RESUMEN
The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. We therefore hypothesized that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. We identified three distinct low-complexity regions in Ph based on sequence composition. We systematically tested the role of each of these sequences in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. Our data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. Our results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. Our observations highlight the complexity of regulation of biological condensates housed in single proteins.
RESUMEN
Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Complejo Represivo Polycomb 1/química , Proteínas del Grupo Polycomb/metabolismo , Motivo alfa Estéril/fisiología , Animales , Compartimento Celular , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Polimerizacion , Motivo alfa Estéril/genética , UbiquitinaciónRESUMEN
Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation.
Asunto(s)
ADN/metabolismo , Proteínas de Drosophila/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , ARN/metabolismo , Animales , Drosophila , Proteínas de Drosophila/genética , Embrión no Mamífero/metabolismo , Silenciador del Gen/fisiología , N-Metiltransferasa de Histona-Lisina/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: The symptoms of numerous diseases result from genetic mutations that disrupt the homeostasis maintained by the appropriate integration of signaling gene activities. The relationships between signaling genes suggest avenues through which homeostasis can be restored and disease symptoms subsequently reduced. Specifically, disease symptoms caused by loss-of-function mutations in a particular gene may be reduced by concomitant perturbations in genes with antagonistic activities. METHODOLOGY/PRINCIPAL FINDINGS: Here we use network-neighborhood analyses to predict genetic interactions in Caenorhabditis elegans towards mapping antagonisms and synergisms between genes in an animal model. Most of the predicted interactions are novel, and the experimental validation establishes that our approach provides a gain in accuracy compared to previous efforts. In particular, we identified genetic interactors of gdi-1, the orthologue of GDI1, a gene associated with mental retardation in human. Interestingly, some gdi-1 interactors have human orthologues with known neurological functions, and upon validation of the interactions in mammalian systems, these orthologues would be potential therapeutic targets for GDI1-associated neurological disorders. We also observed the conservation of a gdi-1 interaction between different cellular systems in C. elegans, suggesting the involvement of GDI1 in human muscle degeneration. CONCLUSIONS/SIGNIFICANCE: We developed a novel predictor of genetic interactions that may have the ability to significantly streamline the identification of therapeutic targets for monogenic disorders involving genes conserved between human and C. elegans.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/genética , Transducción de Señal , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Epistasis Genética , Genes de Helminto/genética , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Humanos , Músculos/metabolismo , Músculos/patología , Fenotipo , Interferencia de ARN , Reproducibilidad de los ResultadosRESUMEN
Study of Caenorhabditis elegans embryonic development has been useful to dissect the molecular mechanisms controlling cell proliferation, cell polarization, cell differentiation, and morphogenic events also involved in embryogenesis in human (1, 2). The strength of this organism for developmental research consists in its amenability to large-scale genetic screening, its simple morphology, and transparency enabling study of developmental processes at a single cell level. Large-scale genetic screening targeting embryonic development in C. elegans is usually poorly sensitive and non-quantitative (3, 4).In this chapter we detail a novel approach enabling genetic dissection of C. elegans embryogenesis in a quantitative and semi-automated manner. This approach based on RNAi and flow cytometry enables the measurement of discrete embryonic lethal phenotypes and staging of arrested embryos.