos, cork, and roots) with the functions already known for their closest homologs in other species was also established. Therefore, the data generated here will be important for future studies exploring the role of epigenetic regulators in this economically important species.

Modifications of DNA and histones, including methylation and acetylation, are critical for the epigenetic regulation of gene expression during plant development, particularly during environmental adaptation processes. However, information on the enzymes catalyzing all these modifications in perennial trees, such as Quercus suber , is still not available. In this study, several epigenetic modifier proteins, including eight DNA methyltransferases (DNA Mtases), three DNA demethylases (DDMEs) and ninety-one histone modifiers including thirty-five histone methyltransferases (HMTs), twenty-six histone demethylases (HDMTs), eight histone acetyltransferases (HATs) and twenty-two histone acetylases (HDACs) were identified in Q. suber . Phylogenetic analyses of the DNA and histone modifier proteins were performed using several plant species homologs, enabling the classification of the Q. suber proteins. Additional in silico analysis showed that some Q. suber DNA Mtases, DMEs and histone modifiers have the typical domains found in the plant model Arabidopsis, which might suggest a conserved functional role. A link between the expression levels of each gene in different Q. suber tissues (buds, flowers, acorns, embryos, cork and roots) with the functions already known for their closest homologs in other species was also established. Therefore, the data generated here are important for future studies exploring the role of epigenetic regulators in this economically important species.