RESUMO
Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.
RESUMO
Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.
RESUMO
The segregation of eukaryotic genomes into euchromatin and heterochromatin represents a fundamental and poorly understood process. Here, we demonstrate that genome-wide establishment of heterochromatin is triggered by the maternal to zygotic transition (MZT) during zebrafish embryogenesis. We find that prior to MZT, zebrafish lack hallmarks of heterochromatin including histone H3 lysine 9 trimethylation (H3K9me3) and condensed chromatin ultrastructure. Global establishment of heterochromatic features occurs following MZT and requires both activation of the zygotic genome and degradation of maternally deposited RNA. Mechanistically, we demonstrate that zygotic transcription of the micro RNA miR-430 promotes degradation of maternal RNA encoding the chromatin remodeling protein Smarca2, and that clearance of Smarca2 is required for global heterochromatin establishment in the early embryo. Our results identify MZT as a key developmental regulator of heterochromatin establishment during vertebrate embryogenesis and uncover functions for Smarca2 in protecting the embryonic genome against heterochromatinization.
Assuntos
Desenvolvimento Embrionário/genética , Heterocromatina/genética , Peixe-Zebra/embriologia , Animais , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Transcrição Gênica , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Pericentromeric satellite repeats are enriched in 5-methylcytosine (5mC). Loss of 5mC at these sequences is common in cancer and is a hallmark of Immunodeficiency, Centromere and Facial abnormalities (ICF) syndrome. While the general importance of 5mC is well-established, the specific functions of 5mC at pericentromeres are less clear. To address this deficiency, we generated a viable animal model of pericentromeric hypomethylation through mutation of the ICF-gene ZBTB24. Deletion of zebrafish zbtb24 caused a progressive loss of 5mC at pericentromeres and ICF-like phenotypes. Hypomethylation of these repeats triggered derepression of pericentromeric transcripts and activation of an interferon-based innate immune response. Injection of pericentromeric RNA is sufficient to elicit this response in wild-type embryos, and mutation of the MDA5-MAVS dsRNA-sensing machinery blocks the response in mutants. These findings identify activation of the innate immune system as an early consequence of pericentromeric hypomethylation, implicating derepression of pericentromeric transcripts as a trigger of autoimmunity. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Assuntos
Centrômero , Metilação de DNA , Face/anormalidades , Síndromes de Imunodeficiência/patologia , Interferons/metabolismo , Animais , Modelos Animais de Doenças , Face/patologia , Técnicas de Inativação de Genes , Imunidade Inata , Doenças da Imunodeficiência Primária , Peixe-ZebraRESUMO
Kupffer's vesicle (KV) is the zebrafish organ of laterality, patterning the embryo along its left-right (LR) axis. Regional differences in cell shape within the lumen-lining KV epithelium are essential for its LR patterning function. However, the processes by which KV cells acquire their characteristic shapes are largely unknown. Here, we show that the notochord induces regional differences in cell shape within KV by triggering extracellular matrix (ECM) accumulation adjacent to anterior-dorsal (AD) regions of KV. This localized ECM deposition restricts apical expansion of lumen-lining epithelial cells in AD regions of KV during lumen growth. Our study provides mechanistic insight into the processes by which KV translates global embryonic patterning into regional cell shape differences required for its LR symmetry-breaking function.